Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive [Ca.sup.2+] channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for [Ca.sup.2+] influx and determining the bulk average cytoplasmic Ca concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to [Ca.sup.2+] signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (-70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent [Ca.sup.2+] current ([I.sub.Ca]) and evoked a rise in [[Ca.sup.2+]] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [[Ca.sup.2+]] increase ([[[Ca.sup.2+]].sub.c]) was approximately uniform, whereas that of the subplasma membrane space ([[[Ca.sup.2+]].sub.PM]) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active [Ca.sup.2+] channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive [Ca.sup.2+] channels were not normally constitutively active. The repetitive localized [[[Ca.sup.2+]].sub.PM] rises ('persistent [Ca.sup.2+] sparklets') that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active [Ca.sup.2+] channels regulate the bulk average [[[Ca.sup.2+]].sub.c], A dihydropyridine blocker of [Ca.sup.2+] channels, nimodipine, which blocked [I.sub.Ca] and accompanying [[[Ca.sup.2+]].sub.c] rise, reduced neither the resting bulk average [[[Ca.sup.2+]].sub.c] (at -70 mV) nor the rise in [[[Ca.sup.2+]].sub.c], which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent [Ca.sup.2+] channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [[[Ca.sup.2+]].sub.c] regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [[Ca.sup.2+]].