New insights into adenosine's chronoselective atrial depressant effect - The interplay of adenosine A1 receptor with ion channels and signaling enzymes

In supraventricular tissues adenosine decreases spontaneous pacemaker activity (negative chronotropy), atrioventricular nodal conduction (negative dromotropy) and myocardium contraction force (negative inotropy) through the activation of adenosine A1 receptor (A1R) (Headrick et al., 2011). A1R is a Gαi/o-protein coupled receptor. Once activated, A1R may inhibits catecholamine-stimulated adenylyl cyclase (anti-β-adrenergic effect), increases hyperpolarizing K currents carried through GIRK/KIR3 channels (Belardinelli et al., 1995) and stimulates phospholipase Cβ activity and PKC translocation (Parsons et al., 2000; Yang et al., 2009b). Preliminary data from our lab showed that adenosine is a “chronoselective” atrial depressant via the activation of A1R, yet this property is not shared by other cardiodepressant agents, like acetylcholine acting through muscarinic M2 receptor (M2R) activation (Oliveira-Monteiro et al., unpublished observations). The mechanism(s) underlying adenosine “chronoselectivity” remains to be identified. Given their involvement in atrial physiology, we sought it would be interesting to investigate the expression and function of subtype-selective K and Ca channels in the spontaneously beating rat atria. The putative enrolment of PLC/PKC signaling pathway on A1R chronoselectivity was also investigated. Both the A1R agonist, R-PIA (0.001–1 μM), and the muscarinic M2 receptor agonist, oxotremorine (0.01–3 μM), decreased in a concentration-dependent manner the rate (negative chronotropic effect) and the strength of contractions (negative inotropic effect) in the spontaneously beating rat atria. Unlike muscarinic M2 receptor activation with oxotremorine, the A1 receptor-mediated negative chronotropic effect of R-PIA was evidenced at much lower concentrations than its negative inotropic action (chronoselective effect). The effects of oxotremorine and of R-PIA were prevented upon blocking M2Rs with AF-DX 116 (10 μM) and A1Rs with DPCPX (100 nM), respectively. Blockade of GIRK/KIR3 channels with tertiapin Q (300 nM) prevented negative chronotropism and inotropism of both A1 and M2 receptor agonists, respectively R-PIA and oxotremorine. Inhibition of Kv and KATP/KIR6 currents, respectively with 4-aminopyridine and glibenclamide, were devoid of effect. Blockade of KCa2/SK channels with apamin and CaV1 (L-type) channels with verapamil, sensitized atria to the negative inotropic action of R-PIA, without affecting the nucleoside chronotropic effect and the oxotremorine effect. The same occurred when rat atria were pre-treated with PLC and PKC inhibitors, such as U73122 (3 μM) and chelerythrine (3 μM), respectively. Immunolocalization studies showed that differences in the distribution of A1 receptors between the sinoatrial node and surrounding cardiomyocytes might not afford a rationale for adenosine chronoselectivity. ICBAS/FCUP Abstract ix Immunolabeling of KIR3.1, KCa2.2, KCa2.3 and Cav1 was also observed throughout the right atria. Functional data support Gβγ/PLC/PKC signaling axis as an important pathway to counteract the negative inotropic effects of GIRK/KIR3 opening following A1R stimulation. After GIRK/KIR3 activation, Gβγ subunits released from the vicinity of A1R may negatively regulate these channels through PIP2 hydrolysis. Decrease of K current carried by GIRK/KIR3 channel along with lower KCa2/SK channel conductance embrace a compensatory mechanism for A1R, which is not present with M2R activation. As CaV1 (Ltype) channel blockade sensitized atria to contractile depression promoted via Ca influx, these channels connect inhibition of hyperpolarizing K currents to greater time available for Ca influx. This study strengthens the differences between A1R and M2R signaling in the heart, particularly with respect to PLC/PKC modulation, providing new insights into adenosine’s chronoselectivity.