Cyclic nucleotides regulate the activity of L-type calcium channels in smooth muscle cells from rat portal vein

Abstract

In order to advance our previous findings that the macroscopic slow Ca2+ currents of vascular smooth muscle (VSM) cells are regulated by cyclic nucleotides, the effects of cAMP and cGMP on the activity of single slow (L-type) Ca2+ channels were investigated using cell-attached patch clamp (22-25 degrees C). Freshly isolated VSM cells were obtained from adult male rat portal vein. For the single-channel recordings, the pipette was filled with a solution containing 90 mM Ba2+ and 1 microM Bay-K-8644 solution, and the bath contained 140 mM KCl to "zero" the membrane potential. Depolarizing pulses to 0 mV, from a holding potential (HP) of -80 mV, elicited inward unitary currents. The activity of these channels was completely blocked by superfusion of 10 microM nifedipine. Extracellular perfusion of the single cells with membrane-permeable cGMP and cAMP analogs (8Br-cGMP and 8Br-cAMP) at 1 mM caused a slight inhibition, but higher doses (3 mM), clearly showed an inhibitory effect on the single-channel activity. cAMP (100 microM) stimulated one out of five patches tested, and 100 microM cGMP showed no effect in three patches tested. Compared with control, both cyclic nucleotides at 3 mM decreased the ensemble-averaged currents by 26.7 +/- 4.1% and 37.3 +/- 2.1%, respectively. Unit amplitude and slope conductance were not changed. The normal conductance of the Ca2+ channel was 20.8 +/- 0.04 pS (n = 9), and the conductances in the presence of cAMP (n = 5) and cGMP (n = 6) were 19.3 +/- 0.04 and 20.5 +/- 0.05 pS, respectively. Single-channel kinetic analysis showed that cAMP did not affect the mean open-time, and cGMP slightly decreased the mean open-time. However, both cAMP and cGMP increased the mean closed-time. In addition, cAMP decreased the open probability (NPo) by a factor of 1.7, from 0.26 +/- 0.04 to 0.15 +/- 0.03 (P < 0.05, Student's t-test) and cGMP decreased NPo by a factor of 2.5, from 0.24 +/- 0.08 to 0.10 +/- 0.02 (P < 0.05). H-7, a non-specific protein kinase inhibitor, prevented the inhibitory effects of both cAMP and cGMP on the activity of single Ca2+ channels in rat portal vein cells. The results demonstrate that both cAMP and cGMP inhibit L-type Ca2+ channel activities in VSM cells from rat portal vein. This inhibition may be mediated by the cAMP and cGMP-dependent protein kinase phosphorylation of the L-type Ca2+ channels (or an associated regulatory protein).