Increased Gi protein signaling potentiates the negative chronotropic effect of adenosine in the SHR right atrium

  • Juliano Q.D. Rodrigues
  • Henrique Camara
  • Aron Jurkiewicz
  • Rosely O. Godinho
Original Article


Hypertension is a risk factor for cardiovascular diseases, which have been associated with dysfunction of sympathetic and purinergic neurotransmission. Therefore, herein, we evaluated whether modifications of adenosine receptor signaling may contribute to the cardiac dysfunction observed in hypertension. Isolated right atria from spontaneously hypertensive (SHR) or normotensive Wistar rats (NWR) were used to investigate the influence of adenosine receptor signaling cascade in the cardiac chronotropism. Our results showed that adenosine, the endogenous agonist of adenosine receptors, and CPA, a selective agonist of A1 receptor, decreased the atrial chronotropism of NWR and SHR in a concentration- and time-dependent manner, culminating in cardiac arrest (0 bpm). Interestingly, a 3-fold lower concentration of adenosine was required to induce the negative chronotropic effect in SHR atria. Pre-incubation of tissues from both strains with DPCPX, a selective A1 receptor antagonist, inhibited the negative chronotropic effect of CPA, while simultaneous inhibition of A2 and A3 receptors, with ZM241385 and MRS1523, did not change the adenosine chronotropic effects. Moreover, 1 μg/ml pertussis toxin, which inactivates the Gαi protein subunit, reduced by 80% the negative chronotropic effects of adenosine in the NWR atrium, with minor effects in SHR tissue. These data indicate that the negative chronotropic effect of adenosine in right atrium depends exclusively on the activation of A1 receptors. Moreover, the distinct responsiveness of NWR and SHR atria to pertussis toxin reveals that the enhanced negative chronotropic response of SHR right atrium is probably due to an increased activity of Gαi protein-mediated.


Adenosine receptor G protein Heart rate SHR Right atria 



We thank Enio S. A. Pacini and Edilson D. S. Junior for helpful suggestions on the experimental design.

Funding information

This work was supported by research grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to A. Jurkiewicz (2013/20402-6) and R.O. Godinho (2015/07019-4) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 0309428/2015-7) to R.O. Godinho. J.Q.D. Rodrigues (12/22763-3) was a PhD fellow and H. Camara (2012/24828-5) an undergraduate student fellow from Fapesp.

Compliance with ethical standards

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Supplementary material

210_2018_1482_Fig8_ESM.gif (73 kb)
Supplementary Fig. 1

Chronotropic effect of adenosine and CPA is unchanged after consecutive agonist curves. Adenosine (0.1 – 1000 μM) (a and b) or the adenosine A1 receptor agonist CPA (0.1 nM – 30 μM) (c and d) effect on chronotropism was evaluated in right atria from normotensive (NWR, a and c) and spontaneously hypertensive rats (SHR, b and d) after two consecutive agonist curves. After the first curve (open circle), preparation was washed out with nutritive solution and the second curve (black circle) was repeated after 1 hour of equilibration time. Potency (pEC50) of the agonists was compared using Student’s t-test. Chronotropism is expressed as mean ± S.E.M. of basal value (100%) obtained before addition of adenosine (n=6) or CPA (n=3) (GIF 73 kb)

210_2018_1482_MOESM1_ESM.tif (99 kb)
High Resolution Image (TIFF 98 kb)


  1. Anand-Srivastava MB (1992) Enhanced expression of inhibitory guanine nucleotide regulatory protein in spontaneously hypertensive rats. Relationship to adenylate cyclase inhibition. The Biochemical Journal 288(Pt 1):79–85CrossRefPubMedPubMedCentralGoogle Scholar
  2. Anand-Srivastava MB (1996) G-proteins and adenylyl cyclase signalling in hypertension. Mol Cell Biochem 157:163–170CrossRefPubMedGoogle Scholar
  3. Arunlakshana O, Schild HO (1959) Some quantitative uses of drug antagonists. Br J Pharmacol Chemother 14:48–58CrossRefPubMedPubMedCentralGoogle Scholar
  4. Azevedo I, Osswald W (1992) Does adenosine malfunction play a role in hypertension? Pharmacol Res 25:227–236CrossRefPubMedGoogle Scholar
  5. Batkai S, Thum T (2012) MicroRNAs in hypertension: mechanisms and therapeutic targets. Curr Hypertens Rep 14:79–87CrossRefPubMedGoogle Scholar
  6. Benjamin EJ, Levy D, Vaziri SM, D'Agostino RB, Belanger AJ, Wolf PA (1994) Independent risk factors for atrial fibrillation in a population-based cohort. The Framingham Heart Study. JAMA 271:840–844CrossRefPubMedGoogle Scholar
  7. Blinks JR (1966) Field stimulation as a means of effecting the graded release of autonomic transmitters in isolated heart muscle. J Pharmacol Exp Ther 151:221–235PubMedGoogle Scholar
  8. Boknik P, Neumann J, Schmitz W, Scholz H, Wenzlaff H (1997) Characterization of biochemical effects of CGS 21680C, an A2-adenosine receptor agonist, in the mammalian ventricle. J Cardiovasc Pharmacol 30:750–758CrossRefPubMedGoogle Scholar
  9. Borea PA, Caparrotta L, De Biasi M, Fassina G, Froldi G, Pandolfo L, Ragazzi E (1989) Effect of selective agonists and antagonists on atrial adenosine receptors and their interaction with Bay K 8644 and [3H]-nitrendipine. Br J Pharmacol 96:372–378CrossRefPubMedPubMedCentralGoogle Scholar
  10. Brock JA, Van Helden DF (1995) Enhanced excitatory junction potentials in mesenteric arteries from spontaneously hypertensive rats. Pflugers Archiv : Eur J Physiol 430:901–908CrossRefGoogle Scholar
  11. Burnstock G (2009) Purinergic signalling: past, present and future. Brazilian Journal of Medical and Biological Research = Revista brasileira de pesquisas medicas e biologicas/Sociedade Brasileira de Biofisica [et al] 42: 3-8Google Scholar
  12. Burnstock G, Pelleg A (2015) Cardiac purinergic signalling in health and disease. Purinergic signalling 11:1–46CrossRefPubMedGoogle Scholar
  13. Camara H, da Silva Junior ED, Garcia AG, Jurkiewicz A, Rodrigues JQD (2018) Cardiac arrest induced by muscarinic or adenosine receptors agonists is reversed by DPCPX through double mechanism. Eur J Pharmacol 819:9–15CrossRefPubMedGoogle Scholar
  14. Camara H, Rodrigues JQ, Alves GA, da Silva Junior ED, Caricati-Neto A, Garcia AG, Jurkiewicz A (2015) Would calcium or potassium channels be responsible for cardiac arrest produced by adenosine and ATP in the right atria of Wistar rats? Eur J Pharmacol 768:199–206CrossRefPubMedGoogle Scholar
  15. Carr CS, Hill RJ, Masamune H, Kennedy SP, Knight DR, Tracey WR, Yellon DM (1997) Evidence for a role for both the adenosine A1 and A3 receptors in protection of isolated human atrial muscle against simulated ischaemia. Cardiovasc Res 36:52–59CrossRefPubMedGoogle Scholar
  16. Cordeaux Y, Ijzerman AP, Hill SJ (2004) Coupling of the human A1 adenosine receptor to different heterotrimeric G proteins: evidence for agonist-specific G protein activation. Br J Pharmacol 143:705–714CrossRefPubMedPubMedCentralGoogle Scholar
  17. Council NR (2010) Guide for the care and use of laboratory animals. National Academies PressGoogle Scholar
  18. Duarte T, Menezes-Rodrigues FS, Godinho RO (2012) Contribution of the extracellular cAMP-adenosine pathway to dual coupling of beta2-adrenoceptors to Gs and Gi proteins in mouse skeletal muscle. J Pharmacol Exp Ther 341:820–828CrossRefPubMedGoogle Scholar
  19. Erlinge D, Burnstock G (2008) P2 receptors in cardiovascular regulation and disease. Purinergic signalling 4:1–20CrossRefPubMedGoogle Scholar
  20. Fassina G, de Biasi M, Ragazzi E, Caparrotta L (1991) Adenosine: a natural modulator of L-type calcium channels in atrial myocardium? Pharmacol Res 23:319–326CrossRefPubMedGoogle Scholar
  21. Folkow B (1982) Physiological aspects of primary hypertension. Physiol Rev 62:347–504CrossRefPubMedGoogle Scholar
  22. Ford WR, Broadley KJ (1997) Functional classification of P1-purinoceptors in guinea-pig left and right atria: anomalous characteristics of antagonism by cyclopentyltheophylline. Naunyn Schmiedeberg's Arch Pharmacol 355:759–766CrossRefGoogle Scholar
  23. Fredholm BB, AP IJ, Jacobson KA, Klotz KN, Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacological. Reviews 53:527–552Google Scholar
  24. Furchgott RF (1966) The use of β-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes. Adv Drug Res 3:21–55Google Scholar
  25. Gardner NM, Broadley KJ (1999) Resistance to antagonism of atrial P(1) purinoceptor responses in the presence of K(+) channel blockade. Eur J Pharmacol 383:143–153CrossRefPubMedGoogle Scholar
  26. Gergs U, Boknik P, Schmitz W, Simm A, Silber RE, Neumann J (2008) A positive inotropic effect of ATP in the human cardiac atrium. Am J Phys Heart Circ Phys 294:H1716–H1723Google Scholar
  27. Godinho RO, Duarte T, Pacini ES (2015) New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP e ffl ux and extracellular cAMP-adenosine pathway. Front Pharmacol 6:58CrossRefPubMedPubMedCentralGoogle Scholar
  28. Green A, Johnson JL, DiPette DJ (1990) Decrease in A1 adenosine receptors in adipocytes from spontaneously hypertensive rats. Metab Clin Exp 39:1334–1338CrossRefPubMedGoogle Scholar
  29. Grinberg S, Hasko G, Wu D, Leibovich SJ (2009) Suppression of PLCbeta2 by endotoxin plays a role in the adenosine A(2A) receptor-mediated switch of macrophages from an inflammatory to an angiogenic phenotype. Am J Pathol 175:2439–2453CrossRefPubMedPubMedCentralGoogle Scholar
  30. Headrick JP, Ashton KJ, Rose'meyer RB, Peart JN (2013) Cardiovascular adenosine receptors: expression, actions and interactions. Pharmacol Ther 140:92–111CrossRefPubMedGoogle Scholar
  31. Headrick JP, Peart J (2005) A3 adenosine receptor-mediated protection of the ischemic heart. Vasc Pharmacol 42:271–279CrossRefGoogle Scholar
  32. Heaton DA, Lei M, Li D, Golding S, Dawson TA, Mohan RM, Paterson DJ (2006) Remodeling of the cardiac pacemaker L-type calcium current and its beta-adrenergic responsiveness in hypertension after neuronal NO synthase gene transfer. Hypertension 48:443–452CrossRefPubMedGoogle Scholar
  33. Heaton DA, Li D, Almond SC, Dawson TA, Wang L, Channon KM, Paterson DJ (2007) Gene transfer of neuronal nitric oxide synthase into intracardiac Ganglia reverses vagal impairment in hypertensive rats. Hypertension 49:380–388CrossRefPubMedGoogle Scholar
  34. Herring N, Lee CW, Sunderland N, Wright K, Paterson DJ (2011) Pravastatin normalises peripheral cardiac sympathetic hyperactivity in the spontaneously hypertensive rat. J Mol Cell Cardiol 50:99–106CrossRefPubMedPubMedCentralGoogle Scholar
  35. Hove-Madsen L, Prat-Vidal C, Llach A, Ciruela F, Casado V, Lluis C, Bayes-Genis A, Cinca J, Franco R (2006) Adenosine A2A receptors are expressed in human atrial myocytes and modulate spontaneous sarcoplasmic reticulum calcium release. Cardiovasc Res 72:292–302CrossRefPubMedGoogle Scholar
  36. Jacobson KA, Gao ZG (2006) Adenosine receptors as therapeutic targets. Nat Rev Drug Discov 5:247–264CrossRefPubMedPubMedCentralGoogle Scholar
  37. Kenakin T (2011) Functional selectivity and biased receptor signaling. J Pharmacol Exp Ther 336:296–302CrossRefPubMedGoogle Scholar
  38. Lau DH, Shipp NJ, Kelly DJ, Thanigaimani S, Neo M, Kuklik P, Lim HS, Zhang Y, Drury K, Wong CX, Chia NH, Brooks AG, Dimitri H, Saint DA, Brown L, Sanders P (2013) Atrial arrhythmia in ageing spontaneously hypertensive rats: unraveling the substrate in hypertension and ageing. PLoS One 8:e72416CrossRefPubMedPubMedCentralGoogle Scholar
  39. Levay M, Krobert KA, Wittig K, Voigt N, Bermudez M, Wolber G, Dobrev D, Levy FO, Wieland T (2013) NSC23766, a widely used inhibitor of Rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors. J Pharmacol Exp Ther 347:69–79CrossRefPubMedGoogle Scholar
  40. Li D, Wang L, Lee CW, Dawson TA, Paterson DJ (2007) Noradrenergic cell specific gene transfer with neuronal nitric oxide synthase reduces cardiac sympathetic neurotransmission in hypertensive rats. Hypertension 50:69–74CrossRefPubMedGoogle Scholar
  41. Mancia G, Grassi G (2014) The autonomic nervous system and hypertension. Circ Res 114:1804–1814CrossRefPubMedGoogle Scholar
  42. Mancia G, Grassi G, Giannattasio C, Seravalle G (1999) Sympathetic activation in the pathogenesis of hypertension and progression of organ damage. Hypertension 34:724–728CrossRefPubMedGoogle Scholar
  43. Matias A, Albino-Teixeira A, Polonia J, Azevedo I (1991) Long-term administration of 1,3-dipropyl-8-sulfophenylxanthine causes arterial hypertension. Eur J Pharmacol 193:101–104CrossRefPubMedGoogle Scholar
  44. Mustafa SJ, Ansari HR, Abebe W (2009) P1 (adenosine) purinoceptor assays. Current protocols in pharmacology/editorial board, SJ Enna Chapter 4: Unit 4 7Google Scholar
  45. Neubig RR, Spedding M, Kenakin T, Christopoulos A, International Union of Pharmacology Committee on Receptor N, Drug C (2003) International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacological Reviews 55:597–606CrossRefPubMedGoogle Scholar
  46. Ongini E, Dionisotti S, Gessi S, Irenius E, Fredholm BB (1999) Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors. Naunyn Schmiedeberg's Arch Pharmacol 359:7–10CrossRefGoogle Scholar
  47. Palatini P, Julius S (2009) The role of cardiac autonomic function in hypertension and cardiovascular disease. Curr Hypertens Rep 11:199–205CrossRefPubMedGoogle Scholar
  48. Pandey SK, Anand-Srivastava MB (1996) Modulation of G-protein expression by the angiotensin converting enzyme inhibitor captopril in hearts from spontaneously hypertensive rats. Relationship with adenylyl cyclase. Am J Hypertens 9:833–837CrossRefPubMedGoogle Scholar
  49. Pelleg A, Hurt CM, Michelson EL (1990) Cardiac effects of adenosine and ATP. Ann N Y Acad Sci 603:19–30CrossRefPubMedGoogle Scholar
  50. Rubino A, Burnstock G (1995) Changes in sympathetic neurotransmission and adrenergic control of cardiac contractility during 1,3-dipropyl-8-sulfophenylxanthine-induced hypertension. J Pharmacol Exp Ther 275:422–428PubMedGoogle Scholar
  51. Sassi Y, Abi-Gerges A, Fauconnier J, Mougenot N, Reiken S, Haghighi K, Kranias EG, Marks AR, Lacampagne A, Engelhardt S, Hatem SN, Lompre AM, Hulot JS (2012) Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes. FASEB J: Off Publ Fed Am Soc Exp Biol 26:1009–1017CrossRefGoogle Scholar
  52. Schild HO (1947) pA, a new scale for the measurement of drug antagonism. Br J Pharmacol Chemother 2:189–206CrossRefPubMedPubMedCentralGoogle Scholar
  53. Schild HO (1949) pAx and competitive drug antagonism. Br J Pharmacol Chemother 4:277–280CrossRefPubMedPubMedCentralGoogle Scholar
  54. Smrcka AV (2008) G protein betagamma subunits: central mediators of G protein-coupled receptor signaling. Cell Mol Life Sci: CMLS 65:2191–2214CrossRefPubMedPubMedCentralGoogle Scholar
  55. Starling EH, Visscher MB (1927) The regulation of the energy output of the heart. J Physiol 62:243–261CrossRefPubMedPubMedCentralGoogle Scholar
  56. Trincavelli ML, Daniele S, Martini C (2010) Adenosine receptors: what we know and what we are learning. Curr Top Med Chem 10:860–877CrossRefPubMedGoogle Scholar
  57. Tuovinen K, Tarhanen J (2004) Clearance of cyclopentyladenosine and cyclohexyladenosine in rats following a single subcutaneous dose. Pharmacol Res 50:329–334CrossRefPubMedGoogle Scholar
  58. Vassort G (2001) Adenosine 5′-triphosphate: a P2-purinergic agonist in the myocardium. Physiol Rev 81:767–806CrossRefPubMedGoogle Scholar
  59. Vizi ES (2000) Role of high-affinity receptors and membrane transporters in nonsynaptic communication and drug action in the central nervous system. Pharmacol Rev 52:63–89PubMedGoogle Scholar
  60. Voigt N, Abu-Taha I, Heijman J, Dobrev D (2014) Constitutive activity of the acetylcholine-activated potassium current IK,ACh in cardiomyocytes. Adv Pharmacol 70:393–409CrossRefPubMedGoogle Scholar
  61. von Kugelgen I, Kurz K, Starke K (1993) Axon terminal P2-purinoceptors in feedback control of sympathetic transmitter release. Neuroscience 56:263–267CrossRefGoogle Scholar
  62. Wang WW, Zhang FL, Chen JH, Chen XH, Fu FY, Tang MR, Chen LL (2015) Telmisartan reduces atrial arrhythmia susceptibility through the regulation of RAS-ERK and PI3K-Akt-eNOS pathways in spontaneously hypertensive rats. Can J Physiol Pharmacol 93:657–665CrossRefPubMedGoogle Scholar
  63. Whitworth JA (2003) 2003 World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension. J Hypertens 21:1983–1992CrossRefPubMedGoogle Scholar
  64. Wilson AN, Broadley KJ (1989) Analysis of the direct and indirect effects of adenosine on atrial and ventricular cardiac muscle. Can J Physiol Pharmacol 67:294–303CrossRefPubMedGoogle Scholar
  65. Zhai P, Yamamoto M, Galeotti J, Liu J, Masurekar M, Thaisz J, Irie K, Holle E, Yu X, Kupershmidt S, Roden DM, Wagner T, Yatani A, Vatner DE, Vatner SF, Sadoshima J (2005) Cardiac-specific overexpression of AT1 receptor mutant lacking G alpha q/G alpha i coupling causes hypertrophy and bradycardia in transgenic mice. J Clin Invest 115:3045–3056CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmacology, Escola Paulista de MedicinaUniversidade Federal de São Paulo (UNIFESP)São PauloBrazil

Personalised recommendations