G alpha(i2), G alpha(i3) and G alpha(0) are all required for normal muscarinic inhibition of the cardiac calcium channels in nodal/atrial-like cultured cardiocytes

The cardiac L-type calcium current (I-Ca.L) is an important regulator of my ocardial contractility. It is activated by sympathetic stimulation and inhi bited by parasympathetic activity via muscarinic acetylcholine receptors. M uscarinic inhibition of I-Ca.L occurs ria activation of pertussis toxin (PT X)-sensitive heterotrimeric G-proteins. Although recent studies have shown that expression of G(02) is important for this effect in adult mouse ventri cular cells. two other PTX-sensitive G-proteins (C-t2 and G(i3)) are also e xpressed in cardiocytes and are activated. Their role in the regulation of I-Ca.l has not been examined. In addition. it is not known whether nodal/at rial cardiac cells use the same G-proteins. We show that gene inactivation of each of the three PTX-sensitive G(alpha)-proteins (alpha(i2). alpha(i3), and alpha(0)) affects muscarinic inhibition of cardiac I-Ca.L in embryonic stem (ES) cell-derived cardiocytes. Inactivation of either alpha(i2) or al pha(i3) markedly slows the time course of muscarinic inhibition of I-Ca.L, and in cells a here both alpha(i2), and alpha(i3), are inactivated the effe cts are not additive. We also establish an essential role for alpha(0) in t his atrial/nodal-like cardiocyte system and show that alpha(0) acts proxima l to NO generation. NO generation plays a critical role in I-Ca.L regulatio n since the nitric oxide synthase (NOS) antagonist. L-NMMA blocked the inhi bition of I-Ca.L in WT and in alpha(i2)/alpha(i3)-null cells. In WT cells t he NO generating agent SIN-1 inhibited I-Ca.L and the addition of carbachol resulted in faster inhibition. suggesting that pathways in addition to NO are also activated. This study shows that alpha(i2) and ri, play a critical role in the normal inhibition of carjiocyte I-Ca.L. Thus. all muscarinic r eceptor activated G-proteins )G(i2) G(i3) and G(0)) are necessary for norma l inhibition and act through both NO and non-SO signaling pathways. (C) 199 9 Academic Press.