cGMP-Dependent Protein Kinase Regulation of the L-Type Ca ²⁺ Current in Rat Ventricular Myocytes

Abstract

Regulation of L-type Ca2+ channel current [ICa(L)] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. ICa(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated ICa(L) (by 2 mumol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 mumol/L). When ICa(L) was enhanced by Bay K8644 (1 mumol/L), the enhanced basal ICa(L) was also reduced by PK-G. Basal ICa(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n = 36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 mumol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated ICa(L) (7 of 8 cells). When a holding potential of -80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites.

Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cyclic GMP and nitric oxide.

“…This partial (Ͻ 50%) inhibition is selective, i.e., cGMP and NO inhibit only I Ca,L (T-type Ca 2ϩ current is not targeted). The regulation by cGMP resembles that described in rabbit pulmonary artery and in portal vein and rat ventricular cells (4,(49)(50)(51)(52). Our results suggest that the effect of NO on Ca 2ϩ -channel currents of human coronary myocytes is mediated through a rise in intracellular cGMP.…”

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

“…Secondly, we have demonstrated that the stimulation of myocyte guanylyl cyclase and the increase in intracellular cGMP by muscarinic cholinergic agonists are dependent on the activation of myocyte eNOS. Although the effects of increased intracellular cGMP on I Ca-L in myocytes have been reported to range from stimulatory to neutral to inhibitory, depending on the specific species, myocyte phenotype, and level of intracellular cAMP, the data reported here support a growing consensus that cGMP suppresses the magnitude of Ca 2ϩ influx via I Ca-L in cells that have elevated cAMP levels (19,(29)(30)(31)(32). A similar NO͞cGMP-dependent signal transduction pathway also has been demonstrated in cardiac pacemaker cells (33,34) and basophilic cells (35) after exposure to adenosine.…”

Conditional Neuronal Nitric Oxide Synthase Overexpression Impairs Myocardial Contractility

“…27 In agreement with these findings, 8-BrcGMP has been shown to inhibit I Ca,L in ferret right ventricular myocytes, 28 and the cGMP dependent protein kinase (PKG) has been reported to cause a 40% inhibition of I Ca,L in rat ventricular myocytes. 29 Indeed, we found increased cGMP levels in the nNOS overexpressing myocytes. From the inhibitory effect of nNOS on L-type Ca 2ϩ -channel currents, one would predict an attenuation of the ␤-AR response when nNOS activity is increased.…”