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Ortiz-Capisano MC, Atchison DK, Harding P, Lasley RD, Beierwaltes WH. Adenosine inhibits renin release from juxtaglomerular cells via an A1 receptor-TRPC-mediated pathway. Am J Physiol Renal Physiol 305: F1209-F1219, 2013. First published July 24, 2013; doi:10.1152/ajprenal.00710.2012.--Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A1 receptor (A1R) calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized that adenosine inhibits renin release via A1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A2R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A1R in JG cells, but not A2R subtypes, although RT-PCR indicated the presence of mRNA of both A2AR and A2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA) did not change renin. Activating A1R with 0.5 µM N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 ± 0.05 to 0.14 ± 0.03 µg of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) (P < 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 ± 0.08 µg AngI/ml/mg prot; P 0.01), and blocked renin inhibition by CHA (0.28 ± 0.06 µg AngI/ml/mg prot; P < 0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A1R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC isoforms 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus A1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A2 receptors do not regulate renin release. [ABSTRACT FROM AUTHOR]

All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A2A and/or A2B receptors modulate adenosine A1 receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A2A knockout (KO), and A2BKO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A2A receptor agonist CGS-21680 (200 nM) and the A2B agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A2A or A2B receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A2A and A2B receptors are required for adenosine A1 receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes. [ABSTRACT FROM AUTHOR]

The mammalian myocardium expresses four adenosine receptor (AR) subtypes: A1AR, A2aAR, A2bAR, and A3AR. The A1AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of A2aAR and A2bAR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), A2aAR knockout (KO), and A2bAR KO mice. The A2aAR agonist CGS-21680 did not alter basal contractility or β-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in A2aAR KO hearts. However, A2aAR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the A1AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5′-N-ethylcarboxamido adenosine and the selective A2bAR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The A1AR antiadrenergic effect was not potentiated in A2bAR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that A2aAR does not increase cardiac contractility directly but indirectly alters contractility by modulating the A1AR antiadrenergic effect, whereas A2bAR exerts direct contractile effects but does not alter β-adrenergic or A1AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function. [ABSTRACT FROM AUTHOR]

The mechanism by which distinct stimuli activate the same mitogen-activated protein kinases (MAPKs) is unclear. We examined compartmentalized MAPK signaling and altered redox state as possible mechanisms. Adult rat cardiomyocytes were exposed to the adenosine , receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 500 nM) or H202 (100 μM) for 15 min. Nuclear/myofilament, cytosolic, Triton-soluble membrane, and Triton-insoluble membrane fractions were generated. CCPA and H202 activated p38 MAPK and p44/p42 ERKs in cytosolic fractions. In Triton-soluble membrane fractions, H202 activated p38 MAPK and p42 ERK, whereas CCPA had no effect on MAPK activation in this fraction. The greatest difference between H202 and CCPA was in the Triton-insoluble membrane fraction, where H202 increased p38 and p42 activation and CCPA reduced MAPK activation. CCPA also increased protein phosphatase 2A activity in the Triton-insoluble membrane fraction, suggesting that the activation of this phosphatase may mediate CCPA effects in this fraction. The Triton-insoluble membrane fraction was enriched in the caveolae marker caveolin-3, and >85% of p38 MAPK and p42 ERK was bound to this scaffolding protein in these membranes, suggesting that caveolae may play a role in the divergence of MAPK signals from different stimuli. The antioxidant N-2-mercaptopropionyl glycine (300 μM) reduced H202-mediated MAPK activation but failed to attenuate CCPA-induced MAPK activation. H2O2 but not CCPA increased reactive oxygen species (ROS). Thus the adenosine A1 receptor and oxidative stress differentially modulate subcellular MAPKs, with the main site of divergence being the Triton-insoluble membrane fraction. However, the adenosine A, receptor-mediated MAPK activation does not involve ROS formation. [ABSTRACT FROM AUTHOR]

There is increasing evidence for interactions among adenosine receptor subtypes in the brain and heart. The purpose of this study was to determine whether the adenosine A2a receptor modulates the infarct size-reducing effect of preischemic administration of adenosine receptor agonists in intact rat myocardium. Adult male rats were submitted to in vivo regional myocardial ischemia (25 mm) and 2 h reperfusion. Vehicle-treated rats were compared with rats pretreated with the A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 10 µg/kg), the nonselective agonist 5′-N-ethylcarboxamidoadenosine (NECA, 10 µg/kg), or the A2a agonist 2-[4-(2-carboxyethyl)phenethylamino]-5′-N-methylcarboxamidoadenosine (CGS-2 1680, 20 µg/kg). Additional CCPA- and NECA-treated rats were pretreated with the A antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 µg/kg), the A2a/A2b antagonist 4-(-2-[7-amino-2-{2-furyl} {1,2,4) triazolo{2,3-a}{1,3,5) triazin-5-yl-aminolethyl)phenol (ZM-241385, 1.5 mg/kg) or the A3 antagonist 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523, 2 mg/kg). CCPA and NECA reduced myocardial infarct size by 50% and 35%, respectively, versus vehicle, but COS-21680 had no effect. DPCPX blunted the bradycardia associated with CCPA and NECA, whereas ZM-241385 attenuated their hypotensive effects. Both DPCPX and ZM-241385 blocked the protective effects of CCPA and NECA. The A3 antagonist did not alter the hemodynamic effects of CCPA or NECA, nor did it alter adenosine agonist cardioprotection. None of the antagonists alone altered myocardial infarct size. These findings suggest that although preischemic administration of an A2a receptor agonist does not induce cardioprotection, antagonism of the A2a and/or the A2b receptor blocks the cardioprotection associated with adenosine agonist pretreatment. [ABSTRACT FROM AUTHOR]

Adenosine A1 receptor delayed preconditioning (PC) against myocardial infarction has been well described; however, there have been limited investigations of the signaling mechanisms that mediate this phenomenon. In addition, there are multiple conflicting reports on the role of inducible nitric oxide synthase (iNOS) in mediating A1 late-phase PC. The purpose of this study was to determine the roles of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) in in vivo delayed A1 receptor PC and whether this protection at the myocyte level is due to upregulation of iNOS. Myocardial infarct size was measured in open-chest anesthetized rats 24 h after treatment with vehicle or the adenosine A1 agonist 2-chloroN6-cyclopentyladenosine (CCPA; 100 µg/kg ip). Additional rats receiving CCPA were pretreated with the p38 inhibitor SB-203580 (1 mg/kg ip) or the MAPK/ERK kinase (MEK) inhibitor PD-098059 (0.5 mg/kg ip). At 24 h after CCPA administration, a group of animals was given the iNOS inhibitor 1400W 10 min before ischemia. Treatment with CCPA reduced infarct size from 48 ± 2 to 28 ± 2% of the area at risk, an effect that was blocked by both SB-203580 and PD-098059 but not 1400W. Ventricular myocytes isolated 24 h after CCPA injection exhibited significantly reduced oxidative stress during H2O2 exposure compared with myocytes from vehicle-injected animals, and this effect was not blocked by the iNOS inhibitor 1400W. Western blot analysis of whole heart and cardiac myocyte protein samples revealed no expression of iNOS 6 or 24 h after CCPA treatment. These results indicate that adenosine A1 receptor delayed PC in rats is mediated by MAPK-dependent mechanisms, but this phenomenon is not associated with the early or late expression of iNOS. [ABSTRACT FROM AUTHOR]