Buffering of plasmalemmal Ca2+ current by sarcoplasmic reticulum of guinea pig urinary bladder myocytes
Yoshikawa, A.; van Breemen, C.; Isenberg, G.
American Journal of Physiology 271(3 Pt 1): C833-C841
1996
ISSN/ISBN: 0002-9513 PMID: 8843713 Document Number: 462628
The effects of cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca-2+-ATPase (SERCA), on cytosolic Ca-2+ concentration ((Ca-2+)-c) and membrane currents were studied in isolated urinary bladder myocytes to test the hypothesis that the sarcoplasmic reticulum (SR) buffers Ca-2+, 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 (Ca-2+)-c transients: 1) CPA prolonged the 90% decay time from peak to resting (Ca-2+)-c from 2.2 +- 0.3 to 8.3 +- 0.92 s (n = 5), 2) CPA abolished the "undershoot" of the (Ca-2+)-c transient that follows the washout of caffeine, and 3) CPA prevented caffeine from inducing a second (Ca-2+)-c transient. CPA reversibly increased resting (Ca-2+)-c. Starting from a control (Ca-2+)-c of 137 +- 10 nM, 19 of 24 cells responded with a monotonic increase in (Ca-2+)-c to a steady (Ca-2+)-c of 238 +- 10 nM, whereas 5 of 24 cells responded with a transient rise of (Ca-2+)-c to 472 nM (within 2.8 +- 0.5 s) followed by a decay to a steady (Ca-2+)-c of 161 +- 10 nM. The CPA-mediated rise in (Ca-2+)-c was augmented by increasing extracellular Ca-2+ concentration ((Ca-2+)-o), suggesting a "leakage pathway" for Ca-2+ influx that is unmasked by SERCA blockade. CPA reduced (Ca-2+)-c transients and Ca-2+-activated K+ currents (I-K,Ca), induced by depolarizing clamp steps from -60 to 0 mV, compatible with suppression of SR Ca-2+ release on depletion of SR Ca-2+. To reduce the contribution due to Ca-2+-induced Ca-2+ 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 (Ca-2+)-c and I-K,Ca signals. If spontaneous transient outward currents were present, they were suppressed by CPA. CPA reduced the peak L-type Ca-2+ channel current apparently through increased Ca-2+ inactivation of voltage-gated Ca-2+ channels. The current could be restored to control by elevating (Ca-2+)-o from 2.5 to 5 mM. Under these conditions, CPA increased the ramp-induced (Ca-2+)-c transients from 105.1 +- 22 to 162.0 +- 31 nM (n = 9, P lt 0.05). These results suggest that Ca-2+ sequestration by the SR can buffer part of the Ca-2+ influx during slow depolarizations.