RESUMEN
Nitric oxide (NO) is thought to play an important role as a signaling molecule in embryonic and adult cardiomyocytes; however, its involvement in muscarinic signaling is still unclear. The aim of the present work was to analyze the muscarinic modulation of the L-type Ca2+ current (ICa) in early- and late-stage embryonic ventricular cardiomyocytes. Muscarinic stimulation depressed basal ICa by 30.1 +/- 3.2% (n=27) in early-stage cardiomyocytes. Pharmacological evidence suggested that the muscarinic modulation was mediated through generation of NO, activation of cGMP-dependent phosphodiesterase (PDE) 2, and ensuing lowering of cyclic AMP/protein kinase A (cAMP/PKA) levels. Conversely, in late-stage cardiomyocytes, muscarinic regulation of ICa occurred in a NO-independent manner via inhibition of prestimulated adenylyl cyclase (AC). To unequivocally prove the involvement of NO and to identify the nitric oxide synthase (NOS) isoform(s), we analyzed muscarinic signaling in embryonic ventricular cardiomyocytes of NOS2 (-/-) and NOS3 (-/-) mice. The early-stage NOS3 (-/-) cardiomyocytes lacked muscarinic modulation, whereas it was preserved in NOS2 (-/-) cells. Moreover, at the late embryonic stage, muscarinic modulation of ICa was intact in both strains. Thus, NO is the key regulator of muscarinic signaling in the early embryonic ventricle, whereas at later stages, signaling occurs through a NO-independent pathway.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Corazón Fetal/fisiología , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Animales , Animales no Consanguíneos , Carbacol/farmacología , Colinérgicos/farmacología , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Femenino , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/farmacología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/embriología , Transporte Iónico/efectos de los fármacos , Ratones , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Toxina del Pertussis/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/fisiología , Transducción de Señal/efectos de los fármacos , omega-N-Metilarginina/farmacologíaRESUMEN
To investigate the interaction of cytoskeleton with the receptor modulation of ionic currents, we studied the effect of muscarinic and beta-adrenergic stimulation in adult guinea-pig ventricular cardiac myocytes treated with paclitaxel and colchicine, two drugs that respectively stabilize or destabilize microtubules. We observed that the stabilization of microtubules with paclitaxel (1 microM for 1-4 h) did not markedly affect either the kinetics of I(Ca), or the stimulatory effect of isoproterenol (Iso, 1 microM); however paclitaxel significantly blunted the response to carbachol (CCh, 1 microM). In agreement with the electrophysiological measurements, Iso induced a similar enhancement of intracellular cAMP levels in both control and paclitaxel-treated cells, while the response to CCh 1 microM was significantly reduced in paclitaxel-treated cells. The reduction of muscarinic response induced by paclitaxel was also evident in atrial cells, in which the stimulation of I(KACh) by CCh 1 microM was reduced to about 10%. Compared to the muscarinic response, paclitaxel did not have significant effect on the purinergic (adenosine 1-10 microM) modulation of I(Ca). In contrast to paclitaxel, in colchicine-treated cells, I(Ca) was not enhanced by beta-adrenergic stimulation, but instead reduced by CCh, even in the absence of previous stimulation. In conclusion, our data suggest that microtubule stabilization significantly affects the muscarinic modulation of I(Ca), by interacting with the receptor or the G-protein rather than on the intracellular signaling cascade.