Regulation of basal intracellular calcium concentration by the sarcoplasmic reticulum in myocytes from the rat gastric antrum

1. The intracellular calcium concentration ([Ca2+](i)) was monitored ih fura-2-loaded myocytes isolated from the rat gastric antrum and voltage clamped at -60 mV using the perforated patch clamp technique. The rate of quench of fura-2 fluorescence by Mn2+ was used as a measure of capacitative Ca2+ entry. 2. Cyclopiazonic acid (5 muM) did not affect the holding current but produced a sustained elevation in steady-state [Ca2+](i) that was dependent on the presence of external calcium. Cyclopiazonic acid increased Mn2+ influx with physiological external [Ca2+], but not in Ca2+-free conditions. Cyclopiazonic acid increased the rate of [Ca2+](i) rise following a rapid switch from Ca2+-free to physiological [Ca2+] solution. 3. Sustained application of carbachol (10 muM) produced an elevation in steady-state [Ca2+](i) that was associated with an increased rate of Mn2+ influx. application of cyclopiazonic acid in the presence of carbachol further elevated steady-state [Ca2+](i) without changing Mn2+ influx. 4. Ryanodine (10 muM) elevated steady-state [Ca2+](i) either on its own or following a brief application of caffeine (10 mM). Cyclopiazonic acid had no further effect when added to cells pre-treated with ryanodine. Neither caffeine nor ryanodine increased the rate of Mn2+ influx. When brief applications of ionomycin (25 muM) in Ca2+-free solution were used to release stored Ca2+, ryanodine reduced the amplitude of the resulting [Ca2+](i) transients by approximately 30%, indicating that intracellular stores were partially depleted. 5. These findings suggest that continual uptake of Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase into a ryanodine-sensitive store limits the bulk cytoplasmic [Ca2+](i) under resting conditions. This pathway can be short circuited by 10 muM ryanodine, presumably by opening Ca2+ channels in the sarcoplasmic reticulum. Depletion of stores with cyclopiazonic acid or carbachol also activates capacitative Ca-2+ entry.