Regulation of Ca2+ Channels by cAMP and cGMP in Vascular Smooth Muscle Cells

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Whole-cell Ca2+ channel currents in rabbit portal vein cells were recorded using the amphotericin B-perforated patch-clamp technique at 35°C. This technique allowed recording of stable inward currents in the absence of run-down for more than 30 minutes. Depolarizing voltage steps from a holding potential of −70 mV elicited voltage-dependent inward currents. The voltage dependence of inward currents measured in either 2.5 mmol/L Ba2+ or 2.5 mmol/L Ca2+-containing solution were very similar. However, maximum Ba2+ current (obtained at around +10 mV) was approximately 1.5-fold larger than maximum Ca2+ current. Changing the holding potential from −70 to −40 mV decreased inward currents but did not shift the voltage dependence significantly. Inward currents were also completely blocked by the dihydropyridine Ca2+ channel blocker, nicardipine (10 μmol/L), suggesting the presence of predominantly L-type Ca2+ channels in rabbit portal vein cells. Isoproterenol caused small increases in the amplitude of Ba2+ currents in a concentration-dependent manner (10 nmol/L to 1,mol/L), which were reversed with propranolol. Forskolin (1 μmol/L) or 8-bromo-cAMP (0.1 mmol/L) also caused small increases in the amplitude of Ba2+ currents, suggesting that the stimulatory actions of isoproterenol are importantly linked to the production of cAMP. Higher concentrations of isoproterenol (10 μmol/L) or forskolin (10 μmol/L) caused a transient increase in Ba2+ currents followed by a decrease in current amplitude. Higher doses of 8-bromo-cAMP (1 mmol/L) and low doses of 8-bromo-cGMP (0.1 mmol/L) inhibited Ba2+ currents, increased the rate of current inactivation, and produced a negative voltage shift in steady-state availability. These results indicate that low concentrations of intracellular cAMP produce modest increases in Ca2+ channel activity, whereas cGMP and higher concentrations of cAMP result in inhibition of Ca2+ channel activity in vascular smooth muscle cells. The observed similarities of cGMP and high concentrations of cAMP on Ba2+ current amplitude, kinetics, and steady-state inactivation suggest mediation by a common mechanism, possibly involving activation of cGMP-dependent protein kinase.

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