TYROSINE KINASES MODULATE THE ACTIVITY OF SINGLE L-TYPE CALCIUM CHANNELS IN VASCULAR SMOOTH-MUSCLE CELLS FROM RAT PORTAL-VEIN

In a previous study, we demonstrated that extracellular application of the tyrosine kinase inhibitor genistein produced a dose-dependent inh ibition of the macroscopic slow (L-type) Ca2+ currents of vascular smo oth muscle (VSM) cells, and that daidzein, an inactive analog of genis tein, had no such effect. These results suggested that the L-type Ca2 channels in VSM cells may be modulated by endogenous tyrosine kinase activity. To confirm and extend those findings, the effect of genistei n on the activity of single Ca2+ channels was examined in freshly isol ated single VSM cells from rat portal vein, using the cell-attached pa tch-clamp technique. The pipette solution contained 90 mM Ba2+ as char ge carrier and 0.5 mu M Bay K 8644 (to enhance basal activity of the c hannels), and the bath contained 140 mM KCl to ''zero'' the resting me mbrane potential. Depolarizing pulses to 0 mV, from a holding potentia l of -80 mV, elicited inward unitary currents that were blocked by 1 m u M nifedipine (n = 6). The slope conductance of the unitary Ca2+ curr ents gave a value of 21.5 +/- 0.4 pS (n = 9) for the Ca2+ channels. Ba th application of genistein (50 mu M) did not change the unit amplitud e and slope conductance: the conductance in the presence of genistein was 22.2 +/- 0.5 pS (n = 6). However, compared with controls, the acti vity of single Ca2+ channels was significantly inhibited by genistein in a dose-dependent fashion. The ensemble-averaged currents were decre ased by 48.4 +/- 11.2% with 50 mu M genistein; 100 mu M genistein inhi bited the Ca2+ currents by 76.8 +/- 11.8%. The open probability (NP0) was decreased by 50 mu M genistein from 0.24 +/- 0.09 to 0.11 +/- 0.07 . Single-channel kinetic analysis showed that genistein decreased the mean open time and prolonged the mean closed time. The inhibitory effe ct of genistein on the Ca2+ channnel activity occurred within 3 min, a nd it could be reversed by washout within 3-5 min. Daidzein, in concen trations up to 300 mu M, produced no change in the activity of the sin gle Ca2+ channels. These results demonstrate that genistein inhibits t he activity of the L-type Ca2+ channels in VSM cells, suggesting that the availability of the channels for voltage activation may be maintai ned through tonic tyrosine kinase phosphorylation.