6-cyanodopamine as an Endogenous Modulator of Heart Chronotropism and Inotropism

6-cyanodopamine as an Endogenous Modulator of Heart Chronotropism and Inotropism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 6-cyanodopamine as an Endogenous Modulator of Heart Chronotropism and Inotropism José Britto-Júnior, Antonio Tiago Lima, Denis Lima Oliveira, Fernanda V Mariano, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5375589/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose- Rat isolated atria and ventricles releases endothelium-derived 6-nitrodopamine and this novel catecholamine induces a potent endogenous positive chronotropic and inotropic response. 6-Cyanodopamine is released from rabbit isolated atria and ventricles, however it is not known whether this novel catecholamine has any action on the isolated heart. Therefore, it was investigated whether rat isolated atria and ventricles release 6-cyanodopamine and its action on the rat isolated heart. Methods- Basal release of 6-cyanodopamine was assessed by LC-MS/MS and tyrosine hydroxylase by both immunohistochemistry and fluorescence in situ hybridization. Chronotropic and inotropic effects were evaluated in isolated atria and Langendorff’s preparation, respectively. Results- Rat isolated ventricles presented basal release of 6-cyanodopamine, which was unaffected by pre-treatment with tetrodotoxin. Immunohistochemistry and fluorescence in situ hybridization identified tyrosine hydroxylase expression in both the endothelium and in the cardiomyocytes. 6-Cyanodopamine at 10 and 100 pM induced increases in the atrial rate, which were maintained even at 30min after the preparation was washed. In the Langendorff’s preparation, 1min infusion of 6-cyanodopamine (10 and 100pM) significantly increased heart frequency, LVDP, and dP/dt(max). Bolus injection of noradrenaline (1pmol) had no effect on heart frequency, LVDP and dPdt(max). Infusion of 6-cyanodopamine (0.01pM) significantly increased heart frequency, LVDP, and dP/dt(max) when noradrenaline (1pmol) was injected at the end of the infusion. Conclusion- The results indicate that 6-cyanodopamine is a potent endogenous mediator of both chronotropism and inotropism in the rat isolated heart. It has potential therapeutic effect in heart failure and may be useful as a biomarker of pathophysiological processes. Synergism Endothelium-derived catecholamines Tyrosine hydroxylase NADPH oxidase NOX1/4 Inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The autonomic nervous system is classically supposed to play a major role on the control of heart function (Herring et al., 2019 ). Catecholamines such as dopamine, noradrenaline, and adrenaline are reported to be produced only in the central nervous system, adrenergic nerve fibers and adrenal medulla (Ungar & Phillips, 1983 ; Gorain et al., 1983), which upon appropriate sympathetic stimulation act on α- and β-adrenoceptors (Ahlquist, 1948 ; Molinoff, 1948; Motiejunaite et al., 2021 ). However, the identification of endothelium-derived catecholamines has changed this paradigm (Zatz & De Nucci, 2024 ). For instance, rat isolated atria and ventricles releases endothelium-derived 6-nitrodopamine and this novel catecholamine induces a potent endogenous positive chronotropic and inotropic response (Britto-Júnior et al., 2022a; 2023 a; 2023 b). Inhibition of nitric oxide synthase (NOS) by L-NAME reduces both the basal release of 6-nitrodopamine and atrial basal rate (Britto-Júnior et al., 2023 a; 2023 b). The voltage-gated sodium channel blocker tetrodotoxin did not affect the release of 6-nitrodopamine from mouse and rat isolated hearts, indicating a non-neurogenic source for this catecholamine (Britto-Júnior et al., 2022a; 2023 a; 2023 b). Interestingly, 6-nitrodopamine markedly synergizes with the classical catecholamines dopamine, noradrenaline, and adrenaline in the rat right isolated atrium, resulting in long-lasting increases in atrial rate (Britto-Júnior et al., 2023 c). 6-Cyanodopamine belongs to a novel class of catecholamines that is released by rat isolated vas deferens (Dal Pozzo et al., 2024 ) and by rabbit isolated atria and ventricles (Júnior et al., 2023 ), however it is not known whether this novel catecholamine has activity on the cardiovascular system. Here it was investigated whether rat isolated ventricles release 6-cyanodopamine, and whether this novel endogenous catecholamine modulates heart chronotropism and inotropism. 2. Materials and Methods 2.1. Animals Adult male Wistar rats (280 to 320 g) were obtained from the Central Animal House at University of Campinas (CEMIB-UNICAMP; São Paulo, Brazil). All experimental protocols were approved by the Ethics Committee for Animal Use of the UNICAMP (CEUA; Protocol No. 5746-1/2021; 5831-1/2021) following the Brazilian Guidelines for the Production, Maintenance and Use of Animals for Teaching or Research from the National Council of Control in Animal Experimentation (CONCEA; Andersen, 2016 ) as well as by following the ARRIVE guidelines (Percie du Sert et al, 2020 ). Three individuals were housed in each cage placed on ventilated shelters at a humidity of 55 ± 5% and a temperature of 24 ± 1°C under a 12-hour light-dark cycle. Animals received filtered water and standard rodent food ad libitum . 2.2. Basal release of 6-cyanodopamine, 6-nitrodopamine, dopamine, noradrenaline, and adrenaline from rat isolated ventricles by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) Animals were exposed to a concentration greater than 5% isoflurane until 1 min after breathing stopped. Exsanguination was performed to confirm the euthanasia. Next, the chest was opened, and the heart was rapidly excised. The right and left ventricles were isolated en bloc from each rat heart and suspended in a 5 mL organ bath containing Krebs–Henseleit solution (KHS; in mM: NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO 4 1.7, NaHCO 3 24.9, KH 2 PO 4 1.2, dextrose 11, sodium pyruvate 2, pH 7.4) continuously gassed with a mixture (95% O2: 5% CO2) at 37 ◦C, supplemented with ascorbic acid (3 mM) to prevent catecholamine oxidation. After 30 min, two 2-mL aliquots of KHS solution were transferred to black Eppendorf tubes and stored at -20°C until analysis. Release of catecholamines was also evaluated from isolated ventricles obtained from animals chronically treated with L-NAME (20 mg/rat/day for 4 weeks; Ribeiro et al., 1992 ). The effects of the voltage-gated sodium channel blocker tetrodotoxin (TTX; 1 µM), hydrogen peroxide (H 2 O 2 , 100 µM) and the dual NADPH oxidase NOX1/4 Inhibitor GKT137831 (1 µM) on the catecholamine release were also investigated in the isolated ventricles. The levels of catecholamines in KHS were determined by LC-MS/MS. Briefly, the extraction of the catecholamines from 1 mL of KHS was performed by solid phase extraction. A total of 50 µL of the internal standards (100 ng/mL of catecholamines) were added to 1 mL of KHS. The samples were homogenized for 10 sec. The Strata™-X 33 mm Polymeric Reversed SPE cartridges were preconditioned with 1 mL of methanol and then balanced by 2 mL of deionized water. The samples were injected into the cartridge, and the cartridge was subsequently washed 3 times with deionized water. The samples were then eluted with 0.9 mL methanol/water (90/10, v/v) with 0.1% formic acid. The mobile phase perfused a LC ADVp Liquid Chromatography Shimadzu System (Shimadzu Corp., Kyoto, JP) coupled to a Shimadzu 8060 Triple Quadrupole Mass Spectrometer operating in ESP + mode at 350 µL/min. The dissolved residues were injected by a SIL-30AC autoinjector, at 8 ºC. The transitions monitored by electrospray multiple reaction monitoring (MRM), injection volume, run-time, limit of quantitation and method validation following USFDA guidelines for bioanalytical methods (FDA) were described elsewhere (Junior et al., 2023). As an improvement to the original method, two transitions (a quantifier and a qualifier transition) were monitored to increase selectivity (Dal Pozzo et al., 2024 ). 2.3. Rat isolated right atrium preparation and measurement of chronotropic responses Animals were anaesthetized as previously described. The right atrium was isolated from the heart and mounted between two metal hooks in 10-mL custom designed glass chambers containing KHS solution, continuously gassed with a mixture of 95%O 2 : 5%CO 2 at 37°C using a heated circulator (PolyScience, Illinois, USA). Tissues were allowed to equilibrate under a resting tension of 10 mN for one hour, and the isometric tension was registered using a PowerLab system (ADInstruments, Sydney, Australia; Britto-Júnior et al., 2022a). After an equilibration period was achieved, single concentrations of 6-cyanodopamine (1, 10, and 100 pM) were added to the organ bath, and changes in atrial rate were monitored for 30 min. In separate set of experiments, following the 30-min incubation with 6-cyanodopamine (10 and 100 pM), the KHS was replaced to wash out the agonist, and the atrial rate was monitored for an additional 30 min. A separate atrium was used for each drug concentration. In order to investigate the synergism between 6-cyanodopamine with noradrenaline, adrenaline, and dopamine in the rat isolated atrial rate, the following experimental protocols were employed. A single concentration of dopamine (1 pM), noradrenaline (1 pM) or adrenaline (1 pM) was added to the organ bath, and changes in atrial rate were monitored for 30 min. Next, 6-cyanodopamine (0.001 and 0.01 pM) was co-incubated with either noradrenaline, adrenaline or dopamine, (1 pM each), and the resulting changes in atrial rate were observed for 30 min. A separate atrium was used for each drug and each concentration. Data of atria rate was expressed as rate (bpm) before and after the respective stimulation or delta increase of atrial rate (bpm). 2.4. Langendorff’s isolated perfused heart preparation and measurements of heart contractile function Heparin (1000 IU/kg) was injected intraperitoneally in the animals to prevent blood clotting and euthanasia was performed by isoflurane overdose, as previously described. Exsanguination was performed to confirm the euthanasia. The chest was opened, and the heart was rapidly excised, the ascending aorta was cannulated, and the heart was mounted in a nonrecirculating Langendorff apparatus. The isolated heart was perfused with KHS (pH 7.4, 37°C) equilibrated with carbogen gas mixture (95% O 2 : 5% CO 2 ) at constant flow (10 ml/min) and left ventricular end-diastolic pressure (LVEDP) was maintained between 4–8 mmHg during the initial equilibrium of the experiment (Britto-Júnior et al., 2023 a). A water-filled latex balloon, connected to the pressure transducer (MLT1199 BP Transducer, ADInstruments, Inc., Dunedin, NZ) was inserted into the left ventricle (LV) via the mitral valve. Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVeDP) and heart rate (HR) were continuously recorded by a PowerLab System (ADInstruments, Inc., Dunedin, NZ). Only hearts that presented a basal heart rate between 250–300 bpm were employed in the experiments. The hearts were allowed to equilibrate for at least 10 min. Afterward, single bolus (10 µL) of 6-cyanodopamine (0.01, 0.1, 1, or 10 pmol) was injected, with each heart receiving only one bolus. In a separate set of experiments, the effects of a one-minute infusion (100 µL/min) of 6-cyanodopamine (0.1, 1, 10, 100, or 100 pM, final concentration) were evaluated. Each heart was subjected to only one infusion. Changes were monitored for 30 min. In order to investigate the synergism between 6-cyanodopamine with noradrenaline in the Langendorff’s-perfused heart analysis, the following protocols were employed. One-minute (100 µL/min)-infusion of either 6-cyanodopamine (0.001 or 0.01 pM, final concentration) were performed and then a single bolus of noradrenaline (1 pmol) was administered, and the heart was monitored for 15 min. One heart was used for a single drug and a single infusion. Data obtained with the Landendorff preparations (heart rate, LVDP, dP/dt max and RPP) were expressed as left ventricular developed pressure (LVDP) was calculated by the following formula: LVSP-LVeDP and expressed in mmHg. Rate pressure product (RPP) was defined as the product of heart rate (HR) and left ventricular developed pressures (LVDP) RPP = (HR × LVDP). The maximal rate of rise of the left ventricular pressure (+ dP/dt max ) was monitored continuously by a pressure transducer connected to a Powerlab system (AD Instrument, Australia). 2.5. Immunohistochemistry Rat heart samples (N = 5) were fixed in 10% neutral buffered formalin for 24h at 25 o C, dehydrated, embedded in paraffin wax and sectioned at 4µm. Subsequently, these sections were deparaffinized in xylene and rehydrated in a series of ethanol baths of decreasing concentration. They were treated for 10 minutes with 3% H 2 O 2 to neutralize endogenous peroxidase. Then, they were washed and incubated in citrate buffer at pH 6.0 (regardless of primary antibody) in a steamer set for 20min (at approximately 95 o C). Following this, the sections were incubated for 2h at 25 o C with a chicken polyclonal anti - tyrosine hydroxylase (ab76442; 1:500; Abcam, USA). Tissue sections were sequentially incubated with goat anti-chicken gamma immunoglobulin IgG (ab150169; 1:500 diluted in PBS, Abcam, USA), a rabbit anti-goat IgG (AP106P; 1:250 diluted in PBS, Merck/Sigma, USA), for 1h each, and the detection system. The detection system used was the NovoLink™ Max Polymer Detection System (catalog code RE7280-k, Leica Biosystems, UK), following the manufacturer’s instructions, and using diaminobenzidine (liquid DAB, DakoCytomation, Carpenteria, USA) as chromogen (which renders a brown precipitate at the antibody binding site). Subsequently, the sections were counter-stained with Harris´s hematoxylin and coverslipped with Entellan mounting medium. Negative controls consisted in the omission of the primary antibody and incubation with the primary antibody diluents (one negative control per section) to identify any unspecific background staining. All slides were examined using a trinocular Eclipse 50i microscope (Nikon, Tokyo, Japan) coupled to a 10MP CMOS digital camera (AmScope, EUA). 2.6. In situ hybridization (FISH) To confirm and validate TH protein expression and its topography, we futher investigated TH mRNA expression in the same samples (N = 3), using FISH assay. Briefly, sections from 4 rat seminal vesicles were deparaffinized with xylene and rehydrated in graded alcohols for 5 minutes each. Then, they were incubated in a 0.2 N HCl solution for 20 minutes, and subsequently treated with a citrate pH 6.0 buffer (ZytoVision kit, catalog code Z-2028-20, Germany) at 80ºC for 1 hour. After this, they were incubated with pepsin for 8 minutes at room temperature. The slides were washed with 2XSSC (ZytoVision kit, catalog code Z-2028-20, Germany). They were subsequently submitted to a sequence of ethanols (75%, 80%, and 100% ethanol for 2 minutes each), and then air dried. The slides were further incubated with a probe to TH mRNA (at a concentration of 10 µM, in RNAse-free water) for 10 minutes at 75ºC and overnight in a Dako Hybridizer (Dako, Denmark) at 37ºC. The probe sequence was as follows: 5′- AACCGCGGGGACATGATGGCCT-3′ (RNA Tm = 77.8°C) (Batch: WD11655417, Sigma/Merck, Germany). The probe was labeled with fluorescein 6-FAM in the 5’ region. The next day, the slides were placed in a UREA/0,1Xssc solution at 45°C for 30 minutes, and then, they were washed with a 2xSSC solution for 2 minutes. After this, the slides were dehydrated in 75%, 85%, and 100% ethanols for 2 minutes each, and air dried. Finally, the slides were mounted with 15 µL of a DAPI containing mounting medium (from the ZytoVision kit) and cover slipped (the cover slip being sealed with a Fixogum Rubber Cement, from Marabu, Germany) (Britto-Junior et al., 2020). Negative controls consisted of the omission of the probe and were performed in all FISH assays (one negative control per section) to control for any significant autofluorescence. All FISH slides were examined and photomicrographed at the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas, using an upright fluorescence microscope (Axioskop, Carl Zeiss AG, Germany) coupled to a digital camera (AxioCam MRc, Zeiss, Germany). Images were collected using 350–495 nm laser lines for excitation and 465–517 emission filters for two fluorophores (DAPI and 6-FAM) and 200x optical zoom. 2.7. Chemical and reagents Adrenaline, dopamine, GKT137831, noradrenaline, and tetrodotoxin (TTX) were obtained from Cayman Chemicals (Michigan, USA). 6-Nitrodopamine and 6-nitrodopamine-d 4 were acquired from Toronto Research Chemicals (Ontario, CA). N ω -Nitro-L-arginine methyl ester (L-NAME) was obtained from Sigma-Aldrich Chemicals Co (St Louis, Missouri, USA). Hydrogen peroxide (H 2 O 2 ) was bought from Exodo Cientifica (Sumaré, São Paulo, Brazil). Dopamine-d 3 hydrochloride, DL‐noradrenaline‐d 6 hydrochloride and adrenaline‐d 6 hydrochloride were acquired from CDN Isotopes (Quebec, CA). 6-cyanodopamine was synthesized at Rhodes College (Rote et al., 2017 ). Strata™-X 33 mm Polymeric Reversed SPE cartridges were bought from Phenomenex (California, USA) and GIST-HP C 18 columns were obtained from Shimadzu (Duisburg, Germany). Calcium chloride (CaCl 2 ), dextrose, magnesium sulfate (MgSO 4 ), potassium chloride (KCl), sodium bicarbonate (NaHCO 3 ), potassium phosphate monobasic (KH 2 PO 4 ) and sodium chloride (NaCl), were bought from Merck KGaA (Darmstadt, Germany). Acetonitrile and methanol were obtained from J.T. Baker (Phillipsburg, NJ, USA) and formic acid from Mallinckrodt (St Louis, Missouri, USA). The composition of the KHS was in mM: NaCl 118, KCl 4.7, CaCl 2 2.5, MgSO 4 1.2, NaHCO 3 25, KH 2 PO 4 1.2 and dextrose 5.6. 2.9. Statistical Analysis Data represent the mean ± standard error of the mean (SEM). Comparison between baseline values to values obtained during drug stimulation in the same sample was performed by paired t -test. Comparison between two groups was performed by unpaired t -test. Comparisons among three or more groups were evaluated using one-way analysis of variance (ANOVA), followed by Newman-Keuls test. P < 0.05 was taken as statistically significant. 3. Results 3.1. Basal release of 6-cyanodopamine and 6-nitrodopamine from rat isolated ventricles Rat isolated ventricles presented basal release of both 6-cyanodopamine and 6-nitrodopamine, but the levels 6-nitrodopamine were significantly higher than 6-cyanodopamine (Fig. 1 ). In isolated ventricles obtained from animals chronically treated with L-NAME, the basal release of 6-cyanodopamine was unaffected (Fig. 2 A), whereas that of 6-nitrodopamine was significantly reduced (Fig. 2 B). Pre-treatment (30 min) of the ventricles with tetrodotoxin (TTX; 1 µM) affected neither the release of 6-cyanodopamine (Fig. 2 C) nor of 6-nitrodopamine (Fig. 2 D). Pre-treatment (30 min) with either H 2 O 2 (100 µM) or the NOX1/4 inhibitor GKT137,831 (1 µM) significantly decreased the basal release of 6-cyanodopamine (Fig. 2 E and Fig. 2 G, respectively), whereas the levels of 6-nitrodopamine significantly increased by these treatments (Fig. 2 F and Fig. 2 H, respectively). Levels of dopamine, noradrenaline, adrenaline were below the limit of quantitation (LOQ, 0.1 ng/mL). 3.2. Chronotropic effect of 6-cyanodopamine on the rat isolated right atrium Pre-incubation (30 min) of the rat isolated right atrium with 6-cyanodopamine (10 and 100 pM) induced concentration-dependent increases in the atrial rate (Fig. 2 A). The increases in atrial rate induced by 6-cyanodopamine were prolonged, since they are maintained even at 30 min after the preparation was washed (Fig. 2 B). Pre-incubation (30 min) of the atria with low concentrations of noradrenaline (1 pM; Fig. 3 A), adrenaline (1 pM; Fig. 3 B), and dopamine (1 pM; Fig. 3 C) did not affect the atrial basal frequency. Co-incubation (30 min) of the atria with 6-cyanodopamine at 0.001 pM and noradrenaline, adrenaline or dopamine had no effect in the atrial basal rate (Fig. 3 A-C). However, co-incubation of the atria with higher concentrations of 6-cyanodopamine (0.01, 0.1 and 1 pM) resulted in significant increases in atrial rate in response to noradrenaline, adrenaline or dopamine (Fig. 4 A-C). 3.3. Heart contractile effects by 6-cyanodopamine in comparison with 6-nitrodopamine in the Langendorff’s preparation Figure 5 shows data of heart rate (Fig. 5 A-B), LVDP (Fig. 5 C-D), dP/dt max (Fig. 5 E-F) and rate-pressure product (RPP; Fig. 5 G-H) obtained in the Langendorff preparation. Bolus injection of 6-cyanodopamine at 0.01 to 1 pmol had no effect on heart rate, but 10 pmol resulted in a significant increase in heart rate (Fig. 5 A). Bolus injections of 0.1, 1, and 10 pmol of 6-cyanodopamine led to significant increases in both LVDP (Fig. 5 C) and dP/dt (max) (Fig. 5 E). The RPP parameter was significantly elevated at 0.1, 1, and 10 pmol of 6-cyanodopamine (Fig. 5 G). Next, testing of 6-cyanodopamine will proceed as a one-minute infusion instead of a bolus injection. Infusion (1 min) of 6-cyanodopamine (0.1 and 1 pM) did not significantly affect heart rate (Fig. 5 B); however, at higher concentrations (10 and 100 pM), significant increases in heart rate were observed (Fig. 5 B). One-min infusion of 6-cyanodopamine at 0.1 pM had no significant effect on either LVDP (Fig. 5 D) or dP/dt(max) (Fig. 5 F), but the infusion at higher concentrations (1, 10, and 100 pM) provoked significant increases in both LVDP and dP/dt(max) (Fig. 5 D and F). Infusion (1 min) of 6-cyanodopamine (0.1 and 1 pM) had no significant effect on the rate-pressure product (RPP; Fig. 5 H). Nonetheles K s, higher concentrations (10 and 100 pM) resulted in significant increases in RPP (Fig. 5 ). 3.4. Interactions of 6-cyanodopamine with noradrenaline on the rat isolated heart (Langendorff’s preparation) Bolus injection of noradrenaline at 1 pmol had no effect on heart rate frequency (Fig. 6 A), LVDP (Fig. 6 B), dPdt(max) (Fig. 6 D) and RPP (Fig. 6 E). One-min infusion of 6-cyanodopamine (0.001 pM) alone did not alter any of these parameters either. However, infusion of 6-cyanodopamine (0.01 pM) significantly increased the heart rate frequency (Fig. 6 A), LVDP (Fig. 6 B), dP/dt(max) (Fig. 6 C), and RPP (Fig. 6 D) when noradrenaline (1 pmol) was injected at the end of the infusion (1 min). 3.5. Immunohistochemistry and in situ hybridization (FISH) Figure 7 and Table 1 summarize the results of the immunohistochemical detection of tyrosine hydroxylase (TH) in rat heart. Briefly, TH was positive in endocardium (Fig. 7 A), myocardium (Fig. 7 A-C), coronary endothelium (Fig. 7 B) and epicardial nerves (Fig. 7 C). The negative control (omission of primary antibody) is illustrated in Figs. 7 D-F. Occasionally, a weak background staining was observed in intravascular serum, but this nonspecific reaction did not compromise the interpretation of the slides and was probably related to secondary antibody of the detection system, since it was also present in control sections (Table 1 , Fig. 7 A-F). To validate the immunohistochemistry (IHC) findings, we used fluorescence in situ hydridization (FISH) in 3 samples of rat heart. As shown in Fig. 8 , TH mRNA expression was observed in myocardium, endocardium, and coronary endothelial cells, as well as in epicardial nerves of rat heart, thus confirming IHC derived data. Table 1 Detection of Tyrosine hydroxylase (TH) by immunohistochemistry (IHC) and FISH. Rat heart Negative control (omission of the primary antibody) N = 5 TH protein detection by IHC N = 5 TH mRNA detection by FISH N = 3 Endocardium (-) (+) (+) Myocardium (-) (+) (+) Coronary endothelium (-) (+) (+) Epicardium nerves (-) (+) (+) 4. Discussion The results clearly demonstrated that rat isolated ventricles present basal release of 6-cyanodopamine, and this release has some important characteristics. For instance, the levels of 6-cyanodopamine are approximately 20 times smaller than that of 6-nitrodopamine. It is interesting that in the rabbit isolated atria the levels of 6-cyanodopamine were also smaller than that of 6-nitrodopamine, although the difference was smaller (3 times; Júnior et al., 2023 ). In both rabbit isolated ventricles and in rat isolated vas deferens (Dal Pozzo et al., 2024 ), there were no significant differences in the amounts of 6-cyanodopamine and 6-nitrodopamine released. The finding that the release of 6-cyanodopamine was not affected by pre-incubation of the heart with tetrodotoxin, indicates a non-neurogenic source for this novel catecholamine. Indeed, in the mouse isolated atria and ventricles, the release of 6-nitrodopamine was decreased only eNOS −/− mice, but not in nNOS −/− or iNOS −/− mice (Britto-Júnior et al., 2023 b). In contrast, in the rat isolated vas deferens the release of 6-nitrodopamine was virtually abolished when the tissue was pre-treated with tetrodotoxin, whereas that of 6-cyanodopamine was unaffected (Dal Pozzo et al., 2024 ). Thus, it seems that 6-cyanodopamine in the heart is produced mainly by the either the endothelium and/or cardiomyocytes. Indeed, as demonstrated here by both immunohistochemistry and fluorescence in situ hybridization, both types of cells do express tyrosine hydroxylase. It is clearly established that NO synthase activity is important for 6-nitrodopamine biosynthesis, since pre-treatment of the tissues with L-NAME significantly inhibits 6-ND basal release (Zatz & De Nucci et al., 2024). The results here presented show that this enzyme is not directly involved in the biosynthesis of 6-cyanodopamine, since in contrast to 6-nitrodopamine basal release, ventricles obtained from animals chronically treated with L-NAME released similar levels of 6-cyanodopamine as compared to ventricles obtained from control animals. However, there is some relationship apparently between 6-cyanodopamine and 6-nitrodopamine synthesis/release, since the pre-incubation of the ventricles with hydrogen peroxide (H 2 O 2 ) and the NOX1,4 inhibitor GKT-137831 (Jiang et al., 2012 ), that causes significant increases in 6-nitrodopamine release from rat isolated atria (Britto-Júnior et al., 2024 ), induced significant decreases in the basal release of 6-cyanodopamine. One possible explanation is that for these distinct effects observed could be that 6-cyanodopamine takes place mainly inside the cell, whereas the “nitration” responsible for the 6-nitrodopamine biosynthesis occurs in the external plasma membrane of the endothelium. It is interesting that cyanide has been proposed as a potential gasotransmitter in the stomach (Zuhra & Szabo, 2022 ), and healthy volunteers present cyanide levels from 300 nM (NATIONAL RESEARCH COUNCIL et al 2002 ) to 2–8 µM (Fasco et al., 2011 ). However, the metabolic pathway(s) that could lead to synthesis of the cyano-catecholamines is(are) at present unknown. As an endogenous positive chronotropic and inotropic agent per se, 6-cyanodopamine is approximately ten times less potent that 6-nitrodopamine, but more potent than the classical catecholamines dopamine, noradrenaline, and adrenaline (Britto-Júnior 2022a), and it shares both the same prolonged positive chronotropic effect of 6-nitrodopamine and the ability to potentiate the positive chronotropic effect induced by noradrenaline. What could be the mechanism(s) by which 6-cyanodopamine and 6-nitrodopamine facilitate the positive chronotropic effects of noradrenaline? b 1 -adrenoceptor activation in the sinoatrial nodal cells occurs following noradrenaline release from sympathetic nerve terminals in the heart. The primary pathway following b 1 -adrenoceptor stimulation involves Gs protein/adenylyl-cyclase (AC)/cAMP/ protein kinase A (PKA) route (Taskén & Aandahl 2004 ). Interestingly, 6-nitrodopamine potentiates the positive chronotropic effect of dopamine, noradrenaline, and adrenaline, and at 0.01 pM, abolished that induced by phosphodiesterase type 3 (PDE3) inhibitors, such as cilostazol (Saitoh et al., 1993 ), dipyridamole (Clarke et al., 1994 ), and milrinone (Remme wt al., 1992). 6-Nitrodopamine (0.01 pM) did not affect the concentration-dependent positive chronotropic effect induced by the PDE4 inhibitor rolipram (Wachtel et al., 1982) in the rat isolated atria (Britto-Júnior 2022c), indicating a selective action on PDE3. Although apparently paradoxical, this result is not surprising, since PDE3 inhibitors do not affect the positive chronotropic effect induced by catecholamine activation of b 1 -adrenoceptors (Kaumann et al., 2009 ). The remarkable synergism observed with the classical catecholamines and the differential effect on the PDE3 and PD4 inhibitors-induced positive chronotropic effect, suggest that one possible mechanism of action of these novel catecholamines could be a receptor mediated modulation of adenylyl cyclase. The concentration of cyclic AMP in sinoatrial cells is significantly larger when compared to atrial or ventricular myocytes obtained from rabbit hearts (Taniguchi et al., 1977 ), and inhibition of adenylyl cyclase by MDL-12,330A abolishes basal sinoatrial nodal cells beating (Vinogradova et al., 2006 ). Cyclic nucleotide phosphodiesterases (PDEs) are responsible for the metabolism of both cAMP (Butcher & Sutherland, 1962 ) and cGMP (Uckert & Kuczyk, 2011 ) and although eleven PDE families have been identified (Bender & Beavo, 2006 ), in the rat cardiomyocytes cAMP is essentially hydrolysed by PDE 3 and PDE 4 (Rochais et al., 2006 ). Platelets possess PDE2, PDE3 and PDE5 isoforms (Gresele et al., 2015), but incubation of human platelets with 6-ND does not cause increase in either cAMP or cGMP contents (Nash et al., 2022 ), indicating that it is unlikely that 6-ND could act as a PDE inhibitor. The use of an agarose coupled to 6-nitrodopamine for purification of cardiomyocyte membranes revealed selective binding to three proteins that modulate adenylyl cyclase, such as cyclase associated protein 1 (CAP1), cyclase associated protein 2 (CAP2), and stromal interaction protein 1 (STIM1; Sweed et al., 2024 ). Cyclase associated protein 1 (CAP1) binds and activates adenylyl cyclase in mammalian cells (Zhang et al., 2021 ). Ablation of CAP2 in mice causes dilated cardiomyopathy associated with severe reduction of the heart rate (Peche et al., 2017). Stromal interaction protein (STIM1) is expressed in cardiomyocytes (Liu et al., 2023 ), has a single transmembrane domain (Hooper et al., 2000 ), it is located in the sarcoplasmic reticulum and plasma membrane (Soboloff et al., 2012 ), and it is also associated with adenylyl cyclase activation (Motiani et al., 2018 ). These proteins are interesting candidates for the proposed novel catecholamines receptor mediated modulation of adenylyl cyclase. This mechanism could also explain the apparent lack of concentration-dependent effect of both catecholamines as chronotropic and inotropic agents. Investigations of the binding of these novel catecholamines in cardiomyocytes where the expression of these proteins has been blocked may provide some further clues of this novel proposed mechanism. It is interesting that patients with chronic kidney disease present circulating levels of 6-cyanodopamine (Ribeiro et al., 2024 ). The finding here reported that is novel catecholamine acts as a potent positive chronotropic and inotropic agent in the isolated heart suggests that besides its potential therapeutic effect in heart failure, it may be useful as a biomarker of pathophysiological processes. Declarations Ethical Approval All experimental protocols were authorized by the Ethics Committee in Animal Use of UNICAMP (CEUA/UNICAMP, protocol number 5746-1/2021; 5831-1/2021). Consent to Participate Not applicable. Consent to Publish The authors authorize the submission and publication of this article Naunyn-Schmiedeberg's Archives of Pharmacology Code availability section Not applicable Author Contributions Statement Conceptualization: JBJ, GDN. Data curation: JBJ, GDN. Formal analysis: GDN Funding acquisition: EA, GDN. Investigation: JBJ, ATL, DLO, FVM, VBS, AAS, LWP, MS, RF, TW, EA, GDN Methodology: JBJ, AAS, GDN. Project administration: GDN. Supervision: EA. Visualization: EA, GDN. Writing – original draft: JBJ, TW, EA, GDN. The authors declare that all data were generated in-house and that no paper mill was used. Funding JBJ thanks FAPESP for post-doctoral fellowship (2021/14414-8). EA thanks FAPESP (2017/15175-1). GDN thanks FAPESP (2019/16805-4) and CNPq (303839/2019-8). Competing interests The authors declare no competing or financial interests Availability of data and materials The authors authorize the availability of any data used in this study. References Herring N, Kalla M, Paterson DJ. The autonomic nervous system and cardiac arrhythmias: current concepts and emerging therapies. Nat Rev Cardiol. 2019 Dec;16(12):707-726. doi: 10.1038/s41569-019-0221-2. Epub 2019 Jun 13. Erratum in: Nat Rev Cardiol. 2019 Dec;16(12):760. doi: 10.1038/s41569-019-0293-z. Erratum in: Nat Rev Cardiol. 2019 Dec;16(12):760. doi: 10.1038/s41569-019-0297-8. PMID: 31197232. Ungar A, Phillips JH. Regulation of the adrenal medulla. Physiol Rev. 1983 Jul;63(3):787-843. doi: 10.1152/physrev.1983.63.3.787. PMID: 6308693. Gorain, B., Dutta, S., Nandy, U., Sengupta, P., Choudhury, H. (2020). Pharmacology of Adrenaline, Noradrenaline, and Their Receptors. In: Kumar, P., Deb, P.K. (eds) Frontiers in Pharmacology of Neurotransmitters. Springer, Singapore. https://doi.org/10.1007/978-981-15-3556-7_4 Ahlquist RP. A study of the adrenotropic receptors. Am J Physiol. 1948 Jun;153(3):586-600. doi: 10.1152/ajplegacy.1948.153.3.586. PMID: 18882199. Molinoff PB. Alpha- and beta-adrenergic receptor subtypes properties, distribution and regulation. Drugs. 1984;28 Suppl 2:1-15. doi: 10.2165/00003495-198400282-00002. PMID: 6098436. Motiejunaite J, Amar L, Vidal-Petiot E. Adrenergic receptors and cardiovascular effects of catecholamines. Ann Endocrinol (Paris). 2021 Jun;82(3-4):193-197. doi: 10.1016/j.ando.2020.03.012. Epub 2020 Mar 18. PMID: 32473788. Zatz R, De Nucci G. Endothelium-Derived Dopamine and 6-Nitrodopamine in the Cardiovascular System. Physiology (Bethesda). 2024 Jan 1;39(1):44-59. doi: 10.1152/physiol.00020.2023. Epub 2023 Oct 24. PMID: 37874898; PMCID: PMC11283902. Britto-Júnior J, de Oliveira MG, Dos Reis Gati C, Campos R, Moraes MO, Moraes MEA, Mónica FZ, Antunes E, De Nucci G. 6-NitroDopamine is an endogenous modulator of rat heart chronotropism. Life Sci. 2022a Oct 15;307:120879. doi: 10.1016/j.lfs.2022.120879. Epub 2022 Aug 10. PMID: 35963299. Britto-Júnior J, Medeiros-Teixeira LR, Lima AT, Dassow LC, Lopes-Martins RÁB, Campos R, Moraes MO, Moraes MEA, Antunes E, De Nucci G. 6-Nitrodopamine Is the Most Potent Endogenous Positive Inotropic Agent in the Isolated Rat Heart. Life (Basel). 2023a Oct 4;13(10):2012. doi: 10.3390/life13102012. PMID: 37895394; PMCID: PMC10607994. Britto-Júnior J, Pereira do Prado GL, Chiavegatto S, Cunha F, Moraes MO, Elisabete A Moraes M, Monica FZ, Antunes E, De Nucci G. The importance of the endothelial nitric oxide synthase on the release of 6-nitrodopamine from mouse isolated atria and ventricles and their role on chronotropism. Nitric Oxide. 2023b Sep 1;138-139:26-33. doi: 10.1016/j.niox.2023.06.001. Epub 2023 Jun 2. PMID: 37269938. Britto-Júnior J, Lima AT, Fuguhara V, Monica FZ, Antunes E, De Nucci G. Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria. Naunyn Schmiedebergs Arch Pharmacol. 2023c Jun;396(6):1279-1290. doi: 10.1007/s00210-023-02394-9. Epub 2023 Jan 31. PMID: 36719453. Dal Pozzo CFS, Junior JEM, Britto-Júnior J, Badin JFA, de Souza VB, Schenka AA, Peterson LW, Fregonesi A, Antunes E, De Nucci G. Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility. Pflugers Arch. 2024 Aug;476(8):1263-1277. doi: 10.1007/s00424-024-02985-2. Epub 2024 Jul 4. PMID: 38963545. Júnior GQ, Britto-Júnior J, Magalhaes TB, Campos R, Nyamkondiwa KL, Klugh KL, Peterson LW, Corvino A, Sparaco R, Frecentese F, Caliendo G, De Nucci G. Measurement of 6-cyanodopamine, 6-nitrodopa, 6-nitrodopamine and 6-nitroadrenaline by LC-MS/MS in Krebs-Henseleit solution. Assessment of basal release from rabbit isolated right atrium and ventricles. Biomed Chromatogr. 2023 Sep;37(9):e5691. doi: 10.1002/bmc.5691. Epub 2023 Jul 10. PMID: 37431086. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, Clark A, Cuthill IC, Dirnagl U, Emerson M, Garner P, Holgate ST, Howells DW, Karp NA, Lazic SE, Lidster K, MacCallum CJ, Macleod M, Pearl EJ, Petersen OH, Rawle F, Reynolds P, Rooney K, Sena ES, Silberberg SD, Steckler T, Würbel H. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. Br J Pharmacol. 2020 Aug;177(16):3617-3624. doi: 10.1111/bph.15193. Epub 2020 Jul 14. PMID: 32662519; PMCID: PMC7393194. Andersen ML. Guia brasileiro de produção, manutenção ou utilização de animais em atividade de ensino ou pesquisa cientifica, Conselho nacional de controle de experimentação animal. Brasília: Ministério da Ciência, Tecnologia e Inovação (2016) Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension. 1992 Sep;20(3):298-303. doi: 10.1161/01.hyp.20.3.298. PMID: 1516948. Britto-Júnior J, Pinheiro DHA, Justo AFO, Figueiredo Murari GM, Campos R, Mariano FV, de Souza VB, Schenka AA, Mónica FZ, Antunes E, De Nucci G. Endothelium-derived dopamine modulates EFS-induced contractions of human umbilical vessels. Pharmacol Res Perspect. 2020 Aug;8(4):e00612. doi: 10.1002/prp2.612. PMID: 32567793; PMCID: PMC7307250. Rote JC, Malkowski SN, Cochrane CS, Bailey GE, Brown NS, Cafiero M, Peterson LW (2017) Catechol reactivity: Synthesis of dopamine derivatives substituted at the 6-position. Synth Com- mun 47(5):435–441 Jiang JX, Chen X, Serizawa N, Szyndralewiez C, Page P, Schröder K, Brandes RP, Devaraj S, Török NJ. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo. Free Radic Biol Med. 2012 Jul 15;53(2):289-96. doi: 10.1016/j.freeradbiomed.2012.05.007. Epub 2012 May 19. PMID: 22618020; PMCID: PMC3392471. Britto-Júnior J, Furlaneto R, Lima AT, de Oliveira MG, Severino B, Frecentese F, Fiorino F, Caliendo G, Muscará MN, De Nucci G. GKT137831 and hydrogen peroxide increase the release of 6-nitrodopamine from the human umbilical artery, rat-isolated right atrium, and rat-isolated vas deferens. Front Pharmacol. 2024 Apr 5;15:1348876. doi: 10.3389/fphar.2024.1348876. PMID: 38645555; PMCID: PMC11026650. Zuhra K, Szabo C. The two faces of cyanide: an environmental toxin and a potential novel mammalian gasotransmitter. FEBS J. 2022 May;289(9):2481-2515. doi: 10.1111/febs.16135. Epub 2021 Aug 5. PMID: 34297873; PMCID: PMC9291117. NATIONAL RESEARCH COUNCIL et al. Hydrogen Cyanide: Acute Exposure Guideline Levels. In: Acute Exposure Guideline Levels for Selected Airborne Chemicals: Volume 2. National Academies Press (US), 2002 Fasco MJ, Stack RF, Lu S, Hauer CR 3rd, Schneider E, Dailey M, Aldous KM. Unique cyanide adduct in human serum albumin: potential as a surrogate exposure marker. Chem Res Toxicol. 2011 Apr 18;24(4):505-14. doi: 10.1021/tx100344e. Epub 2011 Mar 18. PMID: 21366342; PMCID: PMC3089968. Britto-Júnior J, Lima AT, Fuguhara V, Monica FZ, Antunes E, De Nucci G. Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria. Naunyn Schmiedebergs Arch Pharmacol. 2023c Jun;396(6):1279-1290. doi: 10.1007/s00210-023-02394-9. Epub 2023 Jan 31. PMID: 36719453. Taskén K, Aandahl EM. Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev. 2004 Jan;84(1):137-67. doi: 10.1152/physrev.00021.2003. PMID: 14715913. Saitoh S, Saito T, Otake A, Owada T, Mitsugi M, Hashimoto H, Maruyama Y. Cilostazol, a novel cyclic AMP phosphodiesterase inhibitor, prevents reocclusion after coronary arterial thrombolysis with recombinant tissue-type plasminogen activator. Arterioscler Thromb. 1993 Apr;13(4):563-70. doi: 10.1161/01.atv.13.4.563. PMID: 8385480. Clarke WR, Uezono S, Chambers A, Doepfner P. The type III phosphodiesterase inhibitor milrinone and type V PDE inhibitor dipyridamole individually and synergistically reduce elevated pulmonary vascular resistance. Pulm Pharmacol. 1994 Apr;7(2):81-9. doi: 10.1006/pulp.1994.1009. PMID: 8081075. Remme WJ, van Hoogenhuyze DC, Kruijssen HA, Pieper PG, Bruggeling WA. Preload-dependent hemodynamic effects of milrinone in moderate heart failure. Cardiology. 1992;80(2):132-42. doi: 10.1159/000174991. PMID: 1611632. Kaumann AJ, Galindo-Tovar A, Escudero E, Vargas ML. Phosphodiesterases do not limit beta1-adrenoceptor-mediated sinoatrial tachycardia: evidence with PDE3 and PDE4 in rabbits and PDE1-5 in rats. Naunyn Schmiedebergs Arch Pharmacol. 2009 Nov;380(5):421-30. doi: 10.1007/s00210-009-0445-5. Epub 2009 Aug 20. PMID: 19693491. Wachtel H. Characteristic behavioural alterations in rats induced by rolipram and other selective adenosine cyclic 3', 5'-monophosphate phosphodiesterase inhibitors. Psychopharmacology (Berl). 1982;77(4):309-16. doi: 10.1007/BF00432761. PMID: 6182575. Taniguchi T, Fujiwara M, Ohsumi K. Possible involvement of cyclic adenosine 3':5'-monophosphate in the genesis of catecholamine-induced tachycardia in isolated rabbit sinoatrial node. J Pharmacol Exp Ther. 1977 Jun;201(3):678-88. PMID: 194027. Vinogradova TM, Lyashkov AE, Zhu W, Ruknudin AM, Sirenko S, Yang D, Deo S, Barlow M, Johnson S, Caffrey JL, Zhou YY, Xiao RP, Cheng H, Stern MD, Maltsev VA, Lakatta EG. High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells. Circ Res. 2006 Mar 3;98(4):505-14. doi: 10.1161/01.RES.0000204575.94040.d1. Epub 2006 Jan 19. PMID: 16424365 Butcher RW, Sutherland EW. Adenosine 3',5'-phosphate in biological materials. I. Purification and properties of cyclic 3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3',5'-phosphate in human urine. J Biol Chem. 1962 Apr;237:1244-50. PMID: 13875173. Uckert S, Kuczyk MA. Cyclic nucleotide metabolism including nitric oxide and phosphodiesterase-related targets in the lower urinary tract. Handb Exp Pharmacol. 2011;(202):527-42. doi: 10.1007/978-3-642-16499-6_23. PMID: 21290241. Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev. 2006 Sep;58(3):488-520. doi: 10.1124/pr.58.3.5. PMID: 16968949. Rochais F, Abi-Gerges A, Horner K, Lefebvre F, Cooper DM, Conti M, Fischmeister R, Vandecasteele G. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes. Circ Res. 2006 Apr 28;98(8):1081-8. doi: 10.1161/01.RES.0000218493.09370.8e. Epub 2006 Mar 23. PMID: 16556871; PMCID: PMC2099453. Gresele P, Momi S, Falcinelli E. Anti-platelet therapy: phosphodiesterase inhibitors. Br J Clin Pharmacol. 2011 Oct;72(4):634-46. doi: 10.1111/j.1365-2125.2011.04034.x. PMID: 21649691; PMCID: PMC3195739. Nash CES, Antunes NJ, Coelho-Silva WC, Campos R, De Nucci G. Quantification of cyclic AMP and cyclic GMP levels in Krebs-Henseleit solution by LC-MS/MS: Application in washed platelet aggregation samples. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 15;1211:123472. doi: 10.1016/j.jchromb.2022.123472. Epub 2022 Sep 27. PMID: 36215878. Sweed D, Elhamed SMA, Aiad HAS, Ehsan NA, Hemida AS, Dawoud MM. STIM1/SOX2 proteins are co-expressed in the tumor and microenvironmental stromal cells of pancreatic ductal adenocarcinoma and ampullary carcinoma. World J Surg Oncol. 2024 Mar 26;22(1):84. doi: 10.1186/s12957-024-03356-y. PMID: 38532463; PMCID: PMC10964627. Zhang X, Pizzoni A, Hong K, Naim N, Qi C, Korkhov V, Altschuler DL. CAP1 binds and activates adenylyl cyclase in mammalian cells. Proc Natl Acad Sci U S A. 2021 Jun 15;118(24):e2024576118. doi: 10.1073/pnas.2024576118. PMID: 34099549; PMCID: PMC8214675. Soboloff J, Rothberg BS, Madesh M, Gill DL. STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol. 2012 Sep;13(9):549-65. doi: 10.1038/nrm3414. PMID: 22914293; PMCID: PMC3458427. Ribeiro LF, Babadopulos T, de Oliveira MG, Nishimaru F, Zatz R, Elias RM, Moraes O, Moraes E, Peterson LW, De Nucci G. A LC-MS/MS method for the simultaneous determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma and its clinical application in patients with chronic kidney disease. Biomed Chromatogr. 2024 Aug;38(8):e5896. doi: 10.1002/bmc.5896. Epub 2024 May 26. PMID: 38797868. Motiani RK, Tanwar J, Raja DA, Vashisht A, Khanna S, Sharma S, Srivastava S, Sivasubbu S, Natarajan VT, Gokhale RS. STIM1 activation of adenylyl cyclase 6 connects Ca 2+ and cAMP signaling during melanogenesis. EMBO J. 2018 Mar 1;37(5):e97597. doi: 10.15252/embj.201797597. Epub 2018 Jan 8. PMID: 29311116; PMCID: PMC5830913. Peche VS, Holak TA, Burgute BD, Kosmas K, Kale SP, Wunderlich FT, Elhamine F, Stehle R, Pfitzer G, Nohroudi K, Addicks K, Stöckigt F, Schrickel JW, Gallinger J, Schleicher M, Noegel AA. Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy. Cell Mol Life Sci. 2013 Feb;70(3):527-43. doi: 10.1007/s00018-012-1142-y. Epub 2012 Sep 4. Erratum in: Cell Mol Life Sci. 2017 Nov;74(21):4045. doi: 10.1007/s00018-017-2630-x. PMID: 22945801; PMCID: PMC11113306. Liu P, Yang Z, Wang Y, Sun A. Role of STIM1 in the Regulation of Cardiac Energy Substrate Preference. Int J Mol Sci. 2023 Aug 25;24(17):13188. doi: 10.3390/ijms241713188. PMID: 37685995; PMCID: PMC10487555. Hooper JD, Scarman AL, Clarke BE, Normyle JF, Antalis TM. Localization of the mosaic transmembrane serine protease corin to heart myocytes. Eur J Biochem. 2000 Dec;267(23):6931-7. doi: 10.1046/j.1432-1033.2000.01806.x. PMID: 11082206. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Peterson","email":"","orcid":"","institution":"Rhodes College","correspondingAuthor":false,"prefix":"","firstName":"Larryn","middleName":"W.","lastName":"Peterson","suffix":""},{"id":375847081,"identity":"10b044e8-61a5-4b41-95f2-2a501a5dae30","order_by":7,"name":"Maria Skovbjerg Slot","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Skovbjerg","lastName":"Slot","suffix":""},{"id":375847082,"identity":"14e45ea0-3317-46e5-96a3-516908996b48","order_by":8,"name":"Rebecca Fjord","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Fjord","suffix":""},{"id":375847083,"identity":"a73ef0a4-4a42-4f55-aa21-b151fe25e0e0","order_by":9,"name":"Tobias Wang","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Tobias","middleName":"","lastName":"Wang","suffix":""},{"id":375847084,"identity":"1547b6b1-54c7-4e19-9389-8cbbe01f4103","order_by":10,"name":"Edson Antunes","email":"","orcid":"","institution":"State University of Campinas (UNICAMP)","correspondingAuthor":false,"prefix":"","firstName":"Edson","middleName":"","lastName":"Antunes","suffix":""},{"id":375847085,"identity":"cd04307b-4fa8-4053-a189-31abb609fd1f","order_by":11,"name":"Gilberto De Nucci","email":"","orcid":"","institution":"State University of Campinas (UNICAMP)","correspondingAuthor":false,"prefix":"","firstName":"Gilberto","middleName":"","lastName":"De Nucci","suffix":""}],"badges":[],"createdAt":"2024-11-01 21:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5375589/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5375589/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69926597,"identity":"e552cbd0-fa29-4f93-b584-7d38b5a940e5","added_by":"auto","created_at":"2024-11-26 16:30:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":496069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBasal release of 6-cyanodopamine and 6-nitrodopamine from rat isolated ventricles.\u003c/strong\u003e A pool of isolated ventricles from four animals were used to generate one sample for LC-MS/MS (n= 60/15). Student’s unpaired t-test was used. Limit of quantitation (LOQ).\u003c/p\u003e","description":"","filename":"FIgura01.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/688b987fd664951e71841928.png"},{"id":69927156,"identity":"adc0ab67-13bf-4519-8180-7bab32c4fe72","added_by":"auto","created_at":"2024-11-26 16:38:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":828486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBasal release of 6-cyanodopamine (6-CYD) and 6-nitrodopamine (6-ND) from rat isolated ventricles\u003c/strong\u003e. In isolated ventricles obtained from animals chronically treated with L-NAME, the basal release of 6-CYD was unaffected (Panel A), whereas that of 6-ND was significantly reduced (Panel B). Pre-treatment (30 min) of the ventricles with tetrodotoxin (TTX; 1 mM) did not affect the release of either 6-CYD (Panel C) or 6-ND (Panel D). Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; 100 mM) or the NOX1,4 inhibitor GKT-137,831 (1 mM) significantly decreased the basal release of 6-CYD (Panels E and G, respectively), and increased that of 6-ND (Panels F and H, respectively).\u0026nbsp; Student’s unpaired t-test was used. Limit of quantitation (LOQ). Data represent the mean.\u003c/p\u003e","description":"","filename":"FIgura02.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/fe1056d4b75d07b3b03f7ec2.png"},{"id":69926601,"identity":"c8c92baa-63f2-4f8b-8e24-b3b048e8191f","added_by":"auto","created_at":"2024-11-26 16:30:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":636396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChronotropic responses of 6-cyanodopamine (6-CYD)\u003c/strong\u003e \u003cstrong\u003ein rat isolated right atrium frequency.\u003c/strong\u003e Panel A shows the concentration-response curves of 6-CYD (1, 10 and 100 pM) in the rat isolated right atrial rate. In panel B, note the long-lasting positive chronotropic responses to 6-CYD (100 pM), which was maintained for at least 30 min after the 6-CYD washout. ANOVA followed by the Newman–Keuls post-test was applied in Panel A. \u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 compared to 6-CYD 1 pM, \u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 compared to 6-CYD 10 pM, and \u003csup\u003ec\u003c/sup\u003eP \u0026lt; 0.05 compared to 6-CYD 100 pM In panel B, data represent the mean + standard error of the mean (SEM).\u003c/p\u003e","description":"","filename":"FIgura03.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/ed3bbff932dd7cbbada2ac9f.png"},{"id":69927477,"identity":"cfa76a14-a813-4c09-83a6-25baa96995c7","added_by":"auto","created_at":"2024-11-26 16:46:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":659298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteractions of 6-cyanodopamine (6-CYD) with noradrenaline (NA), adrenaline (ADR) and dopamine (DA) on chronotropic responses in the rat isolated atrium.\u003c/strong\u003e Interaction 6-CYD (0.01 to 1 pM) of the rat isolated atrium with single concentrations of noradrenaline (1 pM; Panel A), adrenaline (1 pM; Panel B), or dopamine (1 pM; Panel C). ANOVA followed by the Newman–Keuls post-test was applied in panels A, B and C. \u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 compared to NA, ADR or DA alone, \u003csup\u003eb\u003c/sup\u003eP \u0026lt; 0.05 compared to CYD at 0.001 pM versus catecholamines alone, \u003csup\u003ec\u003c/sup\u003eP \u0026lt; 0.05 compared to CYD at 0.01 pM versus catecholamines alone, \u003csup\u003ed\u003c/sup\u003eP \u0026lt; 0.05 compared to CYD at 0.1 pM versus catecholamines alone and \u003csup\u003ee\u003c/sup\u003eP \u0026lt; 0.05 compared to CYD at 1 pM versus catecholamines alone.\u003c/p\u003e","description":"","filename":"FIgura04.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/ee0b1ece8c406720d29b0d7a.png"},{"id":69926598,"identity":"acdd81fa-7269-492e-8d9d-0b5a5d6cf9e4","added_by":"auto","created_at":"2024-11-26 16:30:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1190010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of both bolus injections and one-minute infusion of 6-cyanodopamine (6-CYD) in the heart rate (HR), left ventricular developer pressure (LVDP), maximum rate of pressure development (dP/dt\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003emax\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) and rate-pressure product (RPP).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData of heart rate for 6-CYD as bolus injections (0.01–10 pmol) or one-minute infusion (0.1-100 pM; 100 μL/min) are shown in panels A and B. Data of LVDP for 6-CYD are shown in panels D (bolus injection) and E (one-minute infusion). Data of dP/dt\u003csub\u003emax\u003c/sub\u003e are shown in panels E (bolus injection) and F (one-minute infusion). Data of RPP are shown in panels J (bolus injection) and H (one-minute infusion).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eP \u0026lt; 0.05 compared with the lowest concentration / dose of 6-CYD in each panel; \u003csup\u003eb\u003c/sup\u003eP \u0026lt; 0.05 compared with the second concentration / dose of 6-CYD in each panel; \u003csup\u003ec\u003c/sup\u003eP \u0026lt; 0.05 \u0026nbsp;compared with the dose of 1 pmol of 6-CYD.\u003c/p\u003e","description":"","filename":"FIgura05.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/8cbb785a3d1aa39d1bca8460.png"},{"id":69926596,"identity":"923b7ede-8fd9-4d42-b030-3d0d7fc53c90","added_by":"auto","created_at":"2024-11-26 16:30:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":718187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of 6-cyanodopamine (6-CYD) with noradrenaline (NA) on heart rate, left ventricular developed pressure (LVDP), maximum rate of pressure development (dP/dt\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003emax\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e), and rate-pressure product (RPP). \u003c/strong\u003e\u0026nbsp;One-minute infusion (100 mL/min) of either 6-cyanodopamine (0.001 or 0.01 pM; left panels) was performed in the absence and presence of a single bolus of noradrenaline (1 pmol). The following parameters were obtained: heart rate (panels A), LVDP (panels B), dP/dt\u003csub\u003emax\u003c/sub\u003e (panels C) and RPP (panels D). ANOVA followed by the Newman–Keuls post-test were applied.\u003csup\u003e a\u003c/sup\u003eP \u0026lt; 0.05 compared with the NA 1 pmol in each panel; \u003csup\u003eb\u003c/sup\u003eP \u0026lt; 0.05 compared with the 6-CYD 0.001 pM + NA 1 pmol .\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"FIgura06.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/ae4d79ab6d2087877a0bb497.png"},{"id":69926603,"identity":"385329c8-f0ce-4256-a6df-2c88c0b958c8","added_by":"auto","created_at":"2024-11-26 16:30:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3057478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustrative images of tyrosine hydroxylase (TH) detection by immunohistochemistry in rat heart.\u003c/strong\u003e A-C: Notice TH positivity in endocardium (Ec), myocardium (M), coronary endothelium (E), and epicardial nerves (N); D-F: negative control (omission of primary antibody). A minor nonspecific staining (see coronary artery [C] depicted in inset “E”) is sometimes observed in intravascular serum (this background reaction is not affected by DAB exposure time, and is probably related to the secondary antibody of the detection system). Immunoperoxidase/DAB + Haematoxylin, 400x (original magnification).\u003c/p\u003e","description":"","filename":"Figura07.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/d9f4a10deebfe223421f25e4.png"},{"id":69926604,"identity":"56d0032f-6686-4866-ab7c-6f0a9832f860","added_by":"auto","created_at":"2024-11-26 16:30:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4222890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTH mRNA detection by fluorescence \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein situ \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehydridization (FISH) in rat heart sections\u003c/strong\u003e. A: TH mRNA is dimly to moderately positive in myocardium (M), and endocardium (Ec) cell. B: TH mRNA expression seen in A, in detail. C: TH mRNA is positive in myocardium (M), endocardium (Ec), and coronary endothelial (Et) cells. D: same as B, in detail. E: TH mRNA expression in myocardium (M) cells, and epicardium nerves (N). F: same as E, in detail. A-F: notice a strong signal observed in red blood cells (R), which is likely due to autofluorescence. Also, in all images, nuclei are identified in dark blue (DAPI). A, C, E: 6-FAM (green), DAPI (dark blue), 200X. B, D, F: 6-FAM (green), DAPI (dark blue), 400X.\u003c/p\u003e","description":"","filename":"Figura08.png","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/eee10b351d821f586ad1ca30.png"},{"id":70270072,"identity":"280aeb28-697c-4e85-993c-6bfe8117a11e","added_by":"auto","created_at":"2024-12-01 08:16:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12028897,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5375589/v1/e7c34ee4-7e43-4a8e-bcb3-d6336b4d5002.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e6-cyanodopamine as an Endogenous Modulator of Heart Chronotropism and Inotropism\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe autonomic nervous system is classically supposed to play a major role on the control of heart function (Herring et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Catecholamines such as dopamine, noradrenaline, and adrenaline are reported to be produced only in the central nervous system, adrenergic nerve fibers and adrenal medulla (Ungar \u0026amp; Phillips, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Gorain et al., 1983), which upon appropriate sympathetic stimulation act on α- and β-adrenoceptors (Ahlquist, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1948\u003c/span\u003e; Molinoff, 1948; Motiejunaite et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the identification of endothelium-derived catecholamines has changed this paradigm (Zatz \u0026amp; De Nucci, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For instance, rat isolated atria and ventricles releases endothelium-derived 6-nitrodopamine and this novel catecholamine induces a potent endogenous positive chronotropic and inotropic response (Britto-J\u0026uacute;nior et al., 2022a; \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea; \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003eb). Inhibition of nitric oxide synthase (NOS) by L-NAME reduces both the basal release of 6-nitrodopamine and atrial basal rate (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea; \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003eb). The voltage-gated sodium channel blocker tetrodotoxin did not affect the release of 6-nitrodopamine from mouse and rat isolated hearts, indicating a non-neurogenic source for this catecholamine (Britto-J\u0026uacute;nior et al., 2022a; \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea; \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003eb). Interestingly, 6-nitrodopamine markedly synergizes with the classical catecholamines dopamine, noradrenaline, and adrenaline in the rat right isolated atrium, resulting in long-lasting increases in atrial rate (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e6-Cyanodopamine belongs to a novel class of catecholamines that is released by rat isolated vas deferens (Dal Pozzo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and by rabbit isolated atria and ventricles (J\u0026uacute;nior et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), however it is not known whether this novel catecholamine has activity on the cardiovascular system. Here it was investigated whether rat isolated ventricles release 6-cyanodopamine, and whether this novel endogenous catecholamine modulates heart chronotropism and inotropism.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eAdult male Wistar rats (280 to 320 g) were obtained from the Central Animal House at University of Campinas (CEMIB-UNICAMP; S\u0026atilde;o Paulo, Brazil). All experimental protocols were approved by the Ethics Committee for Animal Use of the UNICAMP (CEUA; Protocol No. 5746-1/2021; 5831-1/2021) following the Brazilian Guidelines for the Production, Maintenance and Use of Animals for Teaching or Research from the National Council of Control in Animal Experimentation (CONCEA; Andersen, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) as well as by following the ARRIVE guidelines (Percie du Sert et al, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Three individuals were housed in each cage placed on ventilated shelters at a humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5% and a temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under a 12-hour light-dark cycle. Animals received filtered water and standard rodent food \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2. Basal release of 6-cyanodopamine, 6-nitrodopamine, dopamine, noradrenaline, and adrenaline from rat isolated ventricles by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnimals were exposed to a concentration greater than 5% isoflurane until 1 min after breathing stopped. Exsanguination was performed to confirm the euthanasia. Next, the chest was opened, and the heart was rapidly excised. The right and left ventricles were isolated en bloc from each rat heart and suspended in a 5 mL organ bath containing Krebs\u0026ndash;Henseleit solution (KHS; in mM: NaCl 118, KCl 4.7, CaCl\u003csub\u003e2\u003c/sub\u003e 2.5, MgSO\u003csub\u003e4\u003c/sub\u003e 1.7, NaHCO\u003csub\u003e3\u003c/sub\u003e 24.9, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.2, dextrose 11, sodium pyruvate 2, pH 7.4) continuously gassed with a mixture (95% O2: 5% CO2) at 37 ◦C, supplemented with ascorbic acid (3 mM) to prevent catecholamine oxidation. After 30 min, two 2-mL aliquots of KHS solution were transferred to black Eppendorf tubes and stored at -20\u0026deg;C until analysis. Release of catecholamines was also evaluated from isolated ventricles obtained from animals chronically treated with L-NAME (20 mg/rat/day for 4 weeks; Ribeiro et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The effects of the voltage-gated sodium channel blocker tetrodotoxin (TTX; 1 \u0026micro;M), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 100 \u0026micro;M) and the dual NADPH oxidase NOX1/4 Inhibitor GKT137831 (1 \u0026micro;M) on the catecholamine release were also investigated in the isolated ventricles.\u003c/p\u003e \u003cp\u003eThe levels of catecholamines in KHS were determined by LC-MS/MS. Briefly, the extraction of the catecholamines from 1 mL of KHS was performed by solid phase extraction. A total of 50 \u0026micro;L of the internal standards (100 ng/mL of catecholamines) were added to 1 mL of KHS. The samples were homogenized for 10 sec. The Strata\u0026trade;-X 33 mm Polymeric Reversed SPE cartridges were preconditioned with 1 mL of methanol and then balanced by 2 mL of deionized water. The samples were injected into the cartridge, and the cartridge was subsequently washed 3 times with deionized water. The samples were then eluted with 0.9 mL methanol/water (90/10, v/v) with 0.1% formic acid. The mobile phase perfused a LC ADVp Liquid Chromatography Shimadzu System (Shimadzu Corp., Kyoto, JP) coupled to a Shimadzu 8060 Triple Quadrupole Mass Spectrometer operating in ESP\u003csup\u003e+\u003c/sup\u003e mode at 350 \u0026micro;L/min. The dissolved residues were injected by a SIL-30AC autoinjector, at 8 \u0026ordm;C. The transitions monitored by electrospray multiple reaction monitoring (MRM), injection volume, run-time, limit of quantitation and method validation following USFDA guidelines for bioanalytical methods (FDA) were described elsewhere (Junior et al., 2023). As an improvement to the original method, two transitions (a quantifier and a qualifier transition) were monitored to increase selectivity (Dal Pozzo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Rat isolated right atrium preparation and measurement of chronotropic responses\u003c/h2\u003e \u003cp\u003eAnimals were anaesthetized as previously described. The right atrium was isolated from the heart and mounted between two metal hooks in 10-mL custom designed glass chambers containing KHS solution, continuously gassed with a mixture of 95%O\u003csub\u003e2\u003c/sub\u003e: 5%CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C using a heated circulator (PolyScience, Illinois, USA). Tissues were allowed to equilibrate under a resting tension of 10 mN for one hour, and the isometric tension was registered using a PowerLab system (ADInstruments, Sydney, Australia; Britto-J\u0026uacute;nior et al., 2022a).\u003c/p\u003e \u003cp\u003eAfter an equilibration period was achieved, single concentrations of 6-cyanodopamine (1, 10, and 100 pM) were added to the organ bath, and changes in atrial rate were monitored for 30 min. In separate set of experiments, following the 30-min incubation with 6-cyanodopamine (10 and 100 pM), the KHS was replaced to wash out the agonist, and the atrial rate was monitored for an additional 30 min. A separate atrium was used for each drug concentration. In order to investigate the synergism between 6-cyanodopamine with noradrenaline, adrenaline, and dopamine in the rat isolated atrial rate, the following experimental protocols were employed. A single concentration of dopamine (1 pM), noradrenaline (1 pM) or adrenaline (1 pM) was added to the organ bath, and changes in atrial rate were monitored for 30 min. Next, 6-cyanodopamine (0.001 and 0.01 pM) was co-incubated with either noradrenaline, adrenaline or dopamine, (1 pM each), and the resulting changes in atrial rate were observed for 30 min. A separate atrium was used for each drug and each concentration. Data of atria rate was expressed as rate (bpm) before and after the respective stimulation or delta increase of atrial rate (bpm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Langendorff\u0026rsquo;s isolated perfused heart preparation and measurements of heart contractile function\u003c/h2\u003e \u003cp\u003eHeparin (1000 IU/kg) was injected intraperitoneally in the animals to prevent blood clotting and euthanasia was performed by isoflurane overdose, as previously described. Exsanguination was performed to confirm the euthanasia. The chest was opened, and the heart was rapidly excised, the ascending aorta was cannulated, and the heart was mounted in a nonrecirculating Langendorff apparatus. The isolated heart was perfused with KHS (pH 7.4, 37\u0026deg;C) equilibrated with carbogen gas mixture (95% O\u003csub\u003e2\u003c/sub\u003e: 5% CO\u003csub\u003e2\u003c/sub\u003e) at constant flow (10 ml/min) and left ventricular end-diastolic pressure (LVEDP) was maintained between 4\u0026ndash;8 mmHg during the initial equilibrium of the experiment (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea). A water-filled latex balloon, connected to the pressure transducer (MLT1199 BP Transducer, ADInstruments, Inc., Dunedin, NZ) was inserted into the left ventricle (LV) via the mitral valve. Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVeDP) and heart rate (HR) were continuously recorded by a PowerLab System (ADInstruments, Inc., Dunedin, NZ). Only hearts that presented a basal heart rate between 250\u0026ndash;300 bpm were employed in the experiments.\u003c/p\u003e \u003cp\u003eThe hearts were allowed to equilibrate for at least 10 min. Afterward, single bolus (10 \u0026micro;L) of 6-cyanodopamine (0.01, 0.1, 1, or 10 pmol) was injected, with each heart receiving only one bolus. In a separate set of experiments, the effects of a one-minute infusion (100 \u0026micro;L/min) of 6-cyanodopamine (0.1, 1, 10, 100, or 100 pM, final concentration) were evaluated. Each heart was subjected to only one infusion. Changes were monitored for 30 min. In order to investigate the synergism between 6-cyanodopamine with noradrenaline in the Langendorff\u0026rsquo;s-perfused heart analysis, the following protocols were employed. One-minute (100 \u0026micro;L/min)-infusion of either 6-cyanodopamine (0.001 or 0.01 pM, final concentration) were performed and then a single bolus of noradrenaline (1 pmol) was administered, and the heart was monitored for 15 min. One heart was used for a single drug and a single infusion. Data obtained with the Landendorff preparations (heart rate, LVDP, dP/dt max and RPP) were expressed as left ventricular developed pressure (LVDP) was calculated by the following formula: LVSP-LVeDP and expressed in mmHg. Rate pressure product (RPP) was defined as the product of heart rate (HR) and left ventricular developed pressures (LVDP) RPP = (HR \u0026times; LVDP). The maximal rate of rise of the left ventricular pressure (+\u0026thinsp;dP/dt\u003csub\u003emax\u003c/sub\u003e) was monitored continuously by a pressure transducer connected to a Powerlab system (AD Instrument, Australia).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eRat heart samples (N\u0026thinsp;=\u0026thinsp;5) were fixed in 10% neutral buffered formalin for 24h at 25\u003csup\u003eo\u003c/sup\u003eC, dehydrated, embedded in paraffin wax and sectioned at 4\u0026micro;m. Subsequently, these sections were deparaffinized in xylene and rehydrated in a series of ethanol baths of decreasing concentration. They were treated for 10 minutes with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to neutralize endogenous peroxidase. Then, they were washed and incubated in citrate buffer at pH 6.0 (regardless of primary antibody) in a steamer set for 20min (at approximately 95\u003csup\u003eo\u003c/sup\u003e C). Following this, the sections were incubated for 2h at 25\u003csup\u003eo\u003c/sup\u003eC with a chicken polyclonal anti - tyrosine hydroxylase (ab76442; 1:500; Abcam, USA). Tissue sections were sequentially incubated with goat anti-chicken gamma immunoglobulin IgG (ab150169; 1:500 diluted in PBS, Abcam, USA), a rabbit anti-goat IgG (AP106P; 1:250 diluted in PBS, Merck/Sigma, USA), for 1h each, and the detection system. The detection system used was the NovoLink\u0026trade; Max Polymer Detection System (catalog code RE7280-k, Leica Biosystems, UK), following the manufacturer\u0026rsquo;s instructions, and using diaminobenzidine (liquid DAB, DakoCytomation, Carpenteria, USA) as chromogen (which renders a brown precipitate at the antibody binding site). Subsequently, the sections were counter-stained with Harris\u0026acute;s hematoxylin and coverslipped with Entellan mounting medium. Negative controls consisted in the omission of the primary antibody and incubation with the primary antibody diluents (one negative control per section) to identify any unspecific background staining. All slides were examined using a trinocular Eclipse 50i microscope (Nikon, Tokyo, Japan) coupled to a 10MP CMOS digital camera (AmScope, EUA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eIn situ\u003c/em\u003e hybridization (FISH)\u003c/h2\u003e \u003cp\u003eTo confirm and validate TH protein expression and its topography, we futher investigated TH mRNA expression in the same samples (N\u0026thinsp;=\u0026thinsp;3), using FISH assay. Briefly, sections from 4 rat seminal vesicles were deparaffinized with xylene and rehydrated in graded alcohols for 5 minutes each. Then, they were incubated in a 0.2 N HCl solution for 20 minutes, and subsequently treated with a citrate pH 6.0 buffer (ZytoVision kit, catalog code Z-2028-20, Germany) at 80\u0026ordm;C for 1 hour. After this, they were incubated with pepsin for 8 minutes at room temperature. The slides were washed with 2XSSC (ZytoVision kit, catalog code Z-2028-20, Germany). They were subsequently submitted to a sequence of ethanols (75%, 80%, and 100% ethanol for 2 minutes each), and then air dried. The slides were further incubated with a probe to TH mRNA (at a concentration of 10 \u0026micro;M, in RNAse-free water) for 10 minutes at 75\u0026ordm;C and overnight in a Dako Hybridizer (Dako, Denmark) at 37\u0026ordm;C. The probe sequence was as follows: 5\u0026prime;- AACCGCGGGGACATGATGGCCT-3\u0026prime; (RNA Tm\u0026thinsp;=\u0026thinsp;77.8\u0026deg;C) (Batch: WD11655417, Sigma/Merck, Germany). The probe was labeled with fluorescein 6-FAM in the 5\u0026rsquo; region. The next day, the slides were placed in a UREA/0,1Xssc solution at 45\u0026deg;C for 30 minutes, and then, they were washed with a 2xSSC solution for 2 minutes. After this, the slides were dehydrated in 75%, 85%, and 100% ethanols for 2 minutes each, and air dried. Finally, the slides were mounted with 15 \u0026micro;L of a DAPI containing mounting medium (from the ZytoVision kit) and cover slipped (the cover slip being sealed with a Fixogum Rubber Cement, from Marabu, Germany) (Britto-Junior et al., 2020). Negative controls consisted of the omission of the probe and were performed in all FISH assays (one negative control per section) to control for any significant autofluorescence. All FISH slides were examined and photomicrographed at the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas, using an upright fluorescence microscope (Axioskop, Carl Zeiss AG, Germany) coupled to a digital camera (AxioCam MRc, Zeiss, Germany). Images were collected using 350\u0026ndash;495 nm laser lines for excitation and 465\u0026ndash;517 emission filters for two fluorophores (DAPI and 6-FAM) and 200x optical zoom.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Chemical and reagents\u003c/h2\u003e \u003cp\u003eAdrenaline, dopamine, GKT137831, noradrenaline, and tetrodotoxin (TTX) were obtained from Cayman Chemicals (Michigan, USA). 6-Nitrodopamine and 6-nitrodopamine-d\u003csub\u003e4\u003c/sub\u003e were acquired from Toronto Research Chemicals (Ontario, CA). N\u003csup\u003eω\u003c/sup\u003e-Nitro-L-arginine methyl ester (L-NAME) was obtained from Sigma-Aldrich Chemicals Co (St Louis, Missouri, USA). Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was bought from Exodo Cientifica (Sumar\u0026eacute;, S\u0026atilde;o Paulo, Brazil). Dopamine-d\u003csub\u003e3\u003c/sub\u003e hydrochloride, DL‐noradrenaline‐d\u003csub\u003e6\u003c/sub\u003e hydrochloride and adrenaline‐d\u003csub\u003e6\u003c/sub\u003e hydrochloride were acquired from CDN Isotopes (Quebec, CA). 6-cyanodopamine was synthesized at Rhodes College (Rote et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Strata\u0026trade;-X 33 mm Polymeric Reversed SPE cartridges were bought from Phenomenex (California, USA) and GIST-HP C\u003csub\u003e18\u003c/sub\u003e columns were obtained from Shimadzu (Duisburg, Germany). Calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), dextrose, magnesium sulfate (MgSO\u003csub\u003e4\u003c/sub\u003e), potassium chloride (KCl), sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e), potassium phosphate monobasic (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) and sodium chloride (NaCl), were bought from Merck KGaA (Darmstadt, Germany). Acetonitrile and methanol were obtained from J.T. Baker (Phillipsburg, NJ, USA) and formic acid from Mallinckrodt (St Louis, Missouri, USA). The composition of the KHS was in mM: NaCl 118, KCl 4.7, CaCl\u003csub\u003e2\u003c/sub\u003e 2.5, MgSO\u003csub\u003e4\u003c/sub\u003e 1.2, NaHCO\u003csub\u003e3\u003c/sub\u003e 25, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.2 and dextrose 5.6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e \u003cp\u003eData represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Comparison between baseline values to values obtained during drug stimulation in the same sample was performed by paired \u003cem\u003et\u003c/em\u003e-test. Comparison between two groups was performed by unpaired \u003cem\u003et\u003c/em\u003e-test. Comparisons among three or more groups were evaluated using one-way analysis of variance (ANOVA), followed by Newman-Keuls test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was taken as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Basal release of 6-cyanodopamine and 6-nitrodopamine from rat isolated ventricles\u003c/h2\u003e \u003cp\u003eRat isolated ventricles presented basal release of both 6-cyanodopamine and 6-nitrodopamine, but the levels 6-nitrodopamine were significantly higher than 6-cyanodopamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In isolated ventricles obtained from animals chronically treated with L-NAME, the basal release of 6-cyanodopamine was unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whereas that of 6-nitrodopamine was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Pre-treatment (30 min) of the ventricles with tetrodotoxin (TTX; 1 \u0026micro;M) affected neither the release of 6-cyanodopamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) nor of 6-nitrodopamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Pre-treatment (30 min) with either H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (100 \u0026micro;M) or the NOX1/4 inhibitor GKT137,831 (1 \u0026micro;M) significantly decreased the basal release of 6-cyanodopamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, respectively), whereas the levels of 6-nitrodopamine significantly increased by these treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, respectively). Levels of dopamine, noradrenaline, adrenaline were below the limit of quantitation (LOQ, 0.1 ng/mL).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Chronotropic effect of 6-cyanodopamine on the rat isolated right atrium\u003c/h2\u003e \u003cp\u003ePre-incubation (30 min) of the rat isolated right atrium with 6-cyanodopamine (10 and 100 pM) induced concentration-dependent increases in the atrial rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The increases in atrial rate induced by 6-cyanodopamine were prolonged, since they are maintained even at 30 min after the preparation was washed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003ePre-incubation (30 min) of the atria with low concentrations of noradrenaline (1 pM; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), adrenaline (1 pM; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and dopamine (1 pM; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) did not affect the atrial basal frequency. Co-incubation (30 min) of the atria with 6-cyanodopamine at 0.001 pM and noradrenaline, adrenaline or dopamine had no effect in the atrial basal rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). However, co-incubation of the atria with higher concentrations of 6-cyanodopamine (0.01, 0.1 and 1 pM) resulted in significant increases in atrial rate in response to noradrenaline, adrenaline or dopamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Heart contractile effects by 6-cyanodopamine in comparison with 6-nitrodopamine in the Langendorff\u0026rsquo;s preparation\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows data of heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B), LVDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D), dP/dt max (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F) and rate-pressure product (RPP; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-H) obtained in the Langendorff preparation. Bolus injection of 6-cyanodopamine at 0.01 to 1 pmol had no effect on heart rate, but 10 pmol resulted in a significant increase in heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Bolus injections of 0.1, 1, and 10 pmol of 6-cyanodopamine led to significant increases in both LVDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and dP/dt (max) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The RPP parameter was significantly elevated at 0.1, 1, and 10 pmol of 6-cyanodopamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, testing of 6-cyanodopamine will proceed as a one-minute infusion instead of a bolus injection. Infusion (1 min) of 6-cyanodopamine (0.1 and 1 pM) did not significantly affect heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB); however, at higher concentrations (10 and 100 pM), significant increases in heart rate were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). One-min infusion of 6-cyanodopamine at 0.1 pM had no significant effect on either LVDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) or dP/dt(max) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), but the infusion at higher concentrations (1, 10, and 100 pM) provoked significant increases in both LVDP and dP/dt(max) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and F). Infusion (1 min) of 6-cyanodopamine (0.1 and 1 pM) had no significant effect on the rate-pressure product (RPP; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Nonetheles K s, higher concentrations (10 and 100 pM) resulted in significant increases in RPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Interactions of 6-cyanodopamine with noradrenaline on the rat isolated heart (Langendorff\u0026rsquo;s preparation)\u003c/h2\u003e \u003cp\u003eBolus injection of noradrenaline at 1 pmol had no effect on heart rate frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), LVDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), dPdt(max) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) and RPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). One-min infusion of 6-cyanodopamine (0.001 pM) alone did not alter any of these parameters either. However, infusion of 6-cyanodopamine (0.01 pM) significantly increased the heart rate frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), LVDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), dP/dt(max) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), and RPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) when noradrenaline (1 pmol) was injected at the end of the infusion (1 min).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.5. Immunohistochemistry and\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003ehybridization (FISH)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarize the results of the immunohistochemical detection of tyrosine hydroxylase (TH) in rat heart. Briefly, TH was positive in endocardium (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), myocardium (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C), coronary endothelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and epicardial nerves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The negative control (omission of primary antibody) is illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F. Occasionally, a weak background staining was observed in intravascular serum, but this nonspecific reaction did not compromise the interpretation of the slides and was probably related to secondary antibody of the detection system, since it was also present in control sections (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F). To validate the immunohistochemistry (IHC) findings, we used fluorescence \u003cem\u003ein situ\u003c/em\u003e hydridization (FISH) in 3 samples of rat heart. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, TH mRNA expression was observed in myocardium, endocardium, and coronary endothelial cells, as well as in epicardial nerves of rat heart, thus confirming IHC derived data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetection of Tyrosine hydroxylase (TH) by immunohistochemistry (IHC) and FISH.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRat heart\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative control (omission of the primary antibody)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTH protein detection by IHC\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTH mRNA detection by FISH\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndocardium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyocardium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoronary endothelium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpicardium nerves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results clearly demonstrated that rat isolated ventricles present basal release of 6-cyanodopamine, and this release has some important characteristics. For instance, the levels of 6-cyanodopamine are approximately 20 times smaller than that of 6-nitrodopamine. It is interesting that in the rabbit isolated atria the levels of 6-cyanodopamine were also smaller than that of 6-nitrodopamine, although the difference was smaller (3 times; J\u0026uacute;nior et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In both rabbit isolated ventricles and in rat isolated vas deferens (Dal Pozzo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), there were no significant differences in the amounts of 6-cyanodopamine and 6-nitrodopamine released. The finding that the release of 6-cyanodopamine was not affected by pre-incubation of the heart with tetrodotoxin, indicates a non-neurogenic source for this novel catecholamine. Indeed, in the mouse isolated atria and ventricles, the release of 6-nitrodopamine was decreased only eNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, but not in nNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e or iNOS\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003eb). In contrast, in the rat isolated vas deferens the release of 6-nitrodopamine was virtually abolished when the tissue was pre-treated with tetrodotoxin, whereas that of 6-cyanodopamine was unaffected (Dal Pozzo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, it seems that 6-cyanodopamine in the heart is produced mainly by the either the endothelium and/or cardiomyocytes. Indeed, as demonstrated here by both immunohistochemistry and fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization, both types of cells do express tyrosine hydroxylase.\u003c/p\u003e \u003cp\u003eIt is clearly established that NO synthase activity is important for 6-nitrodopamine biosynthesis, since pre-treatment of the tissues with L-NAME significantly inhibits 6-ND basal release (Zatz \u0026amp; De Nucci et al., 2024). The results here presented show that this enzyme is not directly involved in the biosynthesis of 6-cyanodopamine, since in contrast to 6-nitrodopamine basal release, ventricles obtained from animals chronically treated with L-NAME released similar levels of 6-cyanodopamine as compared to ventricles obtained from control animals. However, there is some relationship apparently between 6-cyanodopamine and 6-nitrodopamine synthesis/release, since the pre-incubation of the ventricles with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and the NOX1,4 inhibitor GKT-137831 (Jiang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), that causes significant increases in 6-nitrodopamine release from rat isolated atria (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), induced significant decreases in the basal release of 6-cyanodopamine. One possible explanation is that for these distinct effects observed could be that 6-cyanodopamine takes place mainly inside the cell, whereas the \u0026ldquo;nitration\u0026rdquo; responsible for the 6-nitrodopamine biosynthesis occurs in the external plasma membrane of the endothelium. It is interesting that cyanide has been proposed as a potential gasotransmitter in the stomach (Zuhra \u0026amp; Szabo, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and healthy volunteers present cyanide levels from 300 nM (NATIONAL RESEARCH COUNCIL et al \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) to 2\u0026ndash;8 \u0026micro;M (Fasco et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, the metabolic pathway(s) that could lead to synthesis of the cyano-catecholamines is(are) at present unknown.\u003c/p\u003e \u003cp\u003eAs an endogenous positive chronotropic and inotropic agent per se, 6-cyanodopamine is approximately ten times less potent that 6-nitrodopamine, but more potent than the classical catecholamines dopamine, noradrenaline, and adrenaline (Britto-J\u0026uacute;nior 2022a), and it shares both the same prolonged positive chronotropic effect of 6-nitrodopamine and the ability to potentiate the positive chronotropic effect induced by noradrenaline. What could be the mechanism(s) by which 6-cyanodopamine and 6-nitrodopamine facilitate the positive chronotropic effects of noradrenaline?\u003c/p\u003e \u003cp\u003eb\u003csub\u003e1\u003c/sub\u003e-adrenoceptor activation in the sinoatrial nodal cells occurs following noradrenaline release from sympathetic nerve terminals in the heart. The primary pathway following b\u003csub\u003e1\u003c/sub\u003e-adrenoceptor stimulation involves Gs protein/adenylyl-cyclase (AC)/cAMP/ protein kinase A (PKA) route (Task\u0026eacute;n \u0026amp; Aandahl \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Interestingly, 6-nitrodopamine potentiates the positive chronotropic effect of dopamine, noradrenaline, and adrenaline, and at 0.01 pM, abolished that induced by phosphodiesterase type 3 (PDE3) inhibitors, such as cilostazol (Saitoh et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), dipyridamole (Clarke et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), and milrinone (Remme wt al., 1992). 6-Nitrodopamine (0.01 pM) did not affect the concentration-dependent positive chronotropic effect induced by the PDE4 inhibitor rolipram (Wachtel et al., 1982) in the rat isolated atria (Britto-J\u0026uacute;nior 2022c), indicating a selective action on PDE3. Although apparently paradoxical, this result is not surprising, since PDE3 inhibitors do not affect the positive chronotropic effect induced by catecholamine activation of b\u003csub\u003e1\u003c/sub\u003e-adrenoceptors (Kaumann et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The remarkable synergism observed with the classical catecholamines and the differential effect on the PDE3 and PD4 inhibitors-induced positive chronotropic effect, suggest that one possible mechanism of action of these novel catecholamines could be a receptor mediated modulation of adenylyl cyclase.\u003c/p\u003e \u003cp\u003eThe concentration of cyclic AMP in sinoatrial cells is significantly larger when compared to atrial or ventricular myocytes obtained from rabbit hearts (Taniguchi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), and inhibition of adenylyl cyclase by MDL-12,330A abolishes basal sinoatrial nodal cells beating (Vinogradova et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Cyclic nucleotide phosphodiesterases (PDEs) are responsible for the metabolism of both cAMP (Butcher \u0026amp; Sutherland, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) and cGMP (Uckert \u0026amp; Kuczyk, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and although eleven PDE families have been identified (Bender \u0026amp; Beavo, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), in the rat cardiomyocytes cAMP is essentially hydrolysed by PDE\u003csub\u003e3\u003c/sub\u003e and PDE\u003csub\u003e4\u003c/sub\u003e (Rochais et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Platelets possess PDE2, PDE3 and PDE5 isoforms (Gresele et al., 2015), but incubation of human platelets with 6-ND does not cause increase in either cAMP or cGMP contents (Nash et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), indicating that it is unlikely that 6-ND could act as a PDE inhibitor.\u003c/p\u003e \u003cp\u003eThe use of an agarose coupled to 6-nitrodopamine for purification of cardiomyocyte membranes revealed selective binding to three proteins that modulate adenylyl cyclase, such as cyclase associated protein 1 (CAP1), cyclase associated protein 2 (CAP2), and stromal interaction protein 1 (STIM1; Sweed et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cyclase associated protein 1 (CAP1) binds and activates adenylyl cyclase in mammalian cells (Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ablation of CAP2 in mice causes dilated cardiomyopathy associated with severe reduction of the heart rate (Peche et al., 2017). Stromal interaction protein (STIM1) is expressed in cardiomyocytes (Liu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), has a single transmembrane domain (Hooper et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), it is located in the sarcoplasmic reticulum and plasma membrane (Soboloff et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and it is also associated with adenylyl cyclase activation (Motiani et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These proteins are interesting candidates for the proposed novel catecholamines receptor mediated modulation of adenylyl cyclase. This mechanism could also explain the apparent lack of concentration-dependent effect of both catecholamines as chronotropic and inotropic agents. Investigations of the binding of these novel catecholamines in cardiomyocytes where the expression of these proteins has been blocked may provide some further clues of this novel proposed mechanism.\u003c/p\u003e \u003cp\u003eIt is interesting that patients with chronic kidney disease present circulating levels of 6-cyanodopamine (Ribeiro et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The finding here reported that is novel catecholamine acts as a potent positive chronotropic and inotropic agent in the isolated heart suggests that besides its potential therapeutic effect in heart failure, it may be useful as a biomarker of pathophysiological processes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were authorized by the Ethics Committee in Animal Use of UNICAMP (CEUA/UNICAMP, protocol number 5746-1/2021; 5831-1/2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors authorize the submission and publication of this article Naunyn-Schmiedeberg\u0026apos;s Archives of Pharmacology\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability section\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: JBJ, GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData curation: JBJ, GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFormal analysis: GDN\u003c/p\u003e\n\u003cp\u003eFunding acquisition: EA, GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInvestigation: JBJ, ATL, DLO, FVM, VBS, AAS, LWP, MS, RF, TW, EA, GDN\u003c/p\u003e\n\u003cp\u003eMethodology: JBJ, AAS, GDN.\u003c/p\u003e\n\u003cp\u003eProject administration: GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision: EA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVisualization: EA, GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: JBJ, TW, EA, GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data were generated in-house and that no paper mill was used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJBJ thanks FAPESP for post-doctoral fellowship (2021/14414-8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEA thanks FAPESP (2017/15175-1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGDN thanks FAPESP (2019/16805-4) and CNPq (303839/2019-8). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing or financial interests\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors authorize the availability of any data used in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHerring N, Kalla M, Paterson DJ. The autonomic nervous system and cardiac arrhythmias: current concepts and emerging therapies. Nat Rev Cardiol. 2019 Dec;16(12):707-726. doi: 10.1038/s41569-019-0221-2. Epub 2019 Jun 13. Erratum in: Nat Rev Cardiol. 2019 Dec;16(12):760. doi: 10.1038/s41569-019-0293-z. Erratum in: Nat Rev Cardiol. 2019 Dec;16(12):760. doi: 10.1038/s41569-019-0297-8. PMID: 31197232.\u003c/li\u003e\n \u003cli\u003eUngar A, Phillips JH. Regulation of the adrenal medulla. Physiol Rev. 1983 Jul;63(3):787-843. doi: 10.1152/physrev.1983.63.3.787. PMID: 6308693.\u003c/li\u003e\n \u003cli\u003eGorain, B., Dutta, S., Nandy, U., Sengupta, P., Choudhury, H. (2020). Pharmacology of Adrenaline, Noradrenaline, and Their Receptors. In: Kumar, P., Deb, P.K. (eds) Frontiers in Pharmacology of Neurotransmitters. Springer, Singapore. https://doi.org/10.1007/978-981-15-3556-7_4\u003c/li\u003e\n \u003cli\u003eAhlquist RP. A study of the adrenotropic receptors. Am J Physiol. 1948 Jun;153(3):586-600. doi: 10.1152/ajplegacy.1948.153.3.586. PMID: 18882199.\u003c/li\u003e\n \u003cli\u003eMolinoff PB. Alpha- and beta-adrenergic receptor subtypes properties, distribution and regulation. Drugs. 1984;28 Suppl 2:1-15. doi: 10.2165/00003495-198400282-00002. PMID: 6098436.\u003c/li\u003e\n \u003cli\u003eMotiejunaite J, Amar L, Vidal-Petiot E. Adrenergic receptors and cardiovascular effects of catecholamines.\u0026nbsp;Ann Endocrinol (Paris). 2021 Jun;82(3-4):193-197. doi: 10.1016/j.ando.2020.03.012. Epub 2020 Mar 18. PMID: 32473788.\u003c/li\u003e\n \u003cli\u003eZatz R, De Nucci G. Endothelium-Derived Dopamine and 6-Nitrodopamine in the Cardiovascular System. Physiology (Bethesda). 2024 Jan 1;39(1):44-59. doi: 10.1152/physiol.00020.2023. Epub 2023 Oct 24. PMID: 37874898; PMCID: PMC11283902.\u003c/li\u003e\n \u003cli\u003eBritto-J\u0026uacute;nior J, de Oliveira MG, Dos Reis Gati C, Campos R, Moraes MO, Moraes MEA, M\u0026oacute;nica FZ, Antunes E, De Nucci G. 6-NitroDopamine is an endogenous modulator of rat heart chronotropism.\u0026nbsp;Life Sci. 2022a Oct 15;307:120879. doi: 10.1016/j.lfs.2022.120879. Epub 2022 Aug 10. PMID: 35963299.\u003c/li\u003e\n \u003cli\u003eBritto-J\u0026uacute;nior J, Medeiros-Teixeira LR, Lima AT, Dassow LC, Lopes-Martins R\u0026Aacute;B, Campos R, Moraes MO, Moraes MEA, Antunes E, De Nucci G. 6-Nitrodopamine Is the Most Potent Endogenous Positive Inotropic Agent in the Isolated Rat Heart. Life (Basel). 2023a Oct 4;13(10):2012. doi: 10.3390/life13102012. PMID: 37895394; PMCID: PMC10607994.\u003c/li\u003e\n \u003cli\u003eBritto-J\u0026uacute;nior J, Pereira do Prado GL, Chiavegatto S, Cunha F, Moraes MO, Elisabete A Moraes M, Monica FZ, Antunes E, De Nucci G. The importance of the endothelial nitric oxide synthase on the release of 6-nitrodopamine from mouse isolated atria and ventricles and their role on chronotropism. Nitric Oxide. 2023b Sep 1;138-139:26-33. doi: 10.1016/j.niox.2023.06.001. Epub 2023 Jun 2. PMID: 37269938.\u003c/li\u003e\n \u003cli\u003eBritto-J\u0026uacute;nior J, Lima AT, Fuguhara V, Monica FZ, Antunes E, De Nucci G. Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria. Naunyn Schmiedebergs Arch Pharmacol. 2023c Jun;396(6):1279-1290. doi: 10.1007/s00210-023-02394-9. Epub 2023 Jan 31. PMID: 36719453.\u003c/li\u003e\n \u003cli\u003eDal Pozzo CFS, Junior JEM, Britto-J\u0026uacute;nior J, Badin JFA, de Souza VB, Schenka AA, Peterson LW, Fregonesi A, Antunes E, De Nucci G. Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility. Pflugers Arch. 2024 Aug;476(8):1263-1277. doi: 10.1007/s00424-024-02985-2. Epub 2024 Jul 4. PMID: 38963545.\u003c/li\u003e\n \u003cli\u003eJ\u0026uacute;nior GQ, Britto-J\u0026uacute;nior J, Magalhaes TB, Campos R, Nyamkondiwa KL, Klugh KL, Peterson LW, Corvino A, Sparaco R, Frecentese F, Caliendo G, De Nucci G. Measurement of 6-cyanodopamine, 6-nitrodopa, 6-nitrodopamine and 6-nitroadrenaline by LC-MS/MS in Krebs-Henseleit solution. Assessment of basal release from rabbit isolated right atrium and ventricles. Biomed Chromatogr. 2023 Sep;37(9):e5691. doi: 10.1002/bmc.5691. Epub 2023 Jul 10. PMID: 37431086.\u003c/li\u003e\n \u003cli\u003ePercie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, Clark A, Cuthill IC, Dirnagl U, Emerson M, Garner P, Holgate ST, Howells DW, Karp NA, Lazic SE, Lidster K, MacCallum CJ, Macleod M, Pearl EJ, Petersen OH, Rawle F, Reynolds P, Rooney K, Sena ES, Silberberg SD, Steckler T, W\u0026uuml;rbel H. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. Br J Pharmacol. 2020 Aug;177(16):3617-3624. doi: 10.1111/bph.15193. Epub 2020 Jul 14. PMID: 32662519; PMCID: PMC7393194.\u003c/li\u003e\n \u003cli\u003eAndersen ML. Guia brasileiro de produção, manutenção ou utilização de animais em atividade de ensino ou pesquisa cientifica, Conselho nacional de controle de experimentação animal. Brasília: Ministério da Ciência, Tecnologia e Inovação (2016)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRibeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis.\u0026nbsp;A new model of arterial hypertension. Hypertension. 1992 Sep;20(3):298-303. doi: 10.1161/01.hyp.20.3.298.\u0026nbsp;PMID: 1516948.\u003c/li\u003e\n \u003cli\u003eBritto-J\u0026uacute;nior J, Pinheiro DHA, Justo AFO, Figueiredo Murari GM, Campos R, Mariano FV, de Souza VB, Schenka AA, M\u0026oacute;nica FZ, Antunes E, De Nucci G. Endothelium-derived dopamine modulates EFS-induced contractions of human umbilical vessels. Pharmacol Res Perspect. 2020 Aug;8(4):e00612. doi: 10.1002/prp2.612. PMID: 32567793; PMCID: PMC7307250.\u003c/li\u003e\n \u003cli\u003eRote JC, Malkowski SN, Cochrane CS, Bailey GE, Brown NS, Cafiero M, Peterson LW (2017) Catechol reactivity: Synthesis of dopamine derivatives substituted at the 6-position. Synth Com- mun 47(5):435\u0026ndash;441\u003c/li\u003e\n \u003cli\u003eJiang JX, Chen X, Serizawa N, Szyndralewiez C, Page P, Schr\u0026ouml;der K, Brandes RP, Devaraj S, T\u0026ouml;r\u0026ouml;k NJ. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo. Free Radic Biol Med. 2012 Jul 15;53(2):289-96. doi: 10.1016/j.freeradbiomed.2012.05.007. Epub 2012 May 19. PMID: 22618020; PMCID: PMC3392471.\u003c/li\u003e\n \u003cli\u003eBritto-J\u0026uacute;nior J, Furlaneto R, Lima AT, de Oliveira MG, Severino B, Frecentese F, Fiorino F, Caliendo G, Muscar\u0026aacute; MN, De Nucci G. GKT137831 and hydrogen peroxide increase the release of 6-nitrodopamine from the human umbilical artery, rat-isolated right atrium, and rat-isolated vas deferens. Front Pharmacol. 2024 Apr 5;15:1348876. doi: 10.3389/fphar.2024.1348876. PMID: 38645555; PMCID: PMC11026650.\u003c/li\u003e\n \u003cli\u003eZuhra K, Szabo C. The two faces of cyanide: an environmental toxin and a potential novel mammalian gasotransmitter. FEBS J. 2022 May;289(9):2481-2515. doi: 10.1111/febs.16135. Epub 2021 Aug 5. PMID: 34297873; PMCID: PMC9291117.\u003c/li\u003e\n \u003cli\u003eNATIONAL RESEARCH COUNCIL et al. Hydrogen Cyanide: Acute Exposure Guideline Levels. In:\u0026nbsp;Acute Exposure Guideline Levels for Selected Airborne Chemicals: Volume 2. National Academies Press (US), 2002\u003c/li\u003e\n \u003cli\u003eFasco MJ, Stack RF, Lu S, Hauer CR 3rd, Schneider E, Dailey M, Aldous KM. Unique cyanide adduct in human serum albumin: potential as a surrogate exposure marker. Chem Res Toxicol. 2011 Apr 18;24(4):505-14. doi: 10.1021/tx100344e. Epub 2011 Mar 18. PMID: 21366342; PMCID: PMC3089968.\u003c/li\u003e\n \u003cli\u003eBritto-J\u0026uacute;nior J, Lima AT, Fuguhara V, Monica FZ, Antunes E, De Nucci G. Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria. Naunyn Schmiedebergs Arch Pharmacol. 2023c Jun;396(6):1279-1290. doi: 10.1007/s00210-023-02394-9. Epub 2023 Jan 31. PMID: 36719453.\u003c/li\u003e\n \u003cli\u003eTask\u0026eacute;n K, Aandahl EM. Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev. 2004 Jan;84(1):137-67. doi: 10.1152/physrev.00021.2003. PMID: 14715913.\u003c/li\u003e\n \u003cli\u003eSaitoh S, Saito T, Otake A, Owada T, Mitsugi M, Hashimoto H, Maruyama Y. Cilostazol, a novel cyclic AMP phosphodiesterase inhibitor, prevents reocclusion after coronary arterial thrombolysis with recombinant tissue-type plasminogen activator. Arterioscler Thromb. 1993 Apr;13(4):563-70. doi: 10.1161/01.atv.13.4.563. PMID: 8385480.\u003c/li\u003e\n \u003cli\u003eClarke WR, Uezono S, Chambers A, Doepfner P. The type III phosphodiesterase inhibitor milrinone and type V PDE inhibitor dipyridamole individually and synergistically reduce elevated pulmonary vascular resistance. Pulm Pharmacol. 1994 Apr;7(2):81-9. doi: 10.1006/pulp.1994.1009. PMID: 8081075.\u003c/li\u003e\n \u003cli\u003eRemme WJ, van Hoogenhuyze DC, Kruijssen HA, Pieper PG, Bruggeling WA. Preload-dependent hemodynamic effects of milrinone in moderate heart failure.\u0026nbsp;Cardiology. 1992;80(2):132-42. doi: 10.1159/000174991. PMID: 1611632.\u003c/li\u003e\n \u003cli\u003eKaumann AJ, Galindo-Tovar A, Escudero E, Vargas ML.\u0026nbsp;Phosphodiesterases do not limit beta1-adrenoceptor-mediated sinoatrial tachycardia: evidence with PDE3 and PDE4 in rabbits and PDE1-5 in rats. Naunyn Schmiedebergs Arch Pharmacol. 2009 Nov;380(5):421-30. doi: 10.1007/s00210-009-0445-5. Epub 2009 Aug 20. PMID: 19693491.\u003c/li\u003e\n \u003cli\u003eWachtel H. Characteristic behavioural alterations in rats induced by rolipram and other selective adenosine cyclic 3\u0026apos;, 5\u0026apos;-monophosphate phosphodiesterase inhibitors. Psychopharmacology (Berl). 1982;77(4):309-16. doi: 10.1007/BF00432761. PMID: 6182575.\u003c/li\u003e\n \u003cli\u003eTaniguchi T, Fujiwara M, Ohsumi K. Possible involvement of cyclic adenosine 3\u0026apos;:5\u0026apos;-monophosphate in the genesis of catecholamine-induced tachycardia in isolated rabbit sinoatrial node. J Pharmacol Exp Ther. 1977 Jun;201(3):678-88. PMID: 194027.\u003c/li\u003e\n \u003cli\u003eVinogradova TM, Lyashkov AE, Zhu W, Ruknudin AM, Sirenko S, Yang D, Deo S, Barlow M, Johnson S, Caffrey JL, Zhou YY, Xiao RP, Cheng H, Stern MD, Maltsev VA, Lakatta EG. High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells. Circ Res. 2006 Mar 3;98(4):505-14. doi: 10.1161/01.RES.0000204575.94040.d1. Epub 2006 Jan 19. PMID: 16424365\u003c/li\u003e\n \u003cli\u003eButcher RW, Sutherland EW. Adenosine 3\u0026apos;,5\u0026apos;-phosphate in biological materials. I. Purification and properties of cyclic 3\u0026apos;,5\u0026apos;-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3\u0026apos;,5\u0026apos;-phosphate in human urine. J Biol Chem. 1962 Apr;237:1244-50. PMID: 13875173.\u003c/li\u003e\n \u003cli\u003eUckert S, Kuczyk MA. Cyclic nucleotide metabolism including nitric oxide and phosphodiesterase-related targets in the lower urinary tract. Handb Exp Pharmacol. 2011;(202):527-42. doi: 10.1007/978-3-642-16499-6_23. PMID: 21290241.\u003c/li\u003e\n \u003cli\u003eBender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev. 2006 Sep;58(3):488-520. doi: 10.1124/pr.58.3.5. PMID: 16968949.\u003c/li\u003e\n \u003cli\u003eRochais F, Abi-Gerges A, Horner K, Lefebvre F, Cooper DM, Conti M, Fischmeister R, Vandecasteele G. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes.\u0026nbsp;Circ Res. 2006 Apr 28;98(8):1081-8. doi: 10.1161/01.RES.0000218493.09370.8e. Epub 2006 Mar 23. PMID: 16556871; PMCID: PMC2099453.\u003c/li\u003e\n \u003cli\u003eGresele P, Momi S, Falcinelli E. Anti-platelet therapy: phosphodiesterase inhibitors. Br J Clin Pharmacol. 2011 Oct;72(4):634-46. doi: 10.1111/j.1365-2125.2011.04034.x. PMID: 21649691; PMCID: PMC3195739.\u003c/li\u003e\n \u003cli\u003eNash CES, Antunes NJ, Coelho-Silva WC, Campos R, De Nucci G. Quantification of cyclic AMP and cyclic GMP levels in Krebs-Henseleit solution by LC-MS/MS: Application in washed platelet aggregation samples. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 15;1211:123472. doi: 10.1016/j.jchromb.2022.123472. Epub 2022 Sep 27. PMID: 36215878.\u003c/li\u003e\n \u003cli\u003eSweed D, Elhamed SMA, Aiad HAS, Ehsan NA, Hemida AS, Dawoud MM. STIM1/SOX2 proteins are co-expressed in the tumor and microenvironmental stromal cells of pancreatic ductal adenocarcinoma and ampullary carcinoma. World J Surg Oncol. 2024 Mar 26;22(1):84. doi: 10.1186/s12957-024-03356-y. PMID: 38532463; PMCID: PMC10964627.\u003c/li\u003e\n \u003cli\u003eZhang X, Pizzoni A, Hong K, Naim N, Qi C, Korkhov V, Altschuler DL. CAP1 binds and activates adenylyl cyclase in mammalian cells. Proc Natl Acad Sci U S A. 2021 Jun 15;118(24):e2024576118. doi: 10.1073/pnas.2024576118. PMID: 34099549; PMCID: PMC8214675.\u003c/li\u003e\n \u003cli\u003eSoboloff J, Rothberg BS, Madesh M, Gill DL. STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol. 2012 Sep;13(9):549-65. doi: 10.1038/nrm3414. PMID: 22914293; PMCID: PMC3458427.\u003c/li\u003e\n \u003cli\u003eRibeiro LF, Babadopulos T, de Oliveira MG, Nishimaru F, Zatz R, Elias RM, Moraes O, Moraes E, Peterson LW, De Nucci G. A LC-MS/MS method for the simultaneous determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma and its clinical application in patients with chronic kidney disease. Biomed Chromatogr. 2024 Aug;38(8):e5896. doi: 10.1002/bmc.5896. Epub 2024 May 26. PMID: 38797868.\u003c/li\u003e\n \u003cli\u003eMotiani RK, Tanwar J, Raja DA, Vashisht A, Khanna S, Sharma S, Srivastava S, Sivasubbu S, Natarajan VT, Gokhale RS. STIM1 activation of adenylyl cyclase 6 connects Ca\u003csup\u003e2+\u003c/sup\u003e and cAMP signaling during melanogenesis.\u0026nbsp;EMBO J. 2018 Mar 1;37(5):e97597. doi: 10.15252/embj.201797597. Epub 2018 Jan 8. PMID: 29311116; PMCID: PMC5830913.\u003c/li\u003e\n \u003cli\u003ePeche VS, Holak TA, Burgute BD, Kosmas K, Kale SP, Wunderlich FT, Elhamine F, Stehle R, Pfitzer G, Nohroudi K, Addicks K, St\u0026ouml;ckigt F, Schrickel JW, Gallinger J, Schleicher M, Noegel AA.\u0026nbsp;Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy. Cell Mol Life Sci. 2013 Feb;70(3):527-43. doi: 10.1007/s00018-012-1142-y. Epub 2012 Sep 4. Erratum in: Cell Mol Life Sci. 2017 Nov;74(21):4045. doi: 10.1007/s00018-017-2630-x. PMID: 22945801; PMCID: PMC11113306.\u003c/li\u003e\n \u003cli\u003eLiu P, Yang Z, Wang Y, Sun A. Role of STIM1 in the Regulation of Cardiac Energy Substrate Preference. Int J Mol Sci. 2023 Aug 25;24(17):13188. doi: 10.3390/ijms241713188. PMID: 37685995; PMCID: PMC10487555.\u003c/li\u003e\n \u003cli\u003eHooper JD, Scarman AL, Clarke BE, Normyle JF, Antalis TM. Localization of the mosaic transmembrane serine protease corin to heart myocytes. Eur J Biochem. 2000 Dec;267(23):6931-7. doi: 10.1046/j.1432-1033.2000.01806.x. PMID: 11082206.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Synergism, Endothelium-derived catecholamines, Tyrosine hydroxylase, NADPH oxidase, NOX1/4 Inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-5375589/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5375589/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose-\u003c/strong\u003e Rat isolated atria and ventricles releases endothelium-derived 6-nitrodopamine and this novel catecholamine induces a potent endogenous positive chronotropic and inotropic response. 6-Cyanodopamine is released from rabbit isolated atria and ventricles, however it is not known whether this novel catecholamine has any action on the isolated heart. Therefore, it was investigated whether rat isolated atria and ventricles release 6-cyanodopamine and its action on the rat isolated heart.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods-\u003c/strong\u003e Basal release of 6-cyanodopamine was assessed by LC-MS/MS and tyrosine hydroxylase by both immunohistochemistry and fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization. Chronotropic and inotropic effects were evaluated in isolated atria and Langendorff’s preparation, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults-\u003c/strong\u003e Rat isolated ventricles presented basal release of 6-cyanodopamine, which was unaffected by pre-treatment with tetrodotoxin. Immunohistochemistry and fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization identified tyrosine hydroxylase expression in both the endothelium and in the cardiomyocytes. 6-Cyanodopamine at 10 and 100 pM induced increases in the atrial rate, which were maintained even at 30min after the preparation was washed. In the Langendorff’s preparation, 1min infusion of 6-cyanodopamine (10 and 100pM) significantly increased heart frequency, LVDP, and dP/dt(max). Bolus injection of noradrenaline (1pmol) had no effect on heart frequency, LVDP and dPdt(max). Infusion of 6-cyanodopamine (0.01pM) significantly increased heart frequency, LVDP, and dP/dt(max) when noradrenaline (1pmol) was injected at the end of the infusion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion-\u003c/strong\u003e The results indicate that 6-cyanodopamine is a potent endogenous mediator of both chronotropism and inotropism in the rat isolated heart. It has potential therapeutic effect in heart failure and may be useful as a biomarker of pathophysiological processes.\u003c/p\u003e","manuscriptTitle":"6-cyanodopamine as an Endogenous Modulator of Heart Chronotropism and Inotropism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 16:30:48","doi":"10.21203/rs.3.rs-5375589/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f323b26f-7b51-4c79-afa7-cdc2c3bc2021","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-01T08:08:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-26 16:30:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5375589","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5375589","identity":"rs-5375589","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}