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.

Characteristics of Ca2+ release from stores were investigated in strips from ileum and portal vein and in isolated myocytes from ileum and urinary bladder of the guinea pig with use of caffeine and 9-methyl-7-bromoeudistomin D (MBED), a potent releaser of Ca2+ from skeletal muscle sarcoplasmic reticulum. In skinned strips, 1-30 mM caffeine elicited a transient contraction, but 10-300 microM MBED did not. Pretreatment with 100 microM MBED did not affect the subsequent caffeine-induced contraction. In single cells loaded with indo 1-acetoxymethyl ester, 10 mM caffeine increased cytoplasmic Ca2+ concentration, whereas 100 microM MBED elicited a small or no increase. Under whole cell clamp, spontaneous transient outward currents through Ca(2+)-dependent K+ (BK) channels were first enhanced and then suppressed by 30 microM MBED or 5 mM caffeine. The amplitude of Ca(2+)-dependent transient K+ current on depolarization was reduced by MBED and caffeine (50% inhibitory concentrations = 20 microM and 1 mM, respectively). Other currents and single BK channel activity were not significantly affected by MBED. The Ca2+ release from stores responsible for BK channel activation may be resolved from that for the activation of the contractile system by MBED in these smooth muscle cells.

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ABSTRACT-Ca2+ release by caffeine and 9-methyl-7-bromoeudistomin D (MBED) and the concomitant activation of large conductance Ca 2+-dependent K + (BK) channels were analyzed using confocal Ca 2+ imaging and whole cell voltage-clamp methods in guinea pig urinary bladder smooth muscle cells. Puff application of 3 or 10 mM caffeine for several seconds (2 -5 s) elicited a large increase in intracellular Ca]i) and induced a phasic outward current at a holding potential of -40 mV. The phasic outward current was the summation of spontaneous transient outward currents (STOCs) due to marked activation of BK channels and was followed by a short cessation of STOCs. ]i by MBED occurred mainly in superficial areas. Longer application of 100 mM MBED for 2 min did not induce significant global [Ca 2+ ]i increase but decreased the amount of Ca 2+ release and cell shortening during the subsequent application of 10 mM caffeine. These results indicate that short application of MBED releases Ca 2+ preferentially from superficial storage sites, presumably due to its slow approach to deeper sites. MBED may be a good pharmacological tool to manipulate selectively the superficial Ca 2+ stores related to STOCs.

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