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 inhibition of the macroscopic slow (L-type) Ca2+ currents of vascular smooth muscle (VSM) cells, and that daidzein, an inactive analog of genistein, 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 genistein on the activity of single Ca2+ channels was examined in freshly isolated single VSM cells from rat portal vein, using the cell-attached patch-clamp technique. The pipette solution contained 90 mM Ba2+ as charge carrier and 0.5 microM Bay K 8644 (to enhance basal activity of the channels), and the bath contained 140 mM KCl to "zero" the resting membrane potential. Depolarizing pulses to 0 mV, from a holding potential of -80 mV, elicited inward unitary currents that were blocked by 1 microM nifedipine (n = 6). The slope conductance of the unitary Ca2+ currents gave a value of 21.5 +/- 0.4 pS (n = 9) for the Ca2+ channels. Bath application of genistein (50 microM) did not change the unit amplitude and slope conductance: the conductance in the presence of genistein was 22.2 +/- 0.5 pS (n = 6). However, compared with controls, the activity of single Ca2+ channels was significantly inhibited by genistein in a dose-dependent fashion. The ensemble-averaged currents were decreased by 48.4 +/- 11.2% with 50 microM genistein; 100 microM genistein inhibited the Ca2+ currents by 76.8 +/- 11.8%. The open probability (NPo) was decreased by 50 microM 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 effect of genistein on the Ca2+ channel activity occurred within 3 min, and it could be reversed by washout within 3-5 min. Daidzein, in concentrations up to 300 microM, produced no change in the activity of the single Ca2+ channels. These results demonstrate that genistein inhibits the activity of the L-type Ca2+ channels in VSM cells, suggesting that the availability of the channels for voltage activation may be maintained through tonic tyrosine kinase phosphorylation.
Liu
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Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, OH 45267-0576, USA.
Sperelakis
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D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011169:Portal Vein / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D019833:Genistein; D011505:Protein-Tyrosine Kinases