Caffeine‐induced release and reuptake of Ca2+ by Ca2+ stores in myocytes from guinea‐pig urinary bladder.

Abstract

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 Ca(2+)‐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 Ca(2+)‐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 [Ca2+]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) Ca(2+)‐ATPase (SERCa) by cAMP‐dependent phosphorylation is not the underlying mechanism.(ABSTRACT TRUNCATED AT 400 WORDS) © 1992 The Physiological Society