Isoproterenol modulates the calcium channels through two different mechanisms in smooth-muscle cells from rabbit portal vein

Previous data from our laboratory indicated that the slow Ca²⁺ channel of vascular smooth muscle cells was regulated by cyclic nucleotides. In the present study, the effects of isoproterenol (ISO) on L-type calcium current (I_Ca(L)) were investigated in freshly-isolated single smooth-muscle cells from the rabbit portal vein using the whole-cell voltage-clamp technique. With high-Cs⁺ solution in the pipette and physiolocial salt solution (containing 2.0 mM Ca²⁺) in the bath, (I_Ca(L)) was recorded. At a holding potential of –80 mV, low concentrations of ISO ( 100 nM) increased I_Ca, whereas higher concentrations (1–100 M) transiently increased I_Ca but then inhibited it persistently. At 10 M ISO, I_Ca was initially increased by 44±9%, and was subsequently decreased by 24±3%. Pretreatment of cells with 30 M H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride] caused the first phase to persist and the second inhibitory phase to disappear. Intracellular application of 1 mM GDP[S] (guanosine 5-O-2-thiodiphosphate) abolished both phases of ISO action. In contrast, intracellular application of 100 M GTP caused the initial stimulatory phase of ISO action to be significantly potentiated; the later inhibitory phase was slightly diminished. In addition, the activated G protein subunit (G_s) mimicked the stimulatory effect of ISO. Pertussis toxin had no effect on either phase of the ISO action. These results suggest that ISO modulates the Ca²⁺ channel through mechanisms that involve the pertussis-toxin-insensitive G protein(s). That H-7, a nonspecific inhibitor of protein kinases, blocked the second phase but not the first phase indicates that the actions of ISO are mediated via two different pathways. One pathway (for inhibition) is more indirect, and may be mediated by the adenylate cyclase/cAMP/protein-kinase-A cascade. The other pathway (for stimulation) is more direct, and may reflect a type of G protein gating of the Ca²⁺ channel.