Buffering of plasmalemmal Ca2+ current by sarcoplasmic reticulum of guinea pig urinary bladder myocytes

Abstract

The effects of cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), on cytosolic Ca2+ concentration ([Ca2+]c) and membrane currents were studied in isolated urinary bladder myocytes to test the hypothesis that the sarcoplasmic reticulum (SR) buffers Ca2+, which enters the myocyte at a slow to moderate rate. Inhibition of SERCA by CPA was demonstrated by the following modifications of the caffeine-induced [Ca2+]c transients: 1) CPA prolonged the 90% decay time from peak to resting [Ca2+]c from 2.2 +/- 0.3 to 8.3 +/- 0.92 s (n = 5), 2) CPA abolished the "undershoot" of the [Ca2+]c transient that follows the washout of caffeine, and 3) CPA prevented caffeine from inducing a second [Ca2+]c transient. CPA reversibly increased resting [Ca2+]c. Starting from a control [Ca2+]c of 137 +/- 10 nM, 19 of 24 cells responded with a monotonic increase in [Ca2+]c to a steady [Ca2+]c of 238 +/- 10 nM, whereas 5 of 24 cells responded with a transient rise of [Ca2+]c to 472 nM (within 2.8 +/- 0.5 s) followed by a decay to a steady [Ca2+]c of 161 +/- 10 nM. The CPA-mediated rise in [Ca2+]c was augmented by increasing extracellular Ca2+ concentration ([Ca2+]o), suggesting a "leakage pathway" for Ca2+ influx that is unmasked by SERCA blockade. CPA reduced [Ca2+]c transients and Ca(2+)-activated K+ currents (IK,Ca), induced by depolarizing clamp steps from -60 to 0 mV, compatible with suppression of SR Ca2+ release on depletion of SR Ca2+. To reduce the contribution due to Ca(2+)-induced Ca2+ release, the cells were depolarized with a slow ramplike command (-60 to 0 mV, 15 mV/s). In 12 of these 40 cells, CPA increased [Ca2+]c and IK,Ca signals. If spontaneous transient outward currents were present, they were suppressed by CPA. CPA reduced the peak L-type Ca2+ channel current apparently through increased Ca2+ inactivation of voltage-gated Ca2+ channels. The current could be restored to control by elevating [Ca2+]o from 2.5 to 5 mM. Under these conditions, CPA increased the ramp-induced [Ca2+]c transients from 105.1 +/- 22 to 162.0 +/- 31 nM (n = 9, P < 0.05). These results suggest that Ca2+ sequestration by the SR can buffer part of the Ca2+ influx during slow depolarizations.

Modulation of Ca2+‐activated Cl− currents in rabbit portal vein smooth muscle by an inhibitor of mitochondrial Ca2+ uptake

“…Although CPA and thapsigargin have been shown to increase [Ca¥]é in various smooth muscle cells (Bar o & Eisner, 1992;Sims, Jiao & Zheng, 1996;Yoshikawa, van Breeman & Isenberg, 1996), the data of the present study suggest that any possible increase in [Ca¥]é was insufficient to activate ICl(Ca) in rabbit portal vein smooth muscle cells. Furthermore, in comparison with the data of Sims et al (1996) and Yoshikawa et al (1996) who showed that 10 ìÒ CPA slowed the rate of recovery of caffeine-evoked increases in global [Ca¥], the present data suggest that inhibition of Ca¥ uptake into the SR does not affect subsarcolemmal [Ca¥] sufficiently to modulate the time course of ICl(Ca). In the present study CCCP rapidly abolished STICs that were activated by Ca¥ released from the SR (Wang et al 1992), which suggests that CCCP depleted the Ca¥ content of the SR.…”

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

“…The amount of stored Ca¥ may be substantially reduced by these compounds. The abolition of Ca¥ hot spots cannot be explained simply by the decrease in Ca¥ influx by Ca¥-dependent inactivation of Ca¥ channels (for example, •30% decrease by CPA; Yoshikawa et al 1996). In the present study, substantial ICa was recorded in the presence of these compounds.…”

“…It is possible that Ca¥ influx through voltage-dependent Ca¥ channels near the hot spot results in the accumulation of Ca¥ in the narrow space between superficial SR and plasma membrane. This may therefore suggest that CICR is not involved in the formation of a hot spot and that the superficial SR simply acts as a barrier to Ca¥ diffusion (Van Breemen, Chen & Laher, 1995;Yoshikawa, Van Breemen & Isenberg, 1996). This is, however, very unlikely since [Ca¥] in a hot spot during depolarization to −20 mV reached a maximum within 25 ms and thereafter declined during depolarization.…”

Functionally Separate Intracellular Ca2+ Stores in Smooth Muscle

“…However, the relationship between SR Ca 2ϩ pumps on the one hand and the Ins(1,4,5)P 3 R and RyR on the other could not be presently differentiated by the use of Ca 2ϩ pump inhibitors. SR Ca 2ϩ pumps presumably on both the common Ins(1,4,5)P 3 R/RyR store and the RyR-only store were each inhibited by thapsigargin and CPA (see also 45,46). Differences in the sensitivity of the Ca 2ϩ pumps on the internal SR Ca 2ϩ store to the inhibitors (47,48), which may also vary among different tissues, were presumably responsible for these observations.…”