Increase of L-type calcium current by cGMP-dependent protein kinase regulates in rabbit ventricular myocytes

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 (ICa) 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 ICa by cGMP. However, there is no direct evidence that cGMP-PK can stimulate ICa in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK an ICa in rabbit ventricular myocytes. METHODS AND RESULTS: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of ICa by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. ICa was elicited by a depolarizing pulse to +10 mV from a holding potential of -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 ICa. cGMP-PK also increased basal ICa. The stimulation of basal ICa by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal ICa 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 ICa was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of ICa. In the presence of cGMP-PK, already increased ICa was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. CONCLUSIONS: We present evidence that cGMP-PK stimulated basal ICa by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.

Ionic mechanism underlying chance of L-type Ca~(2+) current in isolated rat ventricular myocytes induced by hypercholesterolemia / 中国药理学通报

Aim To investigate the effect of hypercholesterolemia on L-type Ca2+(ICa-L) current and intracellular calcium concentration ([Ca2+]i) in single ventricular myocytes of hypercholesterolemic rats.Methods 12 male wistar rats were randomly divided into two dietary groups:a group fed a normal diet(n=6)and a group fed high-cholesterol diet(n=6) for 4 weeks,respectively. The level of serum lipid was examined.Zymolytic method was used to isolate single ventricular myocytes from hypercholesterolemic and normal rats,which were loaded with Ca2+-sensitive fluorescent indicator Fluo-3/AM.[Ca2+]i represented by fluorescent intensity(FI)was measured by laser scanning confocal microscope.Whole cell patch clamp technique was used to record ICa-L.Results There was no significant influence exhibited on TG level.However, the serum total cholesterol(TC)level of hypercholesterolemic rats was much higher than that of model control group; at the test potential of 0 mV, ICa-L decreased from(-8.56?1.29)pA/pF(Control)to(-5.24?0.90) pA/pF(HC)(n=6 in each group,P