1. Voltage-clamped isolated smooth muscle cells from guinea-pig urinary bladder were studied with 3.6 mm extracellular Ca2+ at 36-degrees-C. The fluorescence of the Ca2+-sensitive dye Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transient). Fast application of caffeine (10 mM) to the cell was used to release the intracellular Ca2+ from a 'caffeine-sensitive Ca2+ store'. 2. At the holding potential -60 mV, a short (1 s) caffeine application increased [Ca2+]i within less than 1 s from the resting 118 +/- 22 nm to 1490 +/- 332 nm. Following the caffeine wash-out, [Ca2+]i fell from this peak to a subresting level of 47 +/- 12 nm, i.e. an 'undershoot' of [Ca2+]i occurred. Subsequent caffeine-induced [Ca2+]i transients had attenuated peaks suggesting that the caffeine-sensitive Ca2+ store had lost a part of the releasable Ca2+. 3. In the continuous presence of caffeine, [Ca2+]i decayed from its peak to control resting [Ca2+]i values. The wash-out of caffeine following prolonged (10-30 s) treatment also resulted in [Ca2+]i undershoot. Subsequent caffeine-induced [Ca2+]i transients were largely abolished as if the caffeine-sensitive Ca2+ store had lost a large part of releasable Ca2+. During the undershoot, hyperpolarization to -100 mV did not affect [Ca2+]i. In most cells studied, recovery of [Ca2+]i from the undershoot to the resting level required depolarizations inducing Ca2+ influx through L-type Ca2+ channels. 4. Block of plasmalemmal Ca2+-ATPase (PMCa) with extracellular La3+ (3 mm) did not modify the decay of the [Ca2+]i transients induced by depolarization or by a 1 s caffeine application suggesting that decay rate of both is not limited by PMCa rate. La3+ abolished the undershoot of [Ca2+]i. In the continuous presence of caffeine, La3+ largely prevented the decay of [Ca2+]i. 5. When the depolarizing steps from -60 to 0 mV (160 ms duration) were applied during the period of [Ca2+]i undershoot, the half-time of decay of the corresponding [Ca 2+]i transients was up to three times faster than in control. Repetitive depolarizations restored the rate of decay and [Ca2+]i recovered to the resting value. Both processes recovered along a similar time course. 6. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 0.1 mm) or of 8-Br-cAMP (0.1 mm) did not mimic the above caffeine effects suggesting that stimulation of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCa) by cAMP-dependent phosphorylation is not the underlying mechanism. A Ca2+, calmodulin-dependent phosphorylation is also unlikely to play a role since the decay rate of the depolarization-induced [Ca2+]i transients increased at reduced [Ca2+]i. 7. It is suggested that the undershoot of [Ca2+]i is due to a reduction of the Ca2+ content in the cell due to the plasmalemmal Ca2+-ATPase that extrudes part of the Ca2+ released by caffeine. The concomitant reduction of [Ca2+] in the SR is thought to stimulate the rate of the SR Ca2+-ATPase and therefore accelerate the decay of the depolarization-induced [Ca2+]i transient.
