Effects of sodium on A2A adenosine receptor expression and function: in cellulo approach and pathophysiological perspectives

Effects of sodium on A2A adenosine receptor expression and function: in cellulo approach and pathophysiological perspectives | 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 Effects of sodium on A 2A adenosine receptor expression and function: in cellulo approach and pathophysiological perspectives Farid EL Oufir, Guiol Claire, Marion Marlinge, Nathalie Kipson, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4169022/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The expression and function of G-protein coupled receptor is modulated by small molecules including sodium ion, acting at an allosteric site. Using Adonis, an Ig M monoclonal antibody against a linear epitope located in the second loop of the A 2A adenosine receptor (A 2A R) with agonist properties, we evaluate in cellulo the effects of different sodium ion concentrations on the expression and function of A 2A R of peripheral blood mononuclear cells (PBMC). We found that high sodium ion concentration is associated with an increase in A 2A R expression and a decrease in cAMP production evaluated by measuring the half-maximal cAMP production, (EC 50 ), in a dose-dependent manner. When Adonis and sodium ions were added simultaneously in the culture medium, (competitive conditions), the K D and the EC 50 were high compared to non-competitive conditions (mean 27 and 3 folds respectively). These results suggest that sodium ions could promote Adonis binding to A 2A R as well as its activation. We hypothesize that, by steric hindrance, the binding of Adonis to A 2A R extends beyond the orthostatic site and prevents sodium ion from reaching its allosteric pocket. When sodium ion can reach its site, it acts as an allosteric modulator with respect to A 2A R expression and function. Because the action of sodium ions and A 2A -R have opposite effects on blood pressure, our results highlight a possible role of A 2A -R/sodium ions interaction in the regulation of blood pressure. adenosine A2A receptors sodium ion blood pressure Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction A 2A adenosine receptors (A 2A R) belong to the superfamily of G coupled membrane receptors which activation strongly impacts the immune [ 1 ], the nervous [ 2 , 3 ] and the cardiovascular system [ 4 , 5 ]. In the cardiovascular area, A 2A R stimulation leads to cardiac inotropic effects and to vasodilation via cAMP production of target cells, mostly myocytes and endothelial cells of vascular walls [ 4 ], cAMP production and vasodilation being correlated [ 6 ]. A 2A R are strongly implicated in the control of blood pressure. Indeed, high A 2A R expression is associated with a drop in blood pressure [ 7 ]. Conversely, A 2A R KO mice exhibit high systolic blood pressure [ 8 ] confirming that A 2A R are implicated in the regulation of blood pressure. Adenosine, a ubiquitous nucleoside that comes mostly from the dephosphorylation of ATP, is the natural ligand of A 2A R. While high and low affinity of adenosine for A 2A R have been described [ 9 ], it is well established that A 2A R affinity for ligands can be regulated in an allosteric manner through small molecules like amiloride or sodium [ 10 ]. Thus, it was shown that sodium ions increased the affinity of some radioligand for A 2A R [ 10 ]. Interestingly activation of A 2A R leads to vasodilation and a decrease in blood pressure while conversely sodium excess in blood leads to an increase in blood pressure notably by increasing vascular volume through osmosis mechanism. While these effects of sodium on one hand and the effects of A 2A R activation on the other can be independent, it could be that sodium, apart from its effects on vascular volume, impacts A 2A R expression and function by binding in the sodium pocket, at an allosteric site. We have developed a monoclonal antibody (named Adonis) against a linear epitope in the second loop of the human A 2A R which exhibits agonist properties [ 11 ]. Adonis binds to the orthosteric site with a high affinity (with a K D around 0.1–0.2µM) leading to the production of cAMP in a dose dependent manner [ 11 ]. Peripheral blood mononulcear cells (PBMCs) express a great amount of A 2A R and it is well established that the behavior of these receptors in these cells mirrors the behavior of A 2A R expressed in the cardiovascular system [ 12 , 13 ]. Thus, PBMCs are a good model for studying the adenosinergic profile of the cardiovascular system. The aim of this study was, using the PBMCs model and Adonis, to evaluate the influence of sodium on A 2A R expression and function. We hypothesized that A 2A R expression and function could be regulated by sodium ion and that by steric constraints due to its large size (IgM), Adonis may interfere with both orthosteric and allosteric sites of A 2A R. Material and Methods PBMCs isolation and culture The procedure has been described previously [ 14 , 15 ]. PBMCs were cultured 24 hours at 37°C (5%CO 2 ) in complete RPMI 1640 (supplemented with 10% heat inactivated Fœtal Calf Serum, 100 IU /ml each penicillin/streptomycin and 4 mM glutamine) containing 120 mM, 133 mM or 194 mM NaCl. These concentrations were chosen to be compatible with cell life. Blot analysis The particularity of this experiment is that we need to use a ligand (in this case Adonis) which binds irreversibly to the A 2A receptor, “irreversibly” meaning at least during the incubation period, in order to measure K D and cAMP (ie EC 50 ) production at the same time. In this context, Adonis fits the bill perfectly. Samples (equivalent to 0.2 × 10 6 cells pellets) were submitted to standard 12% polyacrylamide gel electrophoresis under reducing conditions followed by transfer onto a PVDF membrane. The filter was then incubated with Adonis (1 µg/mL), a homemade IgM, κ mouse monoclonal antibody against a linear epitope, which reproduces a sequence from the second extra-cellular loop of A 2A R [ 11 ], and blot revelation was performed using alkaline phosphatase-labeled anti-mouse antibodies and p-nitrophenylphosphate substrat. The 45-kDa bands corresponding to A 2A R were submitted to densitometry analysis using the ImageJ 1.42q software (National Institutes of Health) and results were expressed as arbitrary units (AU), the ratio of pixels generated by the A 2A R band to pixels generated by the background signal [ 14 – 16 ]. K D determination The K D was defined as the concentration of Adonis (used as agonist) which leads to 50% of occupied binding sites (A 2A R) [ 17 – 20 ]. We used the visualization of Adonis kappa light chains (25 KDa), which detach during the reducing period, and whose density is proportional to the binding of Adonis to the receptor to determine the K D value as previously described [ 14 – 16 ]. In this perspective, PBMCs (0.75 × 10 6 ) were incubated with increasing concentrations of Adonis (0 to 0.8µM) and then washed to remove free Adonis prior to treatment of the cell pellets with lysis buffer and sonication. Samples were then submitted to standard electrophoresis under reducing conditions prior to transfer onto a PVDF membrane. Semi quantitative measurement was performed using densitometry analysis and expressed as arbitrary units (A.U.), which constitute the ratio of pixels generated by the light kappa chain band (25 KDa) to pixels generated by the background signal as previously described [ 14 – 16 ]. K D values for Adonis binding were estimated using nonlinear regression analysis (Prism software; GraphPad Software). cAMP dosage (EC 50 ) cAMP production was evaluated after incubation of increasing concentrations of Adonis, used as agonist, with PBMCs (0.25 × 10 6 cells) using the Amersham Biotrak Kit (GE Healthcare Bio-Sciences). The incubation step was stopped using Dodecyltrimethylammonium bromide acetate buffer. Measurement were performed in duplicates and results expressed as the percentage of the maximal cAMP production. EC 50 was defined as the concentration of Adonis (uses as agonist) that leads to half maximal stimulation of cAMP production [ 15 ]. Statistical analysis Data were expressed as mean and standard deviations (SD). A variance analysis (ANOVA) was used for the comparison between A 2A R expression in different conditions. A difference in at least 25% in expression was considered as relevant. Results While low concentration (120 mM) in sodium ion did not influence A 2A R expression, high concentration of sodium (194 mM) in the culture medium was associated with an increase in the expression of A 2A R (mean + 54% compared with 133 mM, Fig. 1 ). High concentration (194 mM) in sodium ion decreased both the K D value of Adonis (mean 14.5 folds), and the EC 50 value (mean 2.5 folds) compared with sodium concentration equal to 133 mM. Low concentration in sodium (120 mM) was associated with an increase in the K D value by 2.6 folds, compared with 133mM, while the EC 50 value was poorly modified (see Fig. 2 , Fig. 3 and Table 1 ). When sodium ions and Adonis were added simultaneously in the culture medium (competitive condition), the K D of Adonis for A 2A R was strongly increased (mean 27 folds compared with non-competitive conditions, while EC 50 increased by 3.2 folds (see Fig. 3 and Table 1 ). Table 1 Effects of sodium on affinity (K D ), B max and EC 50 , using Adonis a monoclonal antibody with agonist properties (see methods). All measurements were performed in duplicate. Data are expressed as mean ± SD Sodium (194mM) Sodium (133mM) Sodium (120mM) Sodium (194mM) Competitive conditions K D (µM) 0.011 ± 0.001 0.16 ± 0.014 0.42 ± 0.03 0.3 ± 0.02 Bmax 107 ± 12 123 ± 7.4 140.8 ± 5.3 144.6 ± 4.9 EC 50 (µM) 0.08 ± 0.03 0.20 ± 0.1 0.37 ± 0.19 0.26 ± 0.11 Discussion The main result of this study is that sodium ions increased the expression of A 2A R and decreases K D and EC 50 values (evaluated using Adonis an antibody with agonists properties) in a dose-dependent manner. Furthermore, when Adonis and sodium ions were added simultaneously (competitive conditions), the K D of Adonis increases dramatically. In competitive conditions, it seems that Adonis (an IgM with a very large size) prevents sodium from integrating its allosteric pocket leading to a strong decrease in affinity of Adonis for A 2A R. Indeed, Adonis caps both the orthostatic binding site (on the second extracellualr loop [ 11 ]) and probably the allosteric site (the salt pocket) which is not far from the orthosteric site (on the second transmembrane helix [ 17 ], preventing salt from carrying out its allosteric regulation action. This hypothesis, while plausible, needs further investigations. It's also perfectly possible that the increase in ionic strength linked to the salt concentration reduces electrostatic interactions. Similarly, we can't rule out changes in electrostatic interactions linked to increased ionic strength in competition experiments. While the binding of antagonists and sodium ions to the receptor was non-competitive, the binding of agonists and sodium appears to require mutually exclusive conformational states of the receptor [ 18 ]. It is now established that orthosteric binding pocket of the ligand represent just one among many sites for possible signal modulation in GPCRs and that allosteric binding pocket not only impacts ligand affinity but also plays a major role in receptor signaling [ 19 ]. Thus, molecules different from ligand can modulate binding of natural ligand that binds to the orthosteric site. It was shown that small molecules like amiloride, ions or lipids can act as allosteric modulator of GPCR [ 10 , 20 , 21 ]. The allosteric effect of sodium ion has been previously described in A 1 R [ 21 ] and A 2A R models [ 19 , 20 ]. Modelisation of sodium binding to A 2A R suggests that sodium ion stabilizes A 2A R in its inactive conformation and destabilizes activation-related movements and agonist binding [ 18 ]. It is generally admitted that sodium ion induced an increase in radioligand antagonist binding, but abrogated agonist binding in a dose dependent manner [1!]. Here we found that sodium ion, in competitive conditions decreases the affinity of Adonis for A 2A R but have opposite effects in non-competitive conditions. Our study used an agonist antibody rather than an organic ligand or adenosine itself. Although our antibody seems to behave like an organic agonist, particularly in terms of orthostatic binding site and cAMP production, the interactions between Adonis and the A 2A R receptor may be much more complex than with small organic molecules. While this type of interaction had previously been demonstrated in crystallographic models or in modeling, our results use experimental conditions very close to the blood environment (and PBMCs, whose receptors are known to behave like those of cardiovascular tissue), suggesting, for example, that in response to salt overload, the body might increase the affinity of A 2A R agonists, resulting in compensatory vasodilatation to eventually lower blood pressure. Study limitation Because we used an IgM monoclonal antibody as agonist, it is therefore difficult to extrapolate from what actually happens with a small molecule like adenosine in animals or humans concerning the allosteric effects of sodium ion on adenosine binding. Conclusion and Pathophysiologic Perspectives It's a well established that excess salt increases blood pressure by a whole series of mechanisms. The first of these is the drawing of water into the vessels, resulting in hypervolemia. But salt also stimulates the sympathetic system, resulting in vasoconstriction [ 22 ]. Salt elevation in the CSF also leads to activation of angiotensin II and ouabain-dependent sodium pump, leading to the increase in blood pressure [ 22 , 23 ]. However, it is likely that there are also compensatory mechanisms for the rise in arterial pressure induced by excess salt. Modulation of A 2A R affinity for adenosine could be one such mechanism. Finally, because sodium ion and A 2A -R activation have opposite effects on blood pressure, our results highlight a possible role of A 2A -R/sodium ion interaction in the regulation of blood pressure. However, this hypothesis needs further investigations. Declarations Author Contributions: E. F.; R.G., G.M., and J.R.: conceptualization and writing the manuscript; M.C.C.; C.G.; C.C., N.K, R.Z: W blot and data analysis. S.L.; J.D.; J.F : critical review. All authors have read and agreed to the published version of the manuscript. Funding: Aix Marseille University Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Availability of data: data is available on request from the corresponding author. Ethical approval : NA References Antonioli L, Fornai M, Blandizzi C, Pacher P, Haskó G (2018) Adenosine signaling and the immune system: When a lot could be too much. Immunol Lett 205: 9–15, https://doi.org/10.1016/j.imlet.2018.04.006. Dunwiddie TV, Masino SA (2001) The Role and Regulation of Adenosine in the Central Nervous System. Annu Rev Neurosci 24 : 31–55, https://doi.org/10.1146/annurev.neuro.24.1.31. Ferré S, Ciruela F (2019) Functional and Neuroprotective Role of Striatal Adenosine A2A Receptor Heterotetramers. J Caffeine Adenosine Res ;9(3):89-97. doi: 10.1089/caff.2019.0008. Burnstock, G (2017) Purinergic Signaling in the Cardiovascular System. Circ Res 120: 207–228, https://doi.org/10.1161/circresaha.116.309726. 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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-4169022","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287215656,"identity":"7be9fd5c-c30d-4d9c-b941-d79a861f941e","order_by":0,"name":"Farid EL Oufir","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Farid","middleName":"EL","lastName":"Oufir","suffix":""},{"id":287215658,"identity":"84d1bde3-5e5d-49b7-8a19-0527a6242bee","order_by":1,"name":"Guiol Claire","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Guiol","middleName":"","lastName":"Claire","suffix":""},{"id":287215659,"identity":"706add11-9289-4163-adf9-33f9cde28e4a","order_by":2,"name":"Marion Marlinge","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Marion","middleName":"","lastName":"Marlinge","suffix":""},{"id":287215660,"identity":"5627cbb7-791f-4990-8038-469e6b29412c","order_by":3,"name":"Nathalie Kipson","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"Kipson","suffix":""},{"id":287215661,"identity":"c114ea71-e9c4-4824-a374-25b7e1ce3e39","order_by":4,"name":"Christine Criado","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"","lastName":"Criado","suffix":""},{"id":287215663,"identity":"454c9ed8-93bb-4e52-8807-8ad740a4672c","order_by":5,"name":"Marie C. Chaptal","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"C.","lastName":"Chaptal","suffix":""},{"id":287215666,"identity":"ce1b5a2b-7300-4cc7-b363-e8a886eb096f","order_by":6,"name":"Simon Lledo","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Lledo","suffix":""},{"id":287215668,"identity":"34052f46-95d4-42f6-9297-5dbd96ab8c3f","order_by":7,"name":"Julia Dedoders","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Dedoders","suffix":""},{"id":287215670,"identity":"6eb38243-09cd-4174-afb5-edbfec7b6574","order_by":8,"name":"Zohra Rebaoui","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Zohra","middleName":"","lastName":"Rebaoui","suffix":""},{"id":287215674,"identity":"1c5e4701-306a-4654-84d3-c387689367fb","order_by":9,"name":"Julien Fromonot","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Julien","middleName":"","lastName":"Fromonot","suffix":""},{"id":287215677,"identity":"a2cf8c61-a8a2-4133-bbfa-96195ecf7749","order_by":10,"name":"Jean Ruf","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"","lastName":"Ruf","suffix":""},{"id":287215678,"identity":"a476bb33-4925-4ed2-a2e3-2543482e362f","order_by":11,"name":"Giovanna Mottola","email":"","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Giovanna","middleName":"","lastName":"Mottola","suffix":""},{"id":287215679,"identity":"a07fe94f-5b22-49c1-8b9b-f794f441c84f","order_by":12,"name":"Régis Guieu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYFACxgYIzczA+AAm9oFYLcwGMKEZxNrHJkGUFvmI5MbHPAz35OTd2Z9VfNxjlyfv3sDYXIFHi+GNxGZjHoZiY8PDPGY3ZzxLLjY8c4Cx8Qw+LTMS26R5GBISNzbzsN3mOcCcuHFGAvvDBvxa2n8DtdRvbGZ/VsxzoD5x4/wHjI34tMhLJLYxA7UkyDMzmDHzHDicOF+CAb8WA56HzZJzDBIMNzDzGEvOOHA8cQNPYiN+W9rTH354U5EgL99//OGHDweqE+e3Hz6I35YDYBLGAIvAIheXLQ04GaNgFIyCUTAKoAAA8e9Ouexl4S4AAAAASUVORK5CYII=","orcid":"","institution":"INSERM, INRAE","correspondingAuthor":true,"prefix":"","firstName":"Régis","middleName":"","lastName":"Guieu","suffix":""}],"badges":[],"createdAt":"2024-03-26 10:18:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4169022/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4169022/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54159995,"identity":"691733e7-544e-4d3e-9c3c-d8a7ebfaccc2","added_by":"auto","created_at":"2024-04-05 12:55:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":191658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of sodium ions on A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e adenosine receptor (A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eR) expression of peripheral blood mononuclear cells (PBMCs). \u003c/strong\u003ePBMCs recovered from a healthy subject are cultivated during 24h in 3 concentration conditions of NaCl:\u0026nbsp; 120, 133 and 194 mM. A: Histogram shows variation in the expression of A\u003csub\u003e2A\u003c/sub\u003eR analyzed by western blot (WB:\u0026nbsp; n=5 by concentration) as a function of sodium concentration. Results are expressed in arbitrary unit (A.U. mean ± SD). a: p\u0026lt;0.05;\u0026nbsp; compared with 133 mM; b: p\u0026lt;0.05 compared with 120 mM sodium in a ANOVA. B: examples of WB.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4169022/v1/4d5d64454006cb0e720c723a.png"},{"id":54159990,"identity":"6ae30617-def0-4c76-a6a0-08048ac0e534","added_by":"auto","created_at":"2024-04-05 12:55:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. Effects of sodium concentration on percentage binding of Adonis, a monoclonal antibody with agonist properties. \u003c/strong\u003eThree sodium concentrations are tested: 120, 133 and 194 mM. Data are expressed as the mean of duplicate (see also table 1). \u003cstrong\u003eB \u003c/strong\u003eWestern blot obtained at different concentrations of sodium.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4169022/v1/829d5ce0b4393a2615e615ba.png"},{"id":54159991,"identity":"48e4c3f3-ecce-4553-8dee-5d24511a0eba","added_by":"auto","created_at":"2024-04-05 12:55:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different sodium concentrations on cAMP production in PBMCs incubated with Adonis, an antibody with agonist properties.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4169022/v1/6de9db443acd3b1f57715934.png"},{"id":54160915,"identity":"8fc413e1-4b63-4f24-972a-0cc9b0ec0c0b","added_by":"auto","created_at":"2024-04-05 13:03:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220528,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high sodium ion concentration (194mM) on percentage binding of Adonis, a monoclonal antibody with agonist properties. Black disks: Adonis was added after cells were incubated in high sodium ion concentration condition (non-competitive condition). White disks: Sodium ions and Adonis were added simultaneously in a competitive manner. Data were expressed as the mean of duplicate (see also table 1).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4169022/v1/497e8817827c7e0db90d7ff1.png"},{"id":54558095,"identity":"f6fad16c-baa8-4c02-8b5f-f90cbc695b61","added_by":"auto","created_at":"2024-04-12 09:05:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":796123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4169022/v1/0e22e595-25a9-4713-b63e-edc32261d509.pdf"},{"id":54159993,"identity":"9937c4ac-54df-4731-b3bc-a6402f312182","added_by":"auto","created_at":"2024-04-05 12:55:02","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":399809,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4169022/v1/ecb2df371fd26f0ba9935569.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEffects of sodium on A\u003csub\u003e2A\u003c/sub\u003e adenosine receptor expression and function: in cellulo approach and pathophysiological perspectives\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA\u003csub\u003e2A\u003c/sub\u003e adenosine receptors (A\u003csub\u003e2A\u003c/sub\u003eR) belong to the superfamily of G coupled membrane receptors which activation strongly impacts the immune [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], the nervous [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and the cardiovascular system [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the cardiovascular area, A\u003csub\u003e2A\u003c/sub\u003eR stimulation leads to cardiac inotropic effects and to vasodilation via cAMP production of target cells, mostly myocytes and endothelial cells of vascular walls [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], cAMP production and vasodilation being correlated [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A\u003csub\u003e2A\u003c/sub\u003eR are strongly implicated in the control of blood pressure. Indeed, high A\u003csub\u003e2A\u003c/sub\u003eR expression is associated with a drop in blood pressure [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Conversely, A\u003csub\u003e2A\u003c/sub\u003eR KO mice exhibit high systolic blood pressure [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] confirming that A\u003csub\u003e2A\u003c/sub\u003eR are implicated in the regulation of blood pressure. Adenosine, a ubiquitous nucleoside that comes mostly from the dephosphorylation of ATP, is the natural ligand of A\u003csub\u003e2A\u003c/sub\u003eR. While high and low affinity of adenosine for A\u003csub\u003e2A\u003c/sub\u003eR have been described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], it is well established that A\u003csub\u003e2A\u003c/sub\u003eR affinity for ligands can be regulated in an allosteric manner through small molecules like amiloride or sodium [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, it was shown that sodium ions increased the affinity of some radioligand for A\u003csub\u003e2A\u003c/sub\u003eR [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Interestingly activation of A\u003csub\u003e2A\u003c/sub\u003eR leads to vasodilation and a decrease in blood pressure while conversely sodium excess in blood leads to an increase in blood pressure notably by increasing vascular volume through osmosis mechanism. While these effects of sodium on one hand and the effects of A\u003csub\u003e2A\u003c/sub\u003eR activation on the other can be independent, it could be that sodium, apart from its effects on vascular volume, impacts A\u003csub\u003e2A\u003c/sub\u003eR expression and function by binding in the sodium pocket, at an allosteric site. We have developed a monoclonal antibody (named Adonis) against a linear epitope in the second loop of the human A\u003csub\u003e2A\u003c/sub\u003eR which exhibits agonist properties [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Adonis binds to the orthosteric site with a high affinity (with a K\u003csub\u003eD\u003c/sub\u003e around 0.1\u0026ndash;0.2\u0026micro;M) leading to the production of cAMP in a dose dependent manner [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Peripheral blood mononulcear cells (PBMCs) express a great amount of A\u003csub\u003e2A\u003c/sub\u003eR and it is well established that the behavior of these receptors in these cells mirrors the behavior of A\u003csub\u003e2A\u003c/sub\u003eR expressed in the cardiovascular system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, PBMCs are a good model for studying the adenosinergic profile of the cardiovascular system. The aim of this study was, using the PBMCs model and Adonis, to evaluate the influence of sodium on A\u003csub\u003e2A\u003c/sub\u003eR expression and function. We hypothesized that A\u003csub\u003e2A\u003c/sub\u003eR expression and function could be regulated by sodium ion and that by steric constraints due to its large size (IgM), Adonis may interfere with both orthosteric and allosteric sites of A\u003csub\u003e2A\u003c/sub\u003eR.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003ePBMCs isolation and culture\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe procedure has been described previously [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. PBMCs were cultured 24 hours at 37\u0026deg;C (5%CO\u003csub\u003e2\u003c/sub\u003e) in complete RPMI 1640 (supplemented with 10% heat inactivated F\u0026oelig;tal Calf Serum, 100 IU /ml each penicillin/streptomycin and 4 mM glutamine) containing 120 mM, 133 mM or 194 mM NaCl. These concentrations were chosen to be compatible with cell life.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eBlot analysis\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe particularity of this experiment is that we need to use a ligand (in this case Adonis) which binds irreversibly to the A\u003csub\u003e2A\u003c/sub\u003e receptor, \u0026ldquo;irreversibly\u0026rdquo; meaning at least during the incubation period, in order to measure K\u003csub\u003eD\u003c/sub\u003e and cAMP (ie EC\u003csub\u003e50\u003c/sub\u003e) production at the same time. In this context, Adonis fits the bill perfectly. Samples (equivalent to 0.2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells pellets) were submitted to standard 12% polyacrylamide gel electrophoresis under reducing conditions followed by transfer onto a PVDF membrane. The filter was then incubated with Adonis (1 \u0026micro;g/mL), a homemade IgM, \u0026kappa; mouse monoclonal antibody against a linear epitope, which reproduces a sequence from the second extra-cellular loop of A\u003csub\u003e2A\u003c/sub\u003eR [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], and blot revelation was performed using alkaline phosphatase-labeled anti-mouse antibodies and p-nitrophenylphosphate substrat. The 45-kDa bands corresponding to A\u003csub\u003e2A\u003c/sub\u003eR were submitted to densitometry analysis using the ImageJ 1.42q software (National Institutes of Health) and results were expressed as arbitrary units (AU), the ratio of pixels generated by the A\u003csub\u003e2A\u003c/sub\u003eR band to pixels generated by the background signal [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e determination\u003c/p\u003e\n\u003cp\u003eThe K\u003csub\u003eD\u003c/sub\u003e was defined as the concentration of Adonis (used as agonist) which leads to 50% of occupied binding sites (A\u003csub\u003e2A\u003c/sub\u003eR) [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. We used the visualization of Adonis kappa light chains (25 KDa), which detach during the reducing period, and whose density is proportional to the binding of Adonis to the receptor to determine the K\u003csub\u003eD\u003c/sub\u003e value as previously described [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this perspective, PBMCs (0.75 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were incubated with increasing concentrations of Adonis (0 to 0.8\u0026micro;M) and then washed to remove free Adonis prior to treatment of the cell pellets with lysis buffer and sonication. Samples were then submitted to standard electrophoresis under reducing conditions prior to transfer onto a PVDF membrane. Semi quantitative measurement was performed using densitometry analysis and expressed as arbitrary units (A.U.), which constitute the ratio of pixels generated by the light kappa chain band (25 KDa) to pixels generated by the background signal as previously described [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. K\u003csub\u003eD\u003c/sub\u003e values for Adonis binding were estimated using nonlinear regression analysis (Prism software; GraphPad Software).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003ecAMP dosage (EC\u003csub\u003e50\u003c/sub\u003e)\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003ecAMP production was evaluated after incubation of increasing concentrations of Adonis, used as agonist, with PBMCs (0.25 \u0026times; 10\u003csup\u003e6\u003c/sup\u003ecells) using the Amersham Biotrak Kit (GE Healthcare Bio-Sciences). The incubation step was stopped using Dodecyltrimethylammonium bromide acetate buffer. Measurement were performed in duplicates and results expressed as the percentage of the maximal cAMP production. EC\u003csub\u003e50\u003c/sub\u003e was defined as the concentration of Adonis (uses as agonist) that leads to half maximal stimulation of cAMP production [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eData were expressed as mean and standard deviations (SD). A variance analysis (ANOVA) was used for the comparison between A\u003csub\u003e2A\u003c/sub\u003e R expression in different conditions. A difference in at least 25% in expression was considered as relevant.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eWhile low concentration (120 mM) in sodium ion did not influence A\u003csub\u003e2A\u003c/sub\u003eR expression, high concentration of sodium (194 mM) in the culture medium was associated with an increase in the expression of A\u003csub\u003e2A\u003c/sub\u003eR (mean\u0026thinsp;+\u0026thinsp;54% compared with 133 mM, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eHigh concentration (194 mM) in sodium ion decreased both the K\u003csub\u003eD\u003c/sub\u003e value of Adonis (mean 14.5 folds), and the EC\u003csub\u003e50\u003c/sub\u003e value (mean 2.5 folds) compared with sodium concentration equal to 133 mM. Low concentration in sodium (120 mM) was associated with an increase in the K\u003csub\u003eD\u003c/sub\u003e value by 2.6 folds, compared with 133mM, while the EC\u003csub\u003e50\u003c/sub\u003e value was poorly modified (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eWhen sodium ions and Adonis were added simultaneously in the culture medium (competitive condition), the K\u003csub\u003eD\u003c/sub\u003e of Adonis for A\u003csub\u003e2A\u003c/sub\u003eR was strongly increased (mean 27 folds compared with non-competitive conditions, while EC\u003csub\u003e50\u003c/sub\u003e increased by 3.2 folds (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffects of sodium on affinity (K\u003csub\u003eD\u003c/sub\u003e), B max and EC\u003csub\u003e50\u003c/sub\u003e, using Adonis a monoclonal antibody with agonist properties (see methods). All measurements were performed in duplicate. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSodium (194mM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSodium (133mM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSodium (120mM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSodium\u003c/p\u003e\n\u003cp\u003e(194mM)\u003c/p\u003e\n\u003cp\u003eCompetitive conditions\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.011\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBmax\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e107\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e123\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e140.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e144.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe main result of this study is that sodium ions increased the expression of A\u003csub\u003e2A\u003c/sub\u003eR and decreases K\u003csub\u003eD\u003c/sub\u003e and EC\u003csub\u003e50\u003c/sub\u003e values (evaluated using Adonis an antibody with agonists properties) in a dose-dependent manner. Furthermore, when Adonis and sodium ions were added simultaneously (competitive conditions), the K\u003csub\u003eD\u003c/sub\u003e of Adonis increases dramatically. In competitive conditions, it seems that Adonis (an IgM with a very large size) prevents sodium from integrating its allosteric pocket leading to a strong decrease in affinity of Adonis for A\u003csub\u003e2A\u003c/sub\u003eR. Indeed, Adonis caps both the orthostatic binding site (on the second extracellualr loop [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]) and probably the allosteric site (the salt pocket) which is not far from the orthosteric site (on the second transmembrane helix [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e17\u003c/span\u003e], preventing salt from carrying out its allosteric regulation action. This hypothesis, while plausible, needs further investigations. It's also perfectly possible that the increase in ionic strength linked to the salt concentration reduces electrostatic interactions. Similarly, we can't rule out changes in electrostatic interactions linked to increased ionic strength in competition experiments.\u003c/p\u003e \u003cp\u003eWhile the binding of antagonists and sodium ions to the receptor was non-competitive, the binding of agonists and sodium appears to require mutually exclusive conformational states of the receptor [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is now established that orthosteric binding pocket of the ligand represent just one among many sites for possible signal modulation in GPCRs and that allosteric binding pocket not only impacts ligand affinity but also plays a major role in receptor signaling [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, molecules different from ligand can modulate binding of natural ligand that binds to the orthosteric site. It was shown that small molecules like amiloride, ions or lipids can act as allosteric modulator of GPCR [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The allosteric effect of sodium ion has been previously described in A\u003csub\u003e1\u003c/sub\u003eR [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and A\u003csub\u003e2A\u003c/sub\u003eR models [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Modelisation of sodium binding to A\u003csub\u003e2A\u003c/sub\u003eR suggests that sodium ion stabilizes A\u003csub\u003e2A\u003c/sub\u003eR in its inactive conformation and destabilizes activation-related movements and agonist binding [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eIt is generally admitted that sodium ion induced an increase in radioligand antagonist binding, but abrogated agonist binding in a dose dependent manner [1!]. Here we found that sodium ion, in competitive conditions decreases the affinity of Adonis for A\u003csub\u003e2A\u003c/sub\u003eR but have opposite effects in non-competitive conditions. Our study used an agonist antibody rather than an organic ligand or adenosine itself. Although our antibody seems to behave like an organic agonist, particularly in terms of orthostatic binding site and cAMP production, the interactions between Adonis and the A\u003csub\u003e2A\u003c/sub\u003eR receptor may be much more complex than with small organic molecules. While this type of interaction had previously been demonstrated in crystallographic models or in modeling, our results use experimental conditions very close to the blood environment (and PBMCs, whose receptors are known to behave like those of cardiovascular tissue), suggesting, for example, that in response to salt overload, the body might increase the affinity of A\u003csub\u003e2A\u003c/sub\u003eR agonists, resulting in compensatory vasodilatation to eventually lower blood pressure.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStudy limitation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBecause we used an IgM monoclonal antibody as agonist, it is therefore difficult to extrapolate from what actually happens with a small molecule like adenosine in animals or humans concerning the allosteric effects of sodium ion on adenosine binding.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion and Pathophysiologic Perspectives","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIt's a well established that excess salt increases blood pressure by a whole series of mechanisms. The first of these is the drawing of water into the vessels, resulting in hypervolemia. But salt also stimulates the sympathetic system, resulting in vasoconstriction [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Salt elevation in the CSF also leads to activation of angiotensin II and ouabain-dependent sodium pump, leading to the increase in blood pressure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, it is likely that there are also compensatory mechanisms for the rise in arterial pressure induced by excess salt. Modulation of A\u003csub\u003e2A\u003c/sub\u003eR affinity for adenosine could be one such mechanism. Finally, because sodium ion and A\u003csub\u003e2A\u003c/sub\u003e-R activation have opposite effects on blood pressure, our results highlight a possible role of A\u003csub\u003e2A\u003c/sub\u003e-R/sodium ion interaction in the regulation of blood pressure. However, this hypothesis needs further investigations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eE. F.; R.G., G.M., and J.R.: conceptualization and writing the manuscript; \u0026nbsp; M.C.C.; C.G.; C.C., N.K, R.Z: W blot and data analysis. S.L.; J.D.; J.F : critical review. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eAix Marseille University\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data:\u003c/strong\u003e data is available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e: NA\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n \u003cli\u003eAntonioli L, Fornai M, Blandizzi C, Pacher P, Hask\u0026oacute; G (2018) Adenosine signaling and the immune system: When a lot could be too much. Immunol Lett\u003cem\u003e\u0026nbsp;\u003c/em\u003e 205: 9\u0026ndash;15, https://doi.org/10.1016/j.imlet.2018.04.006.\u003c/li\u003e\n \u003cli\u003eDunwiddie TV, Masino SA (2001) The Role and Regulation of Adenosine in the Central Nervous System. Annu\u003cem\u003e\u0026nbsp;\u003c/em\u003eRev Neurosci\u003cem\u003e\u0026nbsp;\u003c/em\u003e 24 : 31\u0026ndash;55, https://doi.org/10.1146/annurev.neuro.24.1.31.\u003c/li\u003e\n \u003cli\u003eFerr\u0026eacute; S, Ciruela F (2019) Functional and Neuroprotective Role of Striatal Adenosine A2A Receptor Heterotetramers. J Caffeine Adenosine Res ;9(3):89-97. doi: 10.1089/caff.2019.0008.\u003c/li\u003e\n \u003cli\u003eBurnstock, G (2017) Purinergic Signaling in the Cardiovascular System. Circ Res 120: 207\u0026ndash;228, https://doi.org/10.1161/circresaha.116.309726.\u003c/li\u003e\n \u003cli\u003eMustafa SJ, Morrison RR, Teng B, Pelleg A (2009) Adenosine receptors and the heart: role in regulation of coronary blood flow and cardiac electrophysiology. Handb Exp Pharmacol 193:161-88. doi: 10.1007/978-3-540-89615-9_6. PMID: 19639282; PMCID: PMC2913612.\u003c/li\u003e\n \u003cli\u003eCushing DJ, Brown G, Sabouni MH, Mustafa SJ (1991) Adenosine receptor-mediated coronary artery relaxation and cyclic nucleotide production. Am J Physiol Circ Physiol 261: H343\u0026ndash;H348, https://doi.org/10.1152/ajpheart.1991.261.2.h343.\u003c/li\u003e\n \u003cli\u003eGuieu R, Fromonot J, Mottola G, Maille B, Marlinge M, Groppelli A, Conte S, Bechah Y, Lalevee N, Michelet P, Hamdan M, Brignole M, Deharo JC. (2023) Adenosinergic System and Neuroendocrine Syncope: What Is the Link?. Cells 12: 2027, https://doi.org/10.3390/cells12162027.\u003c/li\u003e\n \u003cli\u003eLedent C, Vaugeois JM, Schiffmann SN, Pedrazzini T, El Yacoubi M, Vanderhaeghen JJ, Costentin J, Heath, JK, Vassart G, Parmentier M (1997) Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor. Nature 388: 674\u0026ndash;678, https://doi.org/10.1038/41771.\u003c/li\u003e\n \u003cli\u003eShryock JC, Snowdy S, Baraldi PG, Cacciari B, Spalluto G, Monopoli A, Ongini E, Baker SP, Belardinelli L (1998) A \u003csub\u003e2A\u003c/sub\u003e -Adenosine Receptor Reserve for Coronary Vasodilation. Circulation\u003cem\u003e\u0026nbsp;\u003c/em\u003e98: 711\u0026ndash;718, https://doi.org/10.1161/01.cir.98.7.711.\u003c/li\u003e\n \u003cli\u003eGao ZG, Ijzerman A P (2000) Allosteric modulation of A2A adenosine receptors by amiloride analogues and sodium ions. Biochem Pharmacol\u003cem\u003e\u0026nbsp;\u003c/em\u003e60: 669\u0026ndash;676, https://doi.org/10.1016/s0006-2952(00)00360-9.\u003c/li\u003e\n \u003cli\u003eBy Y, Durand-Gorde JM, Condo J, Lejeune PJ, Mallet B, Carayon P, Guieu R, Ruf J (2009) Production of an agonist-like monoclonal antibody to the human A2A receptor of adenosine for clinical use. Mol Immunol 46: 400\u0026ndash;405, https://doi.org/10.1016/j.molimm.2008.10.017.\u003c/li\u003e\n \u003cli\u003eVarani K, Laghi-Pasini F, Camurri A, Capecchi PL, Maccherini M, Diciolla F, Ceccatelli L, Lazzerini PE, Ulouglu C, Cattabeni F, Borea PA, Abbracchio MP (2003) Changes of peripheral A2A adenosine receptors in chronic heart failure and cardiac transplantation. FASEB J 17(2):280-2. doi: 10.1096/fj.02-0543fje. Epub 2002 Dec 3. PMID: 12475889..\u003c/li\u003e\n \u003cli\u003eGodoy-Mar\u0026iacute;n H, Jim\u0026eacute;nez-S\u0026aacute;bado V, Tarifa C, Ginel A, Santos JLD, Bentzen BH, Hove-Madsen L, Ciruela F (2023) Increased Density of Endogenous Adenosine A2A Receptors in Atrial Fibrillation: From Cellular and Porcine Models to Human Patients. Int J Mol Sci 24(4):3668. doi: 10.3390/ijms24043668. PMID: 36835078; PMCID: PMC9963500.\u003c/li\u003e\n \u003cli\u003eRuf J, Paganelli F, Bonello L, Kipson N, Mottola G, Fromonot J, Condo J, Boussuges A, Bruzzese L, Kerbaul F, J ammes Y, Gariboldi V, Franceschi F, Fenouillet E, Guieu R (2016) Spare Adenosine A2a Receptors Are Associated with Positive Exercise Stress Test in Coronary Artery Disease. Mol Med 22: 530\u0026ndash;536, https://doi.org/10.2119/molmed.2016.00052.\u003c/li\u003e\n \u003cli\u003ePaganelli F, Resseguier N, Marlinge M, Laine M, Malergue F, Kipson N, Armangau P, Pezzoli N, Kerbaul F, Bonello L, Mottola G, Fenouillet E, Guieu R, Ruf J (2018) Specific Pharmacological Profile of A2A Adenosine Receptor Predicts Reduced Fractional Flow Reserve in Patients With Suspected Coronary Artery Disease. J Am Heart Assoc ;7(8):e008290. doi: 10.1161/JAHA.117.008290.\u003c/li\u003e\n \u003cli\u003eRuf J, Vairo D, Paganelli F, Guieu R. Extracellular vesicles withubiquitinated adenosine A\u0026lt;sub\u0026gt;2A\u0026lt;/sub\u0026gt; receptor in plasma of patients with coronary artery disease. J Cell Mol Med. 2019 Oct;23(10):6805-6811. doi:10.1111/jcmm.14564. Epub 2019 Aug 24. PMID: 31444994; PMCID: PMC6787504.\u003c/li\u003e\n \u003cli\u003eParker MS, Wong YY, Parker SL (2008) An ion-responsive motif in the second transmembrane segment of rhodopsin-like receptors. Amino Acids 35:1-15. doi: 10.1007/s00726-008-0637-6.\u003c/li\u003e\n \u003cli\u003eGuti\u0026eacute;rrez-de-Ter\u0026aacute;n H.; Massink A, Rodr\u0026iacute;guez D, Liu W, Han GW, Joseph JS, Heitman LH, Xia L, Ijzerman AP, Cherezov V, Katritch V, Stevens RC. (2013) The role of a sodium ion binding site in the allosteric modulation of the A(2A)adenosine G protein-coupled receptor. Structure.21:2175-85. doi:10.1016/j.str.2013.09.020.\u003c/li\u003e\n \u003cli\u003eMassink A, Guti\u0026eacute;rrez-De-Ter\u0026aacute;n H, Lenselink EB, Ortiz Zacar\u0026iacute;as NV, Xia L, Heitman LH, Katritch V, Stevens RC, Ijzerman AP (2015) Sodium Ion Binding Pocket Mutations and Adenosine A2A Receptor Function. Mol Pharmacol.87: 305\u0026ndash;313, https://doi.org/10.1124/mol.114.095737.\u003c/li\u003e\n \u003cli\u003eChristopoulos A, Kenakin TG (2002) protein-coupled receptor allosterism and complexing. Pharmacol Rev 54:323-74. doi: 10.1124/pr.54.2.323.\u003c/li\u003e\n \u003cli\u003eBarbhaiya H, McClain R, Ijzerman A, Rivkees SA (1996) Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding. Mol Pharmacol 50:1635-42.\u003c/li\u003e\n \u003cli\u003eBlaustein MP, Leenen FH, Chen L, Golovina VA, Hamly JM, Pallone TL, Van Huysse JW, Zhang J, Wier WG (2012) How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension. Am J Physiol Heart Circ Physiol 302(5):H1031-49. doi: 10.1152/ajpheart.00899.2011.\u003c/li\u003e\n \u003cli\u003eLinde CI, Karashima E, Raina H, Zulian A, Wier WG, Hamlyn JM, Ferrari P, Blaustein MP, Golovina, VA (2012) Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats. Am J Physiol Heart Circ Physiol ;302(3):H611-20. doi: 10.1152/ajpheart.00950.2011.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"adenosine A2A receptors, sodium ion, blood pressure","lastPublishedDoi":"10.21203/rs.3.rs-4169022/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4169022/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe expression and function of G-protein coupled receptor is modulated by small molecules including sodium ion, acting at an allosteric site. Using Adonis, an Ig M monoclonal antibody against a linear epitope located in the second loop of the A\u003csub\u003e2A\u003c/sub\u003e adenosine receptor (A\u003csub\u003e2A\u003c/sub\u003eR) with agonist properties, we evaluate in cellulo the effects of different sodium ion concentrations on the expression and function of A\u003csub\u003e2A\u003c/sub\u003eR of peripheral blood mononuclear cells (PBMC). We found that high sodium ion concentration is associated with an increase in A\u003csub\u003e2A\u003c/sub\u003eR expression and a decrease in cAMP production evaluated by measuring the half-maximal cAMP production, (EC\u003csub\u003e50\u003c/sub\u003e), in a dose-dependent manner. When Adonis and sodium ions were added simultaneously in the culture medium, (competitive conditions), the K\u003csub\u003eD\u003c/sub\u003e and the EC\u003csub\u003e50\u003c/sub\u003e were high compared to non-competitive conditions (mean 27 and 3 folds respectively). These results suggest that sodium ions could promote Adonis binding to A\u003csub\u003e2A\u003c/sub\u003eR as well as its activation. We hypothesize that, by steric hindrance, the binding of Adonis to A\u003csub\u003e2A\u003c/sub\u003eR extends beyond the orthostatic site and prevents sodium ion from reaching its allosteric pocket. When sodium ion can reach its site, it acts as an allosteric modulator with respect to A\u003csub\u003e2A\u003c/sub\u003eR expression and function. Because the action of sodium ions and A\u003csub\u003e2A\u003c/sub\u003e-R have opposite effects on blood pressure, our results highlight a possible role of A\u003csub\u003e2A\u003c/sub\u003e-R/sodium ions interaction in the regulation of blood pressure.\u003c/p\u003e","manuscriptTitle":"Effects of sodium on A2A adenosine receptor expression and function: in cellulo approach and pathophysiological perspectives","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-05 12:54:57","doi":"10.21203/rs.3.rs-4169022/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e381b13a-56d7-4c52-b046-6de399bcc3c9","owner":[],"postedDate":"April 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-12T08:57:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-05 12:54:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4169022","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4169022","identity":"rs-4169022","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}