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

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.

Figure 1. Calculation of [Ca²⁺]-insensitive fluorescence (F_i) from fluorescence signals

A, recorded fluorescence output at 340 nm (F₃₄₀) and 380 nm (F₃₈₀) for a single cell which was depolarized from −60 to 0 mV for 1 s. The resulting inward Ca²⁺ current (not shown) raised [Ca²⁺]_i, producing divergent changes in fluorescence output. F_i was calculated from the recorded fluorescence using eqn (1) (see text). B, F₃₄₀ plotted against F₃₈₀ for each data point over the 10 s period immediately following repolarization. The gradient of the linear fit gives the value for α in eqn (1). C, changes in F_i for a single cell voltage clamped at −60 mV. In the absence of external Mn²⁺ there was a slow, linear decline. The rate of decay in F_i increased when external Mn²⁺ was added due to quenching of intracellular fura-2. D, the effects of Mn²⁺ influx can be isolated by correcting F_i to eliminate the Mn²⁺ independent, baseline decay.

Figure 2. Effect of cyclopiazonic acid on basal [Ca²⁺]_i and Mn²⁺ influx

All records were made from gastric myocytes clamped at −60 mV. A, current (upper trace) and [Ca²⁺]_i (lower trace) records under control conditions. Addition of cyclopiazonic acid (CPA) to the superfusate produced a sustained elevation in [Ca²⁺]_i without any obvious change in holding current. B, cyclopiazonic acid did not affect [Ca²⁺]_i when applied after approximately 100 s exposure to zero external Ca²⁺. C, changes in the calculated [Ca²⁺]-independent fluorescence (F_i) in the presence of normal external Ca²⁺. Externally applied Mn²⁺caused a linear decline in fluorescence (the dashed line shows an extrapolated linear fit to these data). Addition of cyclopiazonic acid further increased the rate of decay.

Figure 3. Effects of cyclopiazonic acid on responses to step changes in external [Ca²⁺]

A, tension records from a strip of stomach muscle permeabilized to Ca²⁺ using the ionophore A23187 (present in all solutions at 0.1 μM with 0.001 % DMSO vehicle). External [Ca²⁺] was stepped from 0 to 70 nM, and the resulting increase in tension was measured before (left trace) and during (right trace) perfusion of the organ bath with cyclopiazonic acid (1 μM). B, [Ca²⁺]_i record from an intact, single gastric myocyte voltage clamped at −60 mV. The external [Ca²⁺] was stepped from zero to 1.8 mM (top line). This was repeated in the presence of cyclopiazonic acid. C, F_i plotted for a cell voltage clamped at −60 mV and superfused with a series of Ca²⁺-free solutions. External Mn²⁺ produced a linear decay in fluorescence which was extrapolated using a linear fit (dashed line). Addition of cyclopiazonic acid slightly reduced the decay rate in the absence of external Ca²⁺.

Figure 4. Effects of cyclopiazonic acid on cells pretreated with carbachol

A, current (upper record) and [Ca²⁺]_i (lower record) for a voltage clamped cell exposed first to carbachol (10 μM) and then to cyclopiazonic acid (5 μM) in the maintained presence of carbachol. B, F_i plotted for another cell clamped at −60 mV and superfused with a series of solutions containing physiological [Ca²⁺]. External Mn²⁺ produced a linear decay in fluorescence which was extrapolated using a linear data fit (dashed line). Carbachol increased the rate of this decay but addition of cyclopiazonic acid in the presence of carbachol had no further effect.

Figure 5. Effects of cyclopiazonic acid on cells pretreated with ryanodine

A, current (upper record) and [Ca²⁺]_i (lower record) for a voltage clamped cell (−60 mV) exposed to ryanodine. Brief (< 10 s) applications of caffeine (10 mM) were used to confirm successful blockade of the ryanodine-sensitive channels. Ryanodine alone raised [Ca²⁺]_i but subsequent application of cyclopiazonic acid produced no additional effect. B, records for a second cell exposed to ryanodine and caffeine in which ryanodine produced a sustained rise in [Ca²⁺]_i but only after a brief application of caffeine. Once again, cyclopiazonic acid produced no further effect. C, F_i plotted for a cell voltage clamped at −60 mV in normal [Ca²⁺] solutions. External Mn²⁺ produced a linear decay in fluorescence (dashed line), but this was unaffected by addition of ryanodine alone or in combination with cyclopiazonic acid.

Figure 6. Effect of ryanodine on ionomycin releasable Ca²⁺ stores

A, [Ca²⁺]_i record from a control experiment. A test response to caffeine (10 mM) was followed by a recovery period in normal solution. This was followed by a rapid switch to Ca²⁺-free superfusate containing 25 μM ionomycin (iono; application time < 10 s) which was also washed out in Ca²⁺-free conditions. About 100 s after returning to normal Ca²⁺ solution, a second application of caffeine was followed by another switch to ionomycin in zero Ca²⁺. This produced no measurable rise in [Ca²⁺]_i suggesting that ionomycin and caffeine were acting on the same intracellular Ca²⁺ stores. B, [Ca²⁺]_i transients evoked by ionomycin in zero-Ca²⁺ solution before and during superfusion with ryanodine. Brief application of caffeine in the presence of ryanodine evoked a transient followed by sustained rise in [Ca²⁺]_i.