The effect of the nitric oxide (NO) donor SIN-1 (3-morpholino-sydnonimine) on the calcium current (ICa) was examined in guinea pig ventricular myocytes. SIN-1 had little effect on basal ICa. After moderate stimulation of ICa with 10 nM isoproterenol (ISO), 10 microM SIN-1 caused either stimulation or inhibition of ICa; 100 microM SIN-1 consistently caused inhibition. SIN-1 (1-100 microM) inhibited ICa equally following considerable enhancement of ICa by either 1 microM ISO or 100 microM 3-isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE) inhibitor. SIN-1 (100 microM) also inhibited ICa equally following enhancement by either 10 microM pipette adenosine 3',5'-cyclic monophosphate (cAMP) or hydrolysis-resistant 8-bromo-cAMP. Thus the inhibitory effect of SIN-1 appears independent of PDEs. Addition of LY-83583 (a blocker of guanylate cyclase) to the pipette or superfusion with KT-5823 [a blocker of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase] suppressed the inhibitory effect of SIN-1. We conclude that NO is an important modulator of beta-adrenergic effects on ICa and that the mechanism of NO inhibition of ICa in mammalian cardiac cells involves the cGMP-dependent protein kinase.
The purpose of this study was to investigate sex differences in the functional response of isolated rat heart ventricular myocytes to beta-adrenergic stimulation and in isoproterenol-stimulated signal transduction. Fractional shortening was measured using a video edge-detection system in control- and isoproterenol-stimulated myocytes that had been isolated from weight-matched rats. Number and affinity of the beta-adrenergic receptors and the L-type Ca(2+) channel were measured in ventricular cardiac membranes by radioligand binding studies. Control- and isoproterenol-mediated alteration in Ca(2+) current density (I(Ca)) was determined by patch clamping and cellular cAMP content was determined by radioimmunoassay. Study results demonstrate that female myocytes have higher Ca(2+) channel density and greater I(Ca) than male myocytes. However, isoproterenol elicits a greater beta-adrenergic receptor-mediated increase cell shortening, I(Ca) and cAMP production in male myocytes. Male myocytes were also found to have a higher beta-adrenergic receptor density. These results suggest that cardiac myocytes from male rats have an enhanced response to beta-adrenergic stimulation due to augmented beta-adrenergic signaling that results in a greater transsarcolemmal Ca(2+) influx.
The neonatal rat ventricular action potential has a shape similar to that of most adult mammals. However, shortly after birth, the action potential shortens to a spike-like configuration. The contribution of changes in repolarizing currents to the shortening is unclear. Thus the inwardly rectifying potassium current (IK1) was measured in heart cells from rats of varying ages using patch-clamp techniques. In freshly isolated cells, whole cell IK1 currents increased greatly between ages 3 and 9-13 days and remained constant thereafter. In culture, IK1 disappeared preferentially in older cells, obscuring the developmental increase. Age-dependent differences in single-channel activity were also observed. Adult cells had IK1 channels consisting of two populations (30 and 42 pS), whereas neonatal cells exhibited only the lower conductance channel. The appearance of the 42-pS channel may contribute a part of the developmental increase in IK1. It was concluded that IK1 increases during postnatal development of the rat ventricle and that this increase may contribute to the postnatal shortening of the rat ventricular action potential.
We studied how the nitric oxide (NO*) donor 3-morpholinosydnonimine (SIN-1) alters the response to beta-adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO*, are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of beta-adrenergic stimulation.
1 Cyclic GMP (cGMP) has been shown to be an important modulator of cardiac contractile function. A major component of cGMP regulation of contractility is cGMP-mediated inhibition of the cardiac calcium current (I Ca ). An under-appreciated aspect of cyclic nucleotide signalling is hydrolysis of the cyclic nucleotide (i.e., breakdown by phosphodiesterases (PDEs)). The role of cGMP hydrolysis in regulating I Ca has not been studied. Thus the purpose of this study was to investigate if inhibition of cGMP hydrolysis can modulate I Ca in isolated guinea-pig ventricular myocytes. 2 Zaprinast, a selective inhibitor of cGMP-speci®c PDE (PDE5), caused a signi®cant increase in cGMP levels in myocytes, but was without a ect on basal or b-adrenergic stimulated cAMP levels (consistent with its actions as a speci®c inhibitor of PDE5). 3 Zaprinast inhibited I Ca that was pre-stimulated with cAMP elevating agents (isoproterenol, a badrenergic agonist; or forskolin, a direct activator of adenylate cyclase). The e ect of zaprinast was greatly reduced by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). 4 Zaprinast also signi®cantly inhibited basal I Ca when perforated-patch or whole-cell recording with physiological pipette calcium concentration (10 77 M) was used. However, this e ect was not observed when using standard calcium-free whole-cell recording conditions. 5 These results indicate that inhibition of cGMP hydrolysis can decrease both basal and cAMPstimulated I Ca . Thus, cGMP hydrolysis may likely be an important step for physiological modulation of I Ca . This regulation may also be important in disease states in which cGMP production is increased and PDE5 expression is altered, such as heart failure.
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