The negative chronotropic effects of (±)-propranolol and (±)- 4-NO 2 -propranolol in the rat isolated right atrium are due to blockade of the 6-nitrodopamine receptor

The negative chronotropic effects of (±)-propranolol and (±)- 4-NO 2 -propranolol in the rat isolated right atrium are due to blockade of the 6-nitrodopamine receptor | 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 The negative chronotropic effects of (±)-propranolol and (±)- 4-NO 2 -propranolol in the rat isolated right atrium are due to blockade of the 6-nitrodopamine receptor Denis Lima Oliveira, Vinicius Francisco Cardoso, Jose Britto-Júnior, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4680045/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 7 You are reading this latest preprint version Abstract The positive chronotropic action induced by 6-mitrodopamine (6-ND) is selectively blocked by β 1 -adrenoceptor antagonists at concentrations that do not affect the positive chronotropic effect induced by dopamine, noradrenaline, and adrenaline. Here the effects of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol, were investigated in the rat isolated right atrium. The atrium was mounted in glass chambers containing gassed (95%O 2 :5%CO 2 ) and warmed (37°C) Krebs-Henseleit’s solution, and the isometric tension registered (PowerLab system). (±)-propranolol, (±)-4-NO 2 -propranolol and (±)-7-NO 2 -propranolol, caused concentration-dependent falls in the spontaneous atrial frequency (pIC 50 were 4.80±0.10, 4.64±0.10, and 4.95±0.10, respectively). The calculated pA 2 values for (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranol obtained for noradrenaline-induced positive chronotropic effects were 8.44±0.08, 6.41±0.07, and 9.21±0.29, respectively. The positive chronotropism induced by 6-ND (10pM) was blocked by (±)-propranolol (1mM), and (±)-4-NO 2 -propranolol (30nM). (±)-7-NO 2 -propranol (1mM) had no effect on 6-ND (10pM)-induced increases in atrial rate. The pIC 50 of (±)-propranolol, (±)-4-NO 2 -propranolol and (±)-7-NO 2 -propranolol were significantly shifted to the right in L-NAME treated atria. The discrepancy between pA 2 values of (±)-propranolol and its respective pIC 50 indicates that the falls in atrial rate induced by (±)-propranolol should not be attributed to b-adrenergic antagonism. The reduced chronotropism by (±)-propranolol (10µM) was unaffected by the sodium channel inhibitors tetrodotoxin (1µM) and lidocaine (10µM) but abolished in atria pre-treated with (±)-4-NO 2 -propranolol (10µM). The finding that (±)-propranolol causes falls in spontaneous atrial rate only in concentrations that affect 6-ND positive chronotropic effect, confirms the role of this catecholamine as endogenous modulator of heart chronotropism. (±)-4-NO 2 -propranolol behaves as a selective antagonist of 6-ND in the rat isolated atrium. b-blocker stereoselectivity L-NAME catecholamines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction 6-Nitrodopamine (6-ND) is released from isolated atria of rabbit (Quirino-Junior et al., 2023 ), rat (Britto-Júnior et al., 2022 ) and mouse (Britto-Júnior et al., 2023a ), and presents significant positive chronotropic action. In the rat isolated right atrium, 6-ND is approximately 100 times more potent than noradrenaline and adrenaline, and far more potent than dopamine (~ 10,000 times). 6-Nitrodopamine also synergizes with the classical catecholamines, producing sustained increases in the rat atrial rate (Britto-Júnior et al., 2023b ). In the isolated atria, the stimulation of b 1 -adrenoceptors is associated with increases in atrial rate (Brodde, 1991 ). Interestingly, the positive chronotropic effect induced by 6-ND is selectively blocked by β 1 -adrenoceptor antagonists such as atenolol, betaxolol and metoprolol at concentrations that do not affect the positive chronotropic effect induced by noradrenaline, adrenaline and dopamine (Britto-Júnior et al., 2022 ). Propranolol is a member of the 3-(aryloxy)-1-(alkylamino)-β-blocker family, a class of drugs where the activity is restricted mainly to the S-isomer (Welson and Burke, 1978 ), and is classified as a non-selective, surmountable, b-blocker (Coltart et., 1975). Nitroaromatic compounds are by comparison scarce in nature, and the presence of the nitrogroup in a benzene ring is associated with increased resistance to oxidative degradation (Kamisaki et al., 1998 ). Due to its electron-withdrawing nature, the nitrogroup may provide functional diversity (Kamisaki et al., 1998 ). (±)-4-Nitro-propranolol and (±)-7-nitro-propranolol have been synthetized as racemic mixtures, and the enantiomers purified following chiral high-pressure liquid chromatography (Sparaco et al., 2022 ). Here, the effects of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol and their respective (R)- and (S)-enantiomers were investigated, on both the spontaneous atrial rate and the positive chronotropic effects induced by noradrenaline, adrenaline, and 6-ND. Since the synthesis of 6-ND is significantly reduced by nitric oxide inhibition (Britto-Júnior et al., 2022 ), the negative chronotropic effects of (±)-propranolol and the nitro-derivatives were evaluated in N ω -nitro-L-arginine methyl esther (L-NAME) treated atria. Materials and Methods Animals Adult male Wistar rats (280 to 320 g) were provided by both the Central Animal House of the University of Campinas (CEMIB-UNICAMP; São Paulo, Brazil) and Animais de Laboratório Criação e Com. LTDA (ANILAB; Paulinia, São Paulo, Brazil). All experimental protocols were approved by the Ethics Committee for Animal Use of UNICAMP (CEUA; Protocol No. 5942-1/2022), 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 the ARRIVE guidelines (Percie du Sert et al., 2020 ). Animals were housed in cages (three per cage) located in ventilated cage shelters with a constant humidity of 55 ± 5% and temperature of 24 ± 1°C under a 12-hour light-dark cycle. Animals received filtered water and standard rodent food ad libitum . Rat isolated right atrium preparation Euthanasia was performed by isoflurane overdose, in which animals were exposed to a concentration greater than 5% until 1 min after the breathing stops. Exsanguination was performed to confirm euthanasia. After euthanasia, the heart was removed, and the right atrium was isolated. The right atrium was mounted between two metal hooks in 10-mL custom designed glass chambers containing Krebs-Henseleit’s solution (KHS), 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 1 h, and the isometric tension was registered using a PowerLab system (ADInstruments, Sydney, Australia), according to our previous experience (Britto-Júnior et al., 2022 , 2023b ). The right atrial spontaneous contractions driven by the sinoatrial node were recorded using Powerlab (ADInstruments, Sydney, Australia) and LabChart version 8 for Windows (AD Instruments). Effect of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol in the rat isolated atrial rate Cumulative concentration-response curves to (±)-propranolol (0.001-100 µM), (S)-(-)-propranolol (0.001-100 µM), (R)-(+)-propranolol (0.001-100 µM), (±)-4-NO 2 -propranolol (0.001-100 µM), (S)-(+)-4-NO 2 -propranolol (0.001-100 µM), (R)-(-)-4-NO 2 -propranolol (0.001-100 µM), (±)-7-NO 2 -propranolol (0.001-100 µM), (S)-(+)-7-NO 2 -propranolol (0.001-100 µM), and (R)-(-)-7-NO 2 -propranolol (0.001-300 µM) were obtained in rat isolated atria. In separate experiments, the effects of (±)-propranolol (0.001-100 µM), (±)-4-NO 2 -propranolol (0.001-100 µM) and (±)-7-NO 2 -propranolol (0.001-100 µM) were evaluated in isolated atria pretreated or not with the nitric oxide (NO) synthesis inhibitor N ω -nitro-L-arginine methyl esther (L-NAME, 100 µM, 30 min). The volume added of drugs to the 10-mL Krebs-Henseleit’s solution in the organ bath was 10 to 30 µL for each concentration. The maximum effect of the concentration-response curves was evaluated until the atrium beating stopped. Effect of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 - propranolol on the concentration-response curves to noradrenaline and adrenaline Cumulative concentration-response curves to noradrenaline (0.001-30 µM) and adrenaline (0.001-100 µM) were performed in isolated atria preparations in the absence and the presence of (±)-propranolol (0.001-0.3 µM; 60 min), (±)-4-NO 2 -propranolol (0.1–10 µM; 60 min) and (±)-7-NO 2 -propranolol (0.001 and 3 µM; 60 min). The atria were incubated for 60 min with propranolol and the nitro-derivatives, and the catecholamines were added thereafter. Effect of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 - propranolol on the positive chronotropic effect induced by 6-nitrodopamine (6-ND) The positive chronotropic induced by 6-ND (10 pM) was evaluated in isolated atria preparations in the absence and the presence of (±)-propranolol (0.3 and 1 µM; 60 min), (±)-4-NO 2 -propranolol (10 and 30 nM; 60 min), and (±)-7-NO 2 -propranolol (0.1 and 1 µM; 60 min). Effect of tetrodotoxin and lidocaine on the negative chronotropic effect of (±)-propranolol A single concentration of tetrodotoxin (1 µM) or lidocaine (10 µM) was added to the organ bath, and the changes in atrial rate were monitored for 30 minutes. After 30 minutes, incubation was carried out with (±)-propranolol (10 µM) for an additional 30 min. Effect of co-incubation of (±)-propranolol and (±)-4-NO 2 -propranolol on the basal atrial rate The atria were pre-treated with either (±)-propranolol (10 µM) for 30 min. After 30 minutes, incubation was carried out with (±)-4-NO 2 -propranolol (10 µM) for an additional 30 min. In separate experiments, the effects of (±)-propranolol (10 µM) were evaluated in atria pre-treated with (±)-4-NO 2 -propranolol (10 µM, 30 min). Data analysis Data of atrial rate are presented as beats per minute (bpm) before and after the respective stimulation or as the delta increase of atrial rate. Nonlinear regression analysis to determine the pIC 50 was carried out using GraphPad Prism (GraphPad Software, version 9.4, San Diego, California, USA) with the constraint that F = 0. All concentration–response data were evaluated for a fit to a logistics function in the form: E = E max /([1 + (10c / 10x) n ] + F, where E represents the increase in response contractile induced by the agonist, E max is the effect agonist maximum, c is the logarithm of concentration of the agonist that produces 50% of E max , x is the logarithm of the concentration of the drug; the exponential term, n, is a curve fitting parameter that defines the slope of the concentration–response line, and F is the response observed in the absence of added drug. The values of pIC 50 data represent mean ± standard error of the mean (SEM) of n experiments. Student's two-tail unpaired t-test was employed to compare the statistical differences between groups. The potencies of the antagonists in inhibiting the positive chronotropism of the catecholamines was estimated through the pA 2 (-log of the molar antagonist concentration that produces a two-fold reduction in the potency of the agonist) values as calculated by the equation “pA 2 = -log (antagonist concentration) + log (CR-1) using the lowest effective concentration of the antagonist in the calculations. Additionally, the potencies of propranolol, its nitro-derivatives and enantiomers in inhibiting the spontaneous atrial beating rate were estimated through the pIC 50 values using the equation (Y = Bottom + (Top-Bottom)/(1 + 10^((-pIC50-X)*HillSlope))in the GraphPad Prism Software. Since the study has an exploratory character, the p values should be considered descriptive (Motulsky, 2014 ; Michel et al., 2020 ). Chemicals and reagents (±)-4-NO 2 -propranolol, (±)-7-NO 2 - propranolol, ( S )-(+)-4-NO 2 -propranolol, ( R )-(-)-4-NO 2 -propranolol, ( S )-(+)-7-NO 2 -propranolol and ( R )-(-)-7-NO 2 -propranolol were synthesized as described elsewhere (Sparaco et al., 2023). 6-nitrodopamine was acquired from Toronto Research Chemicals (TRC, Toronto, Canada). (±)-Propranolol, (R)-(+)-propranolol, lidocaine, and N ω -nitro-L-arginine methyl ester (L-NAME) were obtained from Sigma-Aldrich Chemicals Co (Missouri, USA). Adrenaline, noradrenaline, (S)-(-)-propranolol, and tetrodotoxin were purchased from Cayman Chemical Co (Michigan, USA). Sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl 2 ), magnesium sulfate (MgSO 4 ), sodium bicarbonate (NaHCO 3 ), potassium phosphate mono-basic (KH 2 PO 4 ) and glucose were acquired from Merck KGaA (Darmstadt, Germany). The composition of the Krebs-Henseleit’s solution (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. (±)-Propranolol (racemic and enantiomers), (±)-4-NO 2 -propranolol (racemic and enantiomers), (±)-7-NO 2 -propranolol (racemic and enantiomers), 6-nitrodopamine, noradrenaline, and adrenaline were all dissolved in water. Results Effect of (±)-propranolol, (±)-4-NO 2 -propranolol, (±)-7-NO 2 -propranolol and their enantiomers on the basal atrial rate Concentration-response curves to (±)-propranolol, (S)-(-)-propranolol, and (R)-(+)-propranolol in the basal atrial rate are illustrated in Fig. 1 A. The racemic mixture ((±)-propranolol) and its (R)- and (S)-enantiomers (0.001-100 µM) concentration-dependently reduced the spontaneous atrial rate until the atrium stopped beating. Compared with (±)-propranolol, concentration-response curves to both (S)-(+)-propranolol and (R)-(+)-propranolol were displaced to the right with a significant reduction in pIC 50 (Table 1 ), but no differences between (S)-(-)-propranolol and (R)-(+)-propranolol were found (Table 1 ). Table 1 The potency (pIC 50 ) of (±)-propranolol, (S)-(-)-propranolol, (R)-(+)-propranolol, (±)-4-NO 2 -propranolol, (S)-(+)-4-NO 2 -propranolol, (R)-(-)-4-NO 2 -propranolol, (±)-7-NO 2 -propranolol, (S)-(+)-7-NO 2 -propranolol, and (R)-(-)-7-NO 2 -propranolol to reduce the basal atrial rate in the rat isolated atria. Compound pIC 50 (log[M]) p-value n (±)-propranolol 4.80±0.10 7 (S)-(-)-propranolol 4.30±0.10 0.0272 7 (R)-(+)-propranolol 4.34±0.10 0.0220 7 (±)-4-NO 2 -propranolol 4.95±0.13 8 (S)-(+)-4-NO 2 -propranolol 4.78±0.04 0.3197 8 (R)-(-)-4-NO 2 -propranolol 4.09±0.07 0.0009 8 (±)-7-NO 2 -propranolol 4.64±0.09 8 (S)-(-)-7-NO 2 -propranolol 4.68±0.11 0.7122 8 (R)-(-)-7-NO 2 -propranolol 3.84±0.15 0.0054 8 The concentration-response curves to (±)-4-NO 2 -propranolol (0.001-100 µM) and its enantiomers (S)-(+)-4-NO 2 -propranolol (0.001-100 µM) and (R)-(-)-4-NO 2 -propranolol (0.001-300 µM) are shown in Fig. 1 B. All these compounds concentration-dependently reduced the spontaneous atrial rate. The pIC 50 values showed no differences between (±)-4-NO 2 -propranolol and (S)-(+)-4-NO 2 -propranolol (Table 1 ). Compared with (±)-4-NO 2 -propranolol and (S)-(+)-4-NO 2 -propranolol, the concentration-response curve to (R)-(-)-4-NO 2 -propranolol was displaced to the right with a significant reduction in pIC 50 (Table 1 ). The concentration-response curves to (±)-7-NO 2 -propranolol (0.001-100 µM) and its enantiomers (S)-(+)-7-NO 2 -propranolol (0.001-100 µM) and (R)-(-)-7-NO 2 -propranolol (0.001-300 µM) are shown in Fig. 1 C. All these compounds concentration-dependently reduced the spontaneous atrial rate. Compared with (±)-7-NO 2 -propranol and (S)-(-)-7-NO 2 -propranolol, the concentration-response curve to (R)-(-)-7-NO 2 -propranolol was displaced to the right with a significant reduction in pIC 50 (Table 1 ). The pIC 50 values showed no differences between (±)-7-NO 2 -propranolol and (S)-(-)-7-NO 2 -propranolol (Table 1 ). Effect of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol on the positive chronotropic effect induced by noradrenaline and adrenaline To estimate the potencies (pA2) of (±)-propranolol and its nitro-derivatives, concentration-response curves to noradrenaline (NA) were performed in the absence and presence of (±)-propranolol (0.001-0.3 µM; Fig. 2 A), (±)-4-NO 2 -propranolol (0.1–10 µM; Fig. 2 B) and (±)-7-NO 2 -propranolol (0.001-3 µM; Fig. 2 C). Noradrenaline produced concentration-dependent increases in atrial rate, which was significantly displaced to the right by (±)-propranolol at 10 nM, but not by this compound at 1 nM (Fig. 2 A). (±)-4-NO 2 -propranolol (Fig. 2 B) and (±)-7-NO 2 -propranolol (Fig. 2 C) also produced right displacements on the concentration-response of noradrenaline, but the nitro-compound (±)-4-NO 2 -propranolol exhibited the lowest potency, as revealed by its pA 2 values (Table 2 ). Table 2 The values of pA 2 of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol in the concentration-response curves for noradrenaline and adrenaline in rat isolated atria. Compound Noradrenaline (pA 2 ) Adrenaline (pA 2 ) (±)-Propranolol 8.44 ± 0.08 (n = 5) 8.59 ± 0.14 (n = 6) (±)-4-NO 2 -Propranolol 6.41 ± 0.07 (n = 6) 6.98 ± 0.12 (n = 3) (±)-7-NO 2 -Propranolol 9.21 ± 0.29 (n = 4) 8.84 ± 0.24 (n = 4) Similar data on the atrial rate were observed with adrenaline (Fig. 2 D, E and F). The concentration-dependent increases in atrial rate produced by adrenaline were significantly displaced to the right by (±)-propranolol 10 nM (Fig. 2 D) and its nitro-compounds (±)-4-NO 2 -propranolol (Fig. 2 E) and (±)-7-NO 2 -propranolol (Fig. 2 F). Similarly to noradrenaline, the least potent compound at inhibiting the positive chronotropism induced by adrenaline was (±)-4-NO 2 -propranolol, as determined by its pA2 values (Table 2 ). Effect of L-NAME on the negative chronotropic effects induced by (±)-propranolol, (±)-4-NO 2 -propranolol and (±)-7-NO 2 -propranolol Incubation of the rat isolated right atrium with L-NAME (100 µM) caused a small (but significant) reduction in the spontaneous atrial rate (296 ± 6 and 271 ± 5 bpm, for control and L-NAME, respectively; p < 0.01). In addition, L-NAME (100 µM) produced a rightward shift in the concentration-response curves to (±)-propranolol, (±)-4-NO 2 -propranolol and (±)-7-NO 2 -propranolol, significantly reducing the potencies (pIC 50 values) of these compounds in diminishing the spontaneous atrial beat rate (Fig. 3 A, B and C). Table 3 shows the calculated pIC 50 values for these three compounds in control and in L-NAME-treated atria. Table 3 Effect of L-NAME (100 µM) on the potency (pIC 50 ) of (±)-propranolol, (±)-4-NO 2 -propranolol and (±)-7-NO 2 -propranolol in inhibiting the spontaneous rat atria rate. Compound pIC 50 (log[M]) p-value n (±)-propranolol 4.85±0.10 7 (±)-propranolol + L-NAME 4.49±0.08 0.0099 7 (±)-4-NO 2 -propranolol 4.76±0.07 9 (±)-4-NO 2 -propranolol + L-NAME 4.39±0.08 0.0056 9 (±)-7-NO 2 -propranolol 4.37±0.10 8 (±)-7-NO 2 -propranolol + L-NAME 4.05±0.02 0.0021 8 Effect of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol on the positive chronotropic effect of 6-nitrodopamine 6-Nitrodopamine (6-ND; 10 pM) caused a sustained (60 min) increase in the atrial rate (Fig. 4 A-F). The increase in atrial rate induced by 6-ND was unaffected by (±)-propranolol at 300 nM (Fig. 4 A), but it was fully inhibited at a higher concentration (1 µM; Fig. 4 B). Incubation with (±)-4-NO 2 -propranolol (10 nM) caused no significant effect in the increased atrial rate induced by 6-ND (Fig. 4 C); however, at 30 nM, (±)-4-NO 2 -propranolol significantly reduced the increased atrial rate (Fig. 4 D). Incubation with (±)-7-NO 2 -propranolol at 100 nM (Fig. 4 E) or 1 µM (Fig. 4 F) had no effect on the increased atrial rate induced by 6-ND. Effect of tetrodotoxin and lidocaine on the negative chronotropic effect of (±)-propranolol Pre-treatment (30 min) of the isolated right atria with either tetrodotoxin (1 µM) or lidocaine (10 µM) caused significant falls in the atrial basal rate (Fig. 5 A and C, respectively). In tetrodotoxin- and lidocaine-pretreated atria preparations, further incubation of the with (±)-propranolol (10 µM) caused an additional fall in basal rate (Fig. 5 B and D). Effect of co-incubation of (±)-propranolol and (±)-4-NO 2 -propranolol on the basal atrial rate Pre-treatment (30 min) of the isolated right atria with either (±)-propranolol (10 µM) or (±)-4-NO 2 -propranolol (10 µM) caused significant falls in atrial basal rate (Fig. 6 A and 6 C, respectively). In atria pre-incubated with (±)-propranolol (10 µM), further incubation with (±)-4-NO 2 -propranolol (10 µM) did not alter the basal atrial rate (Fig. 6 B). In preparations pre-incubated with (±)-4-NO 2 -propranolol (10 µM), further incubation with (±)-propranolol (10 µM) did not alter basal atrial rate (Fig. 6 D). Discussion The results demonstrate that (±)-4-NO 2 -propranolol causes a fall in spontaneous atrial rate at concentrations that do not affect the positive chronotropic effect induced by noradrenaline and adrenaline but do inhibit the positive chronotropic effect induced by 6-ND. Indeed, the pIC 50 for (±)-4-NO 2 -propranolol and (±)-propranolol is almost identical (4.95 ± 0.13 and 4.80 ± 0.10, respectively), yet (±)-propranolol was approximately one hundred times more potent than (±)-4-NO 2 -propranolol to block the positive chronotropic effect induced by noradrenaline and adrenaline. The calculated pA 2 values obtained in our results for (±)-propranolol (8.2 and 8.5 for noradrenaline and adrenaline, respectively) are like those reported in the isolated cat papillary muscle preparations (8.3) using isoprenaline as an inotropic agent (Lewis et al., 1983), and in the guinea-pig atria (8.2) using noradrenaline as a chronotropic agent (O’Donnell & Wanstall, 1979). Similar discrepancy is observed when the pIC 50 of both (±)-4-NO 2 -propranolol and (±)-7-NO 2 -propranolol is compared; (±)-4-NO 2 -propranolol is more potent than (±)-7-NO 2 -propranolol to cause fall in atrial rate, yet (±)-7-NO 2 -propranolol is one hundred times more potent than (±)-4-NO 2 -propranolol to block the positive chronotropic effect induced by noradrenaline and adrenaline. These discrepancies clearly indicate that the negative chronotropic effects of (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol on the basal atrial rate are not related to their ability to block the positive chronotropic effects of either noradrenaline or adrenaline. Actually, the concentrations that these drugs cause fall in basal atrial rate are the concentrations that they inhibit the positive chronotropic effect of 6-ND. The basal release of 6-ND from rat isolated atria is inhibited by pre-incubation of the atria with the NO synthase inhibitor L-NAME (Britto-Júnior et al., 2022 ); it is interesting that pre-treatment of the atria with L-NAME caused a significant reduction in basal atrial rate (Britto-Júnior et al., 2022 ), and significant right shifts in the concentration-response curves to (±)-propranolol, (±)-4-NO 2 -propranolol, and (±)-7-NO 2 -propranolol. Both findings could be interpreted by inhibition of 6-ND synthesis induced by L-NAME. It is worth mentioning that selective b 1 -adrenoceptor antagonists such as atenolol, betaxolol, and metoprolol, cause significant reduction in basal atrial rate at concentrations that block the positive chronotropic effect of 6-ND, but that do not affect the positive chronotropic effect induced by noradrenaline, adrenaline, and dopamine (Britto-Júnior et al., 2022 ). Propranolol is therapeutically used as a racemic mixture with equal concentrations of (S)-(-)-propranolol and (R)-(+)-propranolol, however the (R)-(+)-propranolol is devoid of blocking activity (Mehvar and Brocks, 2001 ). Our finding that there was no difference in the pIC50 of (S)-(-)-propranolol and (R)-(+)-propranolol, further indicates that the fall in basal atrial rate induced by (±)-propranolol is not related to its effect on the b-adrenoceptors. Propranolol is known to block voltage-gated sodium channels, and this ability is not stereospecific, since both enantiomers of propranolol block cardiac Na v 1.5 channels at equivalent potency (Wang et al., 2010 ). Thus, one possibility would be that the fall in atrial rate induced by (±)-propranolol could be due to blockade of this tetrodotoxin-sensitive sodium channel. However, the finding that provoked significant falls in atrial rate in tetrodotoxin pre-treated atria, indicates that it is unlikely that the falls could be due to blockade of cardiac tetrodotoxin-sensitive Nav channels. Lidocaine is also a Nav 1.5 channel blocker, it has anti-arrhythmic activity, and is supposed to have at least one locus of action at a site other than the TTX blockade site (Desai et al., 1989 ). One possibility would be that propranolol would be acting at this other site. This is also unlikely, since (±)-propranolol was able to induce fall in atrial rate in lidocaine-pretreated atria. Thus, blockade of the 6-ND receptor is the most likely explanation for the fall in atrial rate induced by (±)-propranolol. This concept is further supported by the finding that in atria pre-treated with (±)-4-NO 2 -propranolol, (±)-propranolol fails to induce reduction in atrial rate, and in atria pre-treated with (±)-propranolol, (±)-4-NO 2 -propranolol did not alter basal atrial rate, indicating that both drugs are acting on the same receptor, i.e., the 6-ND receptor. (±)-4-NO 2 -propranolol should be considered a first selective 6-ND receptor antagonist, since it has very weak potency for blocking the positive chronotropic effect induced by both noradrenaline and adrenaline. The autonomic nervous system is believed to be a major regulator in the cardiovascular system and its adaptation to various human body functions (Hirano et al., 1982 ), although this concept has been challenged recently (Zatz & De Nucci, 2023). In patients submitted to orthotopic cardiac transplantation, the heart rate at rest is increased (103 ± 12 bpm) compared to age-matched control (77 ± 13 bpm); however, the peak heart rate following exercise is decreased (137 ± 15 and 176 ± 12, respectively). These patients have no evidence of cardiac innervation (Kavanagh et al., 1988 ). Thus, resting heart seems to be independent of adrenergic catecholamine release. For evaluating the importance of 6-ND as the endogenous modulator of heart chronotropism, it is essential to assess the effect of these nitro-derivatives of propranolol in both anaesthetized and conscious rats. Another possibility is to investigate whether propranolol in humans is metabolized to nitro-derivatives; however, since there are several positions that propranolol can be “nitrated”, synthesis of nitro-atenolol may solve this conundrum provided, unlike propranolol, it has only two positions on its molecule to receive the nitro group. Conclusion The finding that (±)-propranolol and (±)-4-NO 2 -propranolol cause falls in spontaneous atrial rate only in concentrations that affect a 6-ND positive chronotropic effect, confirms the role of this catecholamine as endogenous modulator of heart chronotropism. (±)-4-NO 2 -propranolol behaves as a selective antagonist of 6-ND in the rat isolated atrium. Declarations Ethical Approval All experimental protocols were authorized by the Ethics Committee in Animal Use of UNICAMP (CEUA/UNICAMP, protocol number 5942-1/2022). Consent to Participate Not applicable. Consent to Publish The authors authorize the submission and publication of this article Naunyn-Schmiedeberg's Archives of Pharmacology Author Contributions Statement Conceptualization: JBJ and GDN. Data curation: JBJ and GDN. Formal analysis: GDN Funding acquisition: EA and GDN. Investigation: JBJ, DLO, VFC, VF, GDN. Methodology: JBJ, DLO, VFC, VF, RS, VS, EA, FZM, GDN. Project administration: GDN. Supervision: EA. Visualization: EA and GDN. Writing – original draft: JBJ, FF, GC 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). VF thank FAPESP for PhD fellowship (2022/07737-8). 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 Andersen ML (2016) 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. 11.794/2008, art 22, inciso II Arunlakshana O, Schild HO (1959) Some quantitative uses of drug antagonists. 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Basic Res Cardiol 84:461–468. 10.1007/BF01908198 Wang DW, Mistry AM, Kahlig KM, Kearney JA, Xiang J, George AL Jr (2010) 31;1:144 Propranolol blocks cardiac and neuronal voltage-gated sodium channels. Front Pharmacol. 10.3389/fphar.2010.00144 . PMID: 21833183 Welson WL, Burke TR (1978) Absolute configuration of glycerol derivatives. 5. Oxprenolol enantiomers. J Org Chem 43:3641–3645. 10.1021/jo00413a002 Zatz R, De Nucci G (2024) Endothelium-derived dopamine and 6-nitrodopamine in the cardiovascular system. Physiology. 39:44–59. doi: 10.1152/physiol.00020.2023 Additional Declarations No competing interests reported. Supplementary Files Reviewer103072024d1.docx Reviewer203072024d1.docx Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted Editorial decision: Revision requested 05 Aug, 2024 Reviews received at journal 29 Jul, 2024 Reviewers agreed at journal 09 Jul, 2024 Reviewers invited by journal 09 Jul, 2024 Editor assigned by journal 03 Jul, 2024 Submission checks completed at journal 03 Jul, 2024 First submitted to journal 03 Jul, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4680045","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329338349,"identity":"e7d189dd-fc96-4b61-92d2-15afcae191a0","order_by":0,"name":"Denis Lima Oliveira","email":"","orcid":"","institution":"State University of Campinas (UNICAMP)","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"Lima","lastName":"Oliveira","suffix":""},{"id":329338350,"identity":"b2459515-e4b8-4fd5-82c6-f83df080e13a","order_by":1,"name":"Vinicius Francisco Cardoso","email":"","orcid":"","institution":"State University of Campinas 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11:27:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4680045/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4680045/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-024-03463-3","type":"published","date":"2024-10-09T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61310538,"identity":"01db5232-9188-44eb-bb9a-329199f28f40","added_by":"auto","created_at":"2024-07-29 10:59:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":389427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of (±)-propranolol, (S)-(-)-propranolol, (R)-(+)-propranolol, (±)-4-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol, (S)-(+)-4-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol, (R)-(-)-4-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol, (±)-7-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol, (S)-(+)-7-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol, and (R)-(-)-7-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol on the spontaneous atrial rate.\u003c/strong\u003e Panel A illustrates the concentration-response curves to (±)-propranolol, (S)-(-)-propranolol, and (R)-(+)-propranolol. Panel B illustrates the concentration-response curves to (±)-4-NO2-propranolol, (S)-(+)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (R)-(-)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol. Panel C illustrates the concentration-response curves to (±)-7-NO2-propranolol, (S)-(+)-7-NO2-propranolol, and (R)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol. Data are expressed as mean ± SEM (number of animals are included in each panel).\u003c/p\u003e","description":"","filename":"Figure01.png","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/a9a6eb8d9fdb8045b5b94fec.png"},{"id":61310544,"identity":"55b9b53a-dce2-436f-a8b2-a30a41dc6819","added_by":"auto","created_at":"2024-07-29 10:59:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":766710,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of\u003c/strong\u003e \u003cstrong\u003e(±)-propranolol, (±)-4-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol and (±)-7-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol on the positive chronotropic effect induced by noradrenaline (NA; Panels A, B and C, respectively) and adrenaline (ADR; Panels D, E and F, respectively). \u003c/strong\u003eConcentration-response curves to noradrenaline were performed in the presence of (±)-propranolol (0.001-0.3 mM; Panel A), (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.1-10 mM; Panel B) and (±)-7-NO-propranolol (0.001 and 3 mM; Panel C). Concentration-response curves to adrenaline were performed in the presence of (±)-propranolol (0.001-0.3 mM; Panel D), (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.1-10 mM; Panel E), and (±)-7-NO-propranolol (0.001 and 3 mM; Panel F). Data are expressed as mean ± SEM (number of animals are included in each panel).\u003c/p\u003e","description":"","filename":"Figure02.png","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/4d2fa2eb23671d115205aee3.png"},{"id":61310543,"identity":"a29d0744-cff8-4f4c-98f6-827fd6aff8c5","added_by":"auto","created_at":"2024-07-29 10:59:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":337530,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of L-NAME on the negative chronotropic effect induced by (±)-propranolol, (±)-4-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol and (±)-7-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol. \u003c/strong\u003eIncubation of the rat isolated right atrium with L-NAME (100 mM) caused right shifts in the concentration curves of (±)-propranolol (0.001-100 mM; Panel A), (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 mM; Panel B) and (±)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 mM; Panel C). Data are expressed as mean ± SEM (number of animals are included in each panel).\u003c/p\u003e","description":"","filename":"Figure03.png","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/d7a4c0ebd4b2e693a995064d.png"},{"id":61311331,"identity":"4ed85a5a-d3dd-4774-bb2e-b51a78acc593","added_by":"auto","created_at":"2024-07-29 11:07:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":646786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of (±)-propranolol, (±)-4-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol and (±)-7-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol on the positive chronotropic effect induced by 6-nitrodopamine (6-ND; 10 pM).\u003c/strong\u003e\u0026nbsp; The positive chronotropic effect induced by 6-ND (10 pM) was not affected by incubation with (±)-propranolol at 300 nM (Panel A), but it was abolished when the atria were pre-incubated with (±)-propranolol at 1 mM (Panel B). Incubation of the atria with (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 nM) caused no significant reductions in the increases in atrial frequency induced by 6-ND (Panel C) but it was abolished when the atria were pre-incubated with (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol at 30 nM) (Panel B).The (±)-7NO\u003csub\u003e2\u003c/sub\u003e-propranolol at 100 nM (Panel D) and 1 mM (Panel E) did not affect the positive chronotropic effect induced by 6-ND (10 pM). Data are expressed as mean ± SEM (number of animals are included in each panel).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure04.png","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/3a41832a6e2033049bd44bce.png"},{"id":61310546,"identity":"4fd75cc6-7356-4078-aa68-0877de51f34e","added_by":"auto","created_at":"2024-07-29 10:59:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":220221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of tetrodotoxin and lidocaine on the negative chronotropic effect of (±)-propranolol.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eTetrodotoxin at 1 mM caused significant falls in atrial basal rate (Panel A). The right isolated atria incubation with (±)-propranolol (10 mM) caused a further fall in basal rate of the atria pre-treated with tetrodotoxin (Panel B). Lidocaine at 10 mM caused significant falls in atrial basal rate (Panel C). The right isolated atria incubation with (±)-propranolol (10 mM) caused a further fall in basal rate of the atria pre-treated with either lidocaine (Panel D). Data are expressed as mean ± SEM (number of animals are included in each panel).\u003c/p\u003e","description":"","filename":"Figure05.png","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/9dc11e88b24309d7ba98919d.png"},{"id":61310541,"identity":"3e780c0e-fa0b-41ec-9cb5-db0db61ad8cf","added_by":"auto","created_at":"2024-07-29 10:59:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":247271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of co-incubation of (±)-propranolol and (±)-4-NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-propranolol on the basal atrial rate\u003c/strong\u003e. (±)-Propranolol at 10 mM caused significant falls in atrial basal rate (panel A). In atria pre-incubated with (±)-propranolol (10 mM), further incubation with (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 mM) did not alter basal atrial rate (Panel B). (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol at 10 mM caused significant falls in atrial basal rate (panel C). In atria pre-incubated with (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolo (10 mM), further incubation with (±)-propranolol (10 mM) did not alter basal atrial rate (Panel D). Data are expressed as mean ± SEM (number of animals are included in each panel).\u003c/p\u003e","description":"","filename":"Figure06.png","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/4b8ffd3bdf8e24c0b2b39a47.png"},{"id":66597231,"identity":"6e324dcd-ea00-4bb8-98b1-a3cf394092f1","added_by":"auto","created_at":"2024-10-14 16:08:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3433631,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/e2bb9f14-d499-4f5c-adcb-93a292d5a9e1.pdf"},{"id":61310542,"identity":"cb67c6b7-4c58-4fff-a809-2e7eded56a2b","added_by":"auto","created_at":"2024-07-29 10:59:25","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":238464,"visible":true,"origin":"","legend":"","description":"","filename":"Reviewer103072024d1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/06b84b0ac576d54dbb170c9f.docx"},{"id":61310540,"identity":"7681820b-aaf1-41d3-b87b-6b589b2e3571","added_by":"auto","created_at":"2024-07-29 10:59:24","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":125058,"visible":true,"origin":"","legend":"","description":"","filename":"Reviewer203072024d1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4680045/v1/86251f74f7517cb7d78fd0dc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The negative chronotropic effects of (±)-propranolol and (±)- 4-NO 2 -propranolol in the rat isolated right atrium are due to blockade of the 6-nitrodopamine receptor","fulltext":[{"header":"Introduction","content":"\u003cp\u003e6-Nitrodopamine (6-ND) is released from isolated atria of rabbit (Quirino-Junior et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), rat (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and mouse (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), and presents significant positive chronotropic action. In the rat isolated right atrium, 6-ND is approximately 100 times more potent than noradrenaline and adrenaline, and far more potent than dopamine (~\u0026thinsp;10,000 times). 6-Nitrodopamine also synergizes with the classical catecholamines, producing sustained increases in the rat atrial rate (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). In the isolated atria, the stimulation of b\u003csub\u003e1\u003c/sub\u003e-adrenoceptors is associated with increases in atrial rate (Brodde, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Interestingly, the positive chronotropic effect induced by 6-ND is selectively blocked by β\u003csub\u003e1\u003c/sub\u003e-adrenoceptor antagonists such as atenolol, betaxolol and metoprolol at concentrations that do not affect the positive chronotropic effect induced by noradrenaline, adrenaline and dopamine (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePropranolol is a member of the 3-(aryloxy)-1-(alkylamino)-β-blocker family, a class of drugs where the activity is restricted mainly to the S-isomer (Welson and Burke, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1978\u003c/span\u003e), and is classified as a non-selective, surmountable, b-blocker (Coltart et., 1975). Nitroaromatic compounds are by comparison scarce in nature, and the presence of the nitrogroup in a benzene ring is associated with increased resistance to oxidative degradation (Kamisaki et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Due to its electron-withdrawing nature, the nitrogroup may provide functional diversity (Kamisaki et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). (\u0026plusmn;)-4-Nitro-propranolol and (\u0026plusmn;)-7-nitro-propranolol have been synthetized as racemic mixtures, and the enantiomers purified following chiral high-pressure liquid chromatography (Sparaco et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Here, the effects of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and their respective (R)- and (S)-enantiomers were investigated, on both the spontaneous atrial rate and the positive chronotropic effects induced by noradrenaline, adrenaline, and 6-ND. Since the synthesis of 6-ND is significantly reduced by nitric oxide inhibition (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the negative chronotropic effects of (\u0026plusmn;)-propranolol and the nitro-derivatives were evaluated in N\u003csup\u003eω\u003c/sup\u003e-nitro-L-arginine methyl esther (L-NAME) treated atria.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult male Wistar rats (280 to 320 g) were provided by both the Central Animal House of the University of Campinas (CEMIB-UNICAMP; S\u0026atilde;o Paulo, Brazil) and Animais de Laborat\u0026oacute;rio Cria\u0026ccedil;\u0026atilde;o e Com. LTDA (ANILAB; Paulinia, S\u0026atilde;o Paulo, Brazil). All experimental protocols were approved by the Ethics Committee for Animal Use of UNICAMP (CEUA; Protocol No. 5942-1/2022), 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=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) as well as by the ARRIVE guidelines (Percie du Sert et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Animals were housed in cages (three per cage) located in ventilated cage shelters with a constant humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5% and 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 \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRat isolated right atrium preparation\u003c/h2\u003e \u003cp\u003eEuthanasia was performed by isoflurane overdose, in which animals were exposed to a concentration greater than 5% until 1 min after the breathing stops. Exsanguination was performed to confirm euthanasia. After euthanasia, the heart was removed, and the right atrium was isolated. The right atrium was mounted between two metal hooks in 10-mL custom designed glass chambers containing Krebs-Henseleit\u0026rsquo;s solution (KHS), 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 1 h, and the isometric tension was registered using a PowerLab system (ADInstruments, Sydney, Australia), according to our previous experience (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). The right atrial spontaneous contractions driven by the sinoatrial node were recorded using Powerlab (ADInstruments, Sydney, Australia) and LabChart version 8 for Windows (AD Instruments).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eEffect of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol in the rat isolated atrial rate\u003c/h2\u003e \u003cp\u003eCumulative concentration-response curves to (\u0026plusmn;)-propranolol (0.001-100 \u0026micro;M), (S)-(-)-propranolol (0.001-100 \u0026micro;M), (R)-(+)-propranolol (0.001-100 \u0026micro;M), (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M), (S)-(+)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M), (R)-(-)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M), (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M), (S)-(+)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M), and (R)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-300 \u0026micro;M) were obtained in rat isolated atria. In separate experiments, the effects of (\u0026plusmn;)-propranolol (0.001-100 \u0026micro;M), (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M) and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M) were evaluated in isolated atria pretreated or not with the nitric oxide (NO) synthesis inhibitor N\u003csup\u003eω\u003c/sup\u003e-nitro-L-arginine methyl esther (L-NAME, 100 \u0026micro;M, 30 min). The volume added of drugs to the 10-mL Krebs-Henseleit\u0026rsquo;s solution in the organ bath was 10 to 30 \u0026micro;L for each concentration. The maximum effect of the concentration-response curves was evaluated until the atrium beating stopped.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEffect of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e- propranolol on the concentration-response curves to noradrenaline and adrenaline\u003c/h2\u003e \u003cp\u003eCumulative concentration-response curves to noradrenaline (0.001-30 \u0026micro;M) and adrenaline (0.001-100 \u0026micro;M) were performed in isolated atria preparations in the absence and the presence of (\u0026plusmn;)-propranolol (0.001-0.3 \u0026micro;M; 60 min), (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.1\u0026ndash;10 \u0026micro;M; 60 min) and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001 and 3 \u0026micro;M; 60 min). The atria were incubated for 60 min with propranolol and the nitro-derivatives, and the catecholamines were added thereafter.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e- propranolol on the positive chronotropic effect induced by 6-nitrodopamine (6-ND)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe positive chronotropic induced by 6-ND (10 pM) was evaluated in isolated atria preparations in the absence and the presence of (\u0026plusmn;)-propranolol (0.3 and 1 \u0026micro;M; 60 min), (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 and 30 nM; 60 min), and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.1 and 1 \u0026micro;M; 60 min).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eEffect of tetrodotoxin and lidocaine on the negative chronotropic effect of (\u0026plusmn;)-propranolol\u003c/h2\u003e \u003cp\u003eA single concentration of tetrodotoxin (1 \u0026micro;M) or lidocaine (10 \u0026micro;M) was added to the organ bath, and the changes in atrial rate were monitored for 30 minutes. After 30 minutes, incubation was carried out with (\u0026plusmn;)-propranolol (10 \u0026micro;M) for an additional 30 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eEffect of co-incubation of (\u0026plusmn;)-propranolol and (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol on the basal atrial rate\u003c/h2\u003e \u003cp\u003eThe atria were pre-treated with either (\u0026plusmn;)-propranolol (10 \u0026micro;M) for 30 min. After 30 minutes, incubation was carried out with (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 \u0026micro;M) for an additional 30 min. In separate experiments, the effects of (\u0026plusmn;)-propranolol (10 \u0026micro;M) were evaluated in atria pre-treated with (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 \u0026micro;M, 30 min).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData of atrial rate are presented as beats per minute (bpm) before and after the respective stimulation or as the delta increase of atrial rate. Nonlinear regression analysis to determine the pIC\u003csub\u003e50\u003c/sub\u003e was carried out using GraphPad Prism (GraphPad Software, version 9.4, San Diego, California, USA) with the constraint that F\u0026thinsp;=\u0026thinsp;0. All concentration\u0026ndash;response data were evaluated for a fit to a logistics function in the form: E\u0026thinsp;=\u0026thinsp;E\u003csub\u003emax\u003c/sub\u003e/([1 + (10c / 10x)\u003csup\u003en\u003c/sup\u003e]\u0026thinsp;+\u0026thinsp;F, where E represents the increase in response contractile induced by the agonist, E\u003csub\u003emax\u003c/sub\u003e is the effect agonist maximum, c is the logarithm of concentration of the agonist that produces 50% of E\u003csub\u003emax\u003c/sub\u003e, x is the logarithm of the concentration of the drug; the exponential term, n, is a curve fitting parameter that defines the slope of the concentration\u0026ndash;response line, and F is the response observed in the absence of added drug. The values of pIC\u003csub\u003e50\u003c/sub\u003e data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of \u003cem\u003en\u003c/em\u003e experiments. Student's two-tail unpaired t-test was employed to compare the statistical differences between groups. The potencies of the antagonists in inhibiting the positive chronotropism of the catecholamines was estimated through the pA\u003csub\u003e2\u003c/sub\u003e (-log of the molar antagonist concentration that produces a two-fold reduction in the potency of the agonist) values as calculated by the equation \u0026ldquo;pA\u003csub\u003e2\u003c/sub\u003e = -log (antagonist concentration)\u0026thinsp;+\u0026thinsp;log (CR-1) using the lowest effective concentration of the antagonist in the calculations. Additionally, the potencies of propranolol, its nitro-derivatives and enantiomers in inhibiting the spontaneous atrial beating rate were estimated through the pIC\u003csub\u003e50\u003c/sub\u003e values using the equation (Y\u0026thinsp;=\u0026thinsp;Bottom + (Top-Bottom)/(1\u0026thinsp;+\u0026thinsp;10^((-pIC50-X)*HillSlope))in the GraphPad Prism Software. Since the study has an exploratory character, the \u003cem\u003ep\u003c/em\u003e values should be considered descriptive (Motulsky, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Michel et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003e(\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e- propranolol, (\u003cem\u003eS\u003c/em\u003e)-(+)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, (\u003cem\u003eR\u003c/em\u003e)-(-)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, (\u003cem\u003eS\u003c/em\u003e)-(+)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (\u003cem\u003eR\u003c/em\u003e)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol were synthesized as described elsewhere (Sparaco et al., 2023). 6-nitrodopamine was acquired from Toronto Research Chemicals (TRC, Toronto, Canada). (\u0026plusmn;)-Propranolol, (R)-(+)-propranolol, lidocaine, and N\u003csup\u003eω\u003c/sup\u003e-nitro-L-arginine methyl ester (L-NAME) were obtained from Sigma-Aldrich Chemicals Co (Missouri, USA). Adrenaline, noradrenaline, (S)-(-)-propranolol, and tetrodotoxin were purchased from Cayman Chemical Co (Michigan, USA). Sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), magnesium sulfate (MgSO\u003csub\u003e4\u003c/sub\u003e), sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e), potassium phosphate mono-basic (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) and glucose were acquired from Merck KGaA (Darmstadt, Germany). The composition of the Krebs-Henseleit\u0026rsquo;s solution (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. (\u0026plusmn;)-Propranolol (racemic and enantiomers), (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (racemic and enantiomers), (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (racemic and enantiomers), 6-nitrodopamine, noradrenaline, and adrenaline were all dissolved in water.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and their enantiomers on the basal atrial rate\u003c/h2\u003e \u003cp\u003eConcentration-response curves to (\u0026plusmn;)-propranolol, (S)-(-)-propranolol, and (R)-(+)-propranolol in the basal atrial rate are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The racemic mixture ((\u0026plusmn;)-propranolol) and its (R)- and (S)-enantiomers (0.001-100 \u0026micro;M) concentration-dependently reduced the spontaneous atrial rate until the atrium stopped beating. Compared with (\u0026plusmn;)-propranolol, concentration-response curves to both (S)-(+)-propranolol and (R)-(+)-propranolol were displaced to the right with a significant reduction in pIC\u003csub\u003e50\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but no differences between (S)-(-)-propranolol and (R)-(+)-propranolol were found (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003e\u003cb\u003eThe potency (pIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e) of (\u0026plusmn;)-propranolol, (S)-(-)-propranolol, (R)-(+)-propranolol, (\u0026plusmn;)-4-NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-propranolol, (S)-(+)-4-NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-propranolol, (R)-(-)-4-NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-propranolol, (\u0026plusmn;)-7-NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-propranolol, (S)-(+)-7-NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-propranolol, and (R)-(-)-7-NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-propranolol to reduce the basal atrial rate in the rat isolated atria.\u003c/b\u003e\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epIC\u003csub\u003e50\u003c/sub\u003e (log[M])\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.80\u0026plusmn;0.10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(S)-(-)-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.30\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(R)-(+)-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.34\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.95\u0026plusmn;0.13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(S)-(+)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.78\u0026plusmn;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.3197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(R)-(-)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.09\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4.64\u0026plusmn;0.09\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(S)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.68\u0026plusmn;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.7122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(R)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.84\u0026plusmn;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\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\u003eThe concentration-response curves to (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M) and its enantiomers (S)-(+)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M) and (R)-(-)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-300 \u0026micro;M) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. All these compounds concentration-dependently reduced the spontaneous atrial rate. The pIC\u003csub\u003e50\u003c/sub\u003e values showed no differences between (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (S)-(+)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared with (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (S)-(+)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, the concentration-response curve to (R)-(-)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol was displaced to the right with a significant reduction in pIC\u003csub\u003e50\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe concentration-response curves to (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M) and its enantiomers (S)-(+)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-100 \u0026micro;M) and (R)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-300 \u0026micro;M) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. All these compounds concentration-dependently reduced the spontaneous atrial rate. Compared with (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranol and (S)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, the concentration-response curve to (R)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol was displaced to the right with a significant reduction in pIC\u003csub\u003e50\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The pIC\u003csub\u003e50\u003c/sub\u003e values showed no differences between (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (S)-(-)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e-propranolol on the positive chronotropic effect induced by noradrenaline and adrenaline\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo estimate the potencies (pA2) of (\u0026plusmn;)-propranolol and its nitro-derivatives, concentration-response curves to noradrenaline (NA) were performed in the absence and presence of (\u0026plusmn;)-propranolol (0.001-0.3 \u0026micro;M; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.1\u0026ndash;10 \u0026micro;M; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (0.001-3 \u0026micro;M; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Noradrenaline produced concentration-dependent increases in atrial rate, which was significantly displaced to the right by (\u0026plusmn;)-propranolol at 10 nM, but not by this compound at 1 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) also produced right displacements on the concentration-response of noradrenaline, but the nitro-compound (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol exhibited the lowest potency, as revealed by its pA\u003csub\u003e2\u003c/sub\u003e values (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe values of pA\u003csub\u003e2\u003c/sub\u003e of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol in the concentration-response curves for noradrenaline and adrenaline in rat isolated atria.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNoradrenaline\u003c/p\u003e \u003cp\u003e(pA\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdrenaline\u003c/p\u003e \u003cp\u003e(pA\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-Propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-Propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-Propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 (n\u0026thinsp;=\u0026thinsp;4)\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\u003eSimilar data on the atrial rate were observed with adrenaline (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E and F). The concentration-dependent increases in atrial rate produced by adrenaline were significantly displaced to the right by (\u0026plusmn;)-propranolol 10 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and its nitro-compounds (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Similarly to noradrenaline, the least potent compound at inhibiting the positive chronotropism induced by adrenaline was (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, as determined by its pA2 values (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of L-NAME on the negative chronotropic effects induced by (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/h2\u003e \u003cp\u003eIncubation of the rat isolated right atrium with L-NAME (100 \u0026micro;M) caused a small (but significant) reduction in the spontaneous atrial rate (296\u0026thinsp;\u0026plusmn;\u0026thinsp;6 and 271\u0026thinsp;\u0026plusmn;\u0026thinsp;5 bpm, for control and L-NAME, respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, L-NAME (100 \u0026micro;M) produced a rightward shift in the concentration-response curves to (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, significantly reducing the potencies (pIC\u003csub\u003e50\u003c/sub\u003e values) of these compounds in diminishing the spontaneous atrial beat rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B and C). Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the calculated pIC\u003csub\u003e50\u003c/sub\u003e values for these three compounds in control and in L-NAME-treated atria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of L-NAME (100 \u0026micro;M) on the potency (pIC\u003csub\u003e50\u003c/sub\u003e) of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol in inhibiting the spontaneous rat atria rate.\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epIC\u003csub\u003e50\u003c/sub\u003e (log[M])\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.85\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-propranolol\u0026thinsp;+\u0026thinsp;L-NAME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.49\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.76\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u0026thinsp;+\u0026thinsp;L-NAME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.39\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.37\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol\u0026thinsp;+\u0026thinsp;L-NAME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.05\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol on the positive chronotropic effect of 6-nitrodopamine\u003c/h2\u003e \u003cp\u003e6-Nitrodopamine (6-ND; 10 pM) caused a sustained (60 min) increase in the atrial rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-F). The increase in atrial rate induced by 6-ND was unaffected by (\u0026plusmn;)-propranolol at 300 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), but it was fully inhibited at a higher concentration (1 \u0026micro;M; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Incubation with (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 nM) caused no significant effect in the increased atrial rate induced by 6-ND (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC); however, at 30 nM, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol significantly reduced the increased atrial rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Incubation with (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol at 100 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) or 1 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) had no effect on the increased atrial rate induced by 6-ND.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of tetrodotoxin and lidocaine on the negative chronotropic effect of (\u0026plusmn;)-propranolol\u003c/h2\u003e \u003cp\u003ePre-treatment (30 min) of the isolated right atria with either tetrodotoxin (1 \u0026micro;M) or lidocaine (10 \u0026micro;M) caused significant falls in the atrial basal rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and C, respectively). In tetrodotoxin- and lidocaine-pretreated atria preparations, further incubation of the with (\u0026plusmn;)-propranolol (10 \u0026micro;M) caused an additional fall in basal rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of co-incubation of (\u0026plusmn;)-propranolol and (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol on the basal atrial rate\u003c/h2\u003e \u003cp\u003ePre-treatment (30 min) of the isolated right atria with either (\u0026plusmn;)-propranolol (10 \u0026micro;M) or (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 \u0026micro;M) caused significant falls in atrial basal rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, respectively). In atria pre-incubated with (\u0026plusmn;)-propranolol (10 \u0026micro;M), further incubation with (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 \u0026micro;M) did not alter the basal atrial rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In preparations pre-incubated with (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10 \u0026micro;M), further incubation with (\u0026plusmn;)-propranolol (10 \u0026micro;M) did not alter basal atrial rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results demonstrate that (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol causes a fall in spontaneous atrial rate at concentrations that do not affect the positive chronotropic effect induced by noradrenaline and adrenaline but do inhibit the positive chronotropic effect induced by 6-ND. Indeed, the pIC\u003csub\u003e50\u003c/sub\u003e for (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (\u0026plusmn;)-propranolol is almost identical (4.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 and 4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, respectively), yet (\u0026plusmn;)-propranolol was approximately one hundred times more potent than (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol to block the positive chronotropic effect induced by noradrenaline and adrenaline. The calculated pA\u003csub\u003e2\u003c/sub\u003e values obtained in our results for (\u0026plusmn;)-propranolol (8.2 and 8.5 for noradrenaline and adrenaline, respectively) are like those reported in the isolated cat papillary muscle preparations (8.3) using isoprenaline as an inotropic agent (Lewis et al., 1983), and in the guinea-pig atria (8.2) using noradrenaline as a chronotropic agent (O\u0026rsquo;Donnell \u0026amp; Wanstall, 1979). Similar discrepancy is observed when the pIC\u003csub\u003e50\u003c/sub\u003e of both (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol is compared; (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol is more potent than (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol to cause fall in atrial rate, yet (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol is one hundred times more potent than (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol to block the positive chronotropic effect induced by noradrenaline and adrenaline. These discrepancies clearly indicate that the negative chronotropic effects of (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol on the basal atrial rate are not related to their ability to block the positive chronotropic effects of either noradrenaline or adrenaline. Actually, the concentrations that these drugs cause fall in basal atrial rate are the concentrations that they inhibit the positive chronotropic effect of 6-ND.\u003c/p\u003e \u003cp\u003eThe basal release of 6-ND from rat isolated atria is inhibited by pre-incubation of the atria with the NO synthase inhibitor L-NAME (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); it is interesting that pre-treatment of the atria with L-NAME caused a significant reduction in basal atrial rate (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and significant right shifts in the concentration-response curves to (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (\u0026plusmn;)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol. Both findings could be interpreted by inhibition of 6-ND synthesis induced by L-NAME. It is worth mentioning that selective b\u003csub\u003e1\u003c/sub\u003e-adrenoceptor antagonists such as atenolol, betaxolol, and metoprolol, cause significant reduction in basal atrial rate at concentrations that block the positive chronotropic effect of 6-ND, but that do not affect the positive chronotropic effect induced by noradrenaline, adrenaline, and dopamine (Britto-J\u0026uacute;nior et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePropranolol is therapeutically used as a racemic mixture with equal concentrations of (S)-(-)-propranolol and (R)-(+)-propranolol, however the (R)-(+)-propranolol is devoid of blocking activity (Mehvar and Brocks, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Our finding that there was no difference in the pIC50 of (S)-(-)-propranolol and (R)-(+)-propranolol, further indicates that the fall in basal atrial rate induced by (\u0026plusmn;)-propranolol is not related to its effect on the b-adrenoceptors. Propranolol is known to block voltage-gated sodium channels, and this ability is not stereospecific, since both enantiomers of propranolol block cardiac Na\u003csub\u003ev\u003c/sub\u003e1.5 channels at equivalent potency (Wang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thus, one possibility would be that the fall in atrial rate induced by (\u0026plusmn;)-propranolol could be due to blockade of this tetrodotoxin-sensitive sodium channel. However, the finding that provoked significant falls in atrial rate in tetrodotoxin pre-treated atria, indicates that it is unlikely that the falls could be due to blockade of cardiac tetrodotoxin-sensitive Nav channels. Lidocaine is also a Nav 1.5 channel blocker, it has anti-arrhythmic activity, and is supposed to have at least one locus of action at a site other than the TTX blockade site (Desai et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). One possibility would be that propranolol would be acting at this other site. This is also unlikely, since (\u0026plusmn;)-propranolol was able to induce fall in atrial rate in lidocaine-pretreated atria.\u003c/p\u003e \u003cp\u003eThus, blockade of the 6-ND receptor is the most likely explanation for the fall in atrial rate induced by (\u0026plusmn;)-propranolol. This concept is further supported by the finding that in atria pre-treated with (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, (\u0026plusmn;)-propranolol fails to induce reduction in atrial rate, and in atria pre-treated with (\u0026plusmn;)-propranolol, (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol did not alter basal atrial rate, indicating that both drugs are acting on the same receptor, i.e., the 6-ND receptor. (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol should be considered a first selective 6-ND receptor antagonist, since it has very weak potency for blocking the positive chronotropic effect induced by both noradrenaline and adrenaline.\u003c/p\u003e \u003cp\u003eThe autonomic nervous system is believed to be a major regulator in the cardiovascular system and its adaptation to various human body functions (Hirano et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), although this concept has been challenged recently (Zatz \u0026amp; De Nucci, 2023). In patients submitted to orthotopic cardiac transplantation, the heart rate at rest is increased (103\u0026thinsp;\u0026plusmn;\u0026thinsp;12 bpm) compared to age-matched control (77\u0026thinsp;\u0026plusmn;\u0026thinsp;13 bpm); however, the peak heart rate following exercise is decreased (137\u0026thinsp;\u0026plusmn;\u0026thinsp;15 and 176\u0026thinsp;\u0026plusmn;\u0026thinsp;12, respectively). These patients have no evidence of cardiac innervation (Kavanagh et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Thus, resting heart seems to be independent of adrenergic catecholamine release.\u003c/p\u003e \u003cp\u003eFor evaluating the importance of 6-ND as the endogenous modulator of heart chronotropism, it is essential to assess the effect of these nitro-derivatives of propranolol in both anaesthetized and conscious rats. Another possibility is to investigate whether propranolol in humans is metabolized to nitro-derivatives; however, since there are several positions that propranolol can be \u0026ldquo;nitrated\u0026rdquo;, synthesis of nitro-atenolol may solve this conundrum provided, unlike propranolol, it has only two positions on its molecule to receive the nitro group.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe finding that (\u0026plusmn;)-propranolol and (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol cause falls in spontaneous atrial rate only in concentrations that affect a 6-ND positive chronotropic effect, confirms the role of this catecholamine as endogenous modulator of heart chronotropism. (\u0026plusmn;)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol behaves as a selective antagonist of 6-ND in the rat isolated atrium.\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\u0026nbsp;Ethics Committee in Animal Use of UNICAMP\u0026nbsp;(CEUA/UNICAMP, protocol number 5942-1/2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\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\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: JBJ and GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData curation: JBJ and GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFormal analysis: GDN\u003c/p\u003e\n\u003cp\u003eFunding acquisition: EA and GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInvestigation: JBJ, DLO, VFC, VF, GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: JBJ, DLO, VFC, VF, RS, VS, EA, FZM, GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProject administration: GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision: EA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVisualization: EA and GDN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: JBJ, FF, GC EA, GDN.\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data were generated in-house and that no paper mill was used.\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\u003eVF thank FAPESP for PhD fellowship (2022/07737-8).\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).\u0026nbsp;\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\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\u003cli\u003e\u003cspan\u003eAndersen ML (2016) Guia brasileiro de produ\u0026ccedil;\u0026atilde;o, manuten\u0026ccedil;\u0026atilde;o ou utiliza\u0026ccedil;\u0026atilde;o de animais em atividade de ensino ou pesquisa cientifica, Conselho nacional de controle de experimenta\u0026ccedil;\u0026atilde;o animal. 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Physiology. \u003cem\u003e39:44\u0026ndash;59. doi: 10.1152/physiol.00020.2023\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"b-blocker, stereoselectivity, L-NAME, catecholamines","lastPublishedDoi":"10.21203/rs.3.rs-4680045/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4680045/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe positive chronotropic action induced by 6-mitrodopamine (6-ND) is selectively blocked by β\u003csub\u003e1\u003c/sub\u003e-adrenoceptor antagonists at concentrations that do not affect the positive chronotropic effect induced by dopamine, noradrenaline, and adrenaline. Here the effects of (±)-propranolol, (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (±)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, were investigated in the rat isolated right atrium. The atrium was mounted in glass chambers containing gassed (95%O\u003csub\u003e2\u003c/sub\u003e:5%CO\u003csub\u003e2\u003c/sub\u003e) and warmed (37°C) Krebs-Henseleit’s solution, and the isometric tension registered (PowerLab system). (±)-propranolol, (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (±)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, caused concentration-dependent falls in the spontaneous atrial frequency (pIC\u003csub\u003e50\u003c/sub\u003e were 4.80±0.10, 4.64±0.10, and 4.95±0.10, respectively). The calculated pA\u003csub\u003e2\u003c/sub\u003e values for (±)-propranolol, (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol, and (±)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranol obtained for noradrenaline-induced positive chronotropic effects were 8.44±0.08, 6.41±0.07, and 9.21±0.29, respectively. \u0026nbsp;The positive chronotropism induced by 6-ND (10pM) was blocked by (±)-propranolol (1mM), and (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (30nM). (±)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranol (1mM) had no effect on 6-ND (10pM)-induced increases in atrial rate.\u0026nbsp; The pIC\u003csub\u003e50\u003c/sub\u003e of (±)-propranolol, (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol and (±)-7-NO\u003csub\u003e2\u003c/sub\u003e-propranolol were significantly shifted to the right in L-NAME treated atria. The discrepancy between pA\u003csub\u003e2\u003c/sub\u003e values of (±)-propranolol and its respective pIC\u003csub\u003e50\u003c/sub\u003e indicates that the falls in atrial rate induced by (±)-propranolol should not be attributed to b-adrenergic antagonism. The reduced chronotropism by (±)-propranolol (10µM) was unaffected by the sodium channel inhibitors tetrodotoxin (1µM) and lidocaine (10µM) but abolished in atria pre-treated with (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol (10µM). The finding that (±)-propranolol causes falls in spontaneous atrial rate only in concentrations that affect 6-ND positive chronotropic effect, confirms the role of this catecholamine as endogenous modulator of heart chronotropism. (±)-4-NO\u003csub\u003e2\u003c/sub\u003e-propranolol behaves as a selective antagonist of 6-ND in the rat isolated atrium.\u003c/p\u003e","manuscriptTitle":"The negative chronotropic effects of (±)-propranolol and (±)- 4-NO 2 -propranolol in the rat isolated right atrium are due to blockade of the 6-nitrodopamine receptor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 10:59:17","doi":"10.21203/rs.3.rs-4680045/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-05T18:46:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-29T10:14:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263776308021916663563529010390985508516","date":"2024-07-09T11:24:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-09T10:56:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-04T02:43:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-04T02:42:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2024-07-03T11:26:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f323b26f-7b51-4c79-afa7-cdc2c3bc2021","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:02:27+00:00","versionOfRecord":{"articleIdentity":"rs-4680045","link":"https://doi.org/10.1007/s00210-024-03463-3","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2024-10-09 15:57:46","publishedOnDateReadable":"October 9th, 2024"},"versionCreatedAt":"2024-07-29 10:59:17","video":"","vorDoi":"10.1007/s00210-024-03463-3","vorDoiUrl":"https://doi.org/10.1007/s00210-024-03463-3","workflowStages":[]},"version":"v1","identity":"rs-4680045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4680045","identity":"rs-4680045","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}