Cyclic Nucleotides Regulate the Activity ofL-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°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 mmBa2+and 1μmBay-K-8644 solution, and the bath contained 140 mmKCl 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 10μmnifedipine. Extracellular perfusion of the single cells with membrane-permeable cGMP and cAMP analogs (8Br-cGMP and 8Br-cAMP) at 1 mmcaused a slight inhibition, but higher doses (3 mm), clearly showed an inhibitory effect on the single-channel activity. cAMP (100μm) stimulated one out of five patches tested, and 100μmcGMP showed no effect in three patches tested. Compared with control, both cyclic nucleotides at 3 mmdecreased 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'st-test) and cGMP decreased NPoby 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 inhibitl-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 thel-type Ca2+channels (or an associated regulatory protein).