Section 9
Chapter 8,122

Agonist-independent effects of muscarinic antagonists on calcium and potassium currents in frog and rat cardiac cells

Hanf, R.; Li, Y.; Szabo, G.; Fischmeister, R.

Journal of Physiology (Cambridge) 461: 743-765


ISSN/ISBN: 0022-3751
Accession: 008121696

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The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (I-Ca) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (I-K(ACh)) in frog atrial myocytes. In frog ventricular myocytes, atropine (0.1 nM to 1 mu-M) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on I-Ca previously stimulated by isoprenaline (ISO, 2 mu-M), a beta-adrenergic agonist. However, in the concomitant presence of ISO, ACh and atropine, I-Ca was gt 50% larger than in Iso alone. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of I-Ca with ISO (0.1 or 2 mu-M), atropine induced a dose-dependent stimulation of I-Ca. EC-50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and E-max (i.e. maximal stimulation of I-Ca expressed as percentage increase in I-Ca with respect to the level in ISO alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on I-Ca was not voltage dependent. Atropine (1 mu-M) had no effect on frog I-Ca (i) under basal conditions, (ii) upon stimulation of I-Ca by the dihydropyridine agonist (-)-Bay K 8644 (1 mu-M), or (iii) when I-Ca had been previously stimulated by intracellular perfusion with cyclic AMP (3 mu-M). However, atropine increased I-Ca after a stimulation by forskolin (0.3 mu-M). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on I-Ca. The order of potency of mAChR antagonist to reverse the inhibitory effect of ACh on ISO elevated I-Ca in frog ventricle was atropine gt AF-DIX 116 mchgt pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on ISO elevated I-Ca with the same order of potency. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 mu-M) induced a strong inhibition of frog I-Ca in the presence of ISO (2 mu-M). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), G-i, responsible for adenylyl cyclase inhibition. Atropine (1 mu-M) slowed down by a factor of 2 the rate of I-Ca inhibition induced by Gpp(NH)p. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of I-K(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, G-K. Atropine (1 mu-M) or propylbenzyl choline mustard (1 mu-M) slowed down by a factor of 2-3 the rate of spontaneous activation of I-K(ACh). In rat ventricular cells, atropine (1 mu-M) exerted also a stimulatory effect on I-Ca. However, unlike in frog myocytes, atropine enhanced both basal and ISO-stimulated I-Ca in rat. It is concluded that, in the absence of mAChR agonist, atropine and other 'M2-selective' mAChR antagonists exert a stimulatory effect on I-Ca and an inhibitory effect on I-K(ACh) after binding to the mAChR. Both effects are due to a reduction in the spontaneous activation of the G proteins, G-i and G-K, respectively. Therefore, in addition to displacing the agonist from its binding site, mAChR antagonist may induce a conformational change of the receptor which impairs spontaneous G protein activation by the receptors.

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