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.