Receptor-activated Ca influx in human airway smooth muscle: use of Ca imaging and perforated patch-clamp techniques

Previous studies have demonstrated a dihydropyridine-insensitive, receptor-activated calcium influx pathway in cultured human airway smooth muscle (ASM) cells. To further define the biophysical characteristics of this pathway, the relationship between membrane potential and cytosolic free calcium ([Ca2+]i) was studied with the combined methods of the patch-clamp technique and single cell calcium imaging. The nystatin perforated-patch method was used to maintain normal intracellular calcium buffering and receptor-activated signal transduction processes in voltage-clamped cells. Single voltage-clamped human ASM cells responded to exposure to histamine (200 microM) with an initial transient rise in [Ca2+]i followed by a secondary sustained elevation that was dependent on extracellular calcium. Before agonist activation, step changes in holding potential produced only slight changes in [Ca2+]i, whereas, after activation, cells developed a sustained rise in [Ca2+]i that showed a large variation as a function of membrane potential. Depolarization from -80 to 0 mV caused a fall in the steady-state [Ca2+]i to basal levels or slightly below. Repolarization to -80 mV caused the redevelopment of the sustained phase of the calcium response. When calcium was removed from the extracellular fluid by the addition of a stoichiometric excess of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the voltage dependence of the sustained phase was abolished. In a series of experiments, agonist addition evoked a 54-fold increase in the voltage dependence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)