Ca2+ images and K+ current during depolarization in smooth muscle cells of the guinea-pig vas deferens and urinary bladder

摘要

class="enumerated" style="list-style-type:decimal">Electrical events and intracellular calcium concentration ([Ca²⁺]) imaged using fluo-3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea-pig vas deferens or urinary bladder.Images obtained every 8 ms, during stepping from -60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca²⁺] could be detected within 20 ms of depolarization in five to twenty small (< 2 μm diameter) ‘hot spots’, over 95 % of which were located within 1.5 μm of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places.Cd²⁺ or verapamil abolished both hot spots and Ca²⁺-activated K⁺ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca²⁺], decreased the rise in hot spot [Ca²⁺] and IK,Ca amplitude during depolarization. These results suggest that Ca²⁺ entry caused Ca²⁺-induced Ca²⁺ release (CICR).Under voltage clamp, hot spot [Ca²⁺] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca²⁺] over the whole cell area was much slower. Step depolarization to potentials positive to -20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca²⁺] and contraction. Ca²⁺ hot spots also occurred during the up-stroke of an evoked action potential under current clamp.It is concluded that the entry of Ca²⁺ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca²⁺ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca²⁺-dependent K⁺ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.