Muscarinic cholinergic regulation of cardiac myocyte I(Ca-L) is absent in mice with targeted disruption of endothelial nitric oxide synthase

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (I(Ca-L)) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on I(Ca-L) and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOS(null) mice, with targeted disruption of the eNOS gene. Although both eNOS(null) and wild-type (WT) ventricular myocytes exhibited similar increases in I(Ca-L) in response to ISO, there was no measurable suppression of I(Ca-L) by CCh in cells from eNOS(null) mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOS(null) myocytes. Also, unlike myocytes from WT animals, eNOS(null) myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented I(Ca-L) in eNOS(null) cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K(+) channel by CCh was unaffected in atrial and ventricular eNOS(null) myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of I(Ca-L) in cardiac myocytes.