Increase of L-type Calcium Current by cGMP-dependent Protein Kinase Regulates in Rabbit Ventricular Myocytes

Increase of L-type Calcium Current by cGMP-dependent Protein Kinase Regulates in Rabbit Ventricular Myocytes

<P> <I> </I>Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current (I<SUB>Ca</SUB>) in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of I<SUB>Ca</SUB> by cGMP. However, there is no direct evidence that cGMP-PK can stimulate I<SUB>Ca</SUB> in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on I<SUB>Ca</SUB> in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of I<SUB>Ca</SUB> by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. I<SUB>Ca</SUB> was elicited by a depolarizing pulse to &#8290;10 mV from a holding potential of &#8291;40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3 ,5 -cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal I<SUB>Ca</SUB>. cGMP-PK also increased basal I<SUB>Ca</SUB>. The stimulation of basal I<SUB>Ca</SUB> by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal I<SUB>Ca</SUB> by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8- (4-chlorophenylthio)-guanosine-3 ,5 -cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When I<SUB>Ca</SUB> was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of I<SUB>Ca</SUB>. In the presence of cGMP-PK, already increased I<SUB>Ca</SUB> was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal I<SUB>Ca</SUB> by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.