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

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Abstract
The intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded myocytes isolated from the rat gastric antrum and voltage clamped at -60 1r1rqmV1qusing the perforated patch clamp technique. The rate of quench of fura-2 fluorescence by Mn2+ was used as a measure of capacitative Ca2+ entry. Cyclopiazonic acid (5 microM) 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. Sustained application of carbachol (10 microM) 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. Ryanodine (10 microM) elevated steady-state [Ca2+]i either on its own or following a brief application of caffeine (10 9i1s1sqmMc1q). 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 microM) 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. 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 microM ryanodine, presumably by opening Ca2+ channels in the sarcoplasmic reticulum. Depletion of stores with cyclopiazonic acid or carbachol also activates capacitative Ca2+ entry.