The Influence of Sarcoplasmic Reticulum Ca2+ Concentration on Ca2+ Sparks and Spontaneous Transient Outward Currents in Single Smooth Muscle Cells

Localized, transient elevations in cytosolic Ca2+, known as Ca2+ sparks, caused by Ca2+ release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca2+-activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca2+ concentration within the sarcoplasmic reticulum and a Ca2+ spark and that between a Ca2+ spark and a STOC are not well defined or fully understood. To address these problems, we have employed two approaches using single patch-clamped smooth muscle cells freshly dissociated from toad stomach: a high speed, wide-field imaging system to simultaneously record Ca2+ sparks and STOCs, and a method to simultaneously measure free global Ca2+ concentration in the sarcoplasmic reticulum (Ca2+SR) and in the cytosol (Ca2+CYTO) along with STOCs. At a holding potential of 0 mV, cells displayed Ca2+ sparks and STOCs. Ca2+ sparks were associated with STOCs; the onset of the sparks coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in Ca2+CYTO at the time and location of the spark peak was approximately 100 nM above a resting concentration of approximately 100 nM. The frequency and amplitude of spontaneous Ca2+ sparks recorded at -80 mV were unchanged for a period of 10 min after removal of extracellular Ca2+ (nominally Ca2+-free solution with 50 microM EGTA), indicating that Ca2+ influx is not necessary for Ca2+sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in Ca2+SR in association with a surge in Ca2+CYTO and a fusion of STOCs, followed by a fast restoration of Ca2+CYTO and a gradual recovery of Ca2+SR and STOCs. The return of global Ca2+CYTO to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca2+. After the global Ca2+CYTO was fully restored, recovery of STOC frequency and amplitude were correlated with the level of Ca2+SR, even though the time for refilling varied greatly. STOC frequency did not recover substantially until the Ca2+SR was restored to 60% or more of resting levels. At Ca2+SR levels above 80% of rest, there was a steep relationship between Ca2+SR and STOC frequency. In contrast, the relationship between Ca2+SR and STOC amplitude was linear. The relationship between Ca2+SR and the frequency and amplitude was the same for Ca2+ sparks as it was for STOCs. The results of this study suggest that the regulation of Ca2+SR might provide one mechanism whereby agents could govern Ca2+ sparks and STOCs. The relationship between Ca2+ sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca2+ release site and the Ca2+-activated potassium channels responsible for a STOC.