Ca(2+) regulation in guinea-pig colonic smooth muscle: the role of the Na(+)-Ca(2+) exchanger and the sarcoplasmic reticulum

Abstract

To study the contribution of the Na(+)-Ca(2+) exchanger to Ca(2+) regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca(2+)](c) were evoked by either depolarisation (-70 mV to 0 mV) or by release from the SR by caffeine (10 mM) or flash photolysis of caged InsP(3) (InsP(3)). Depletion of the SR of Ca(2+) (verified by the absence of a response to caffeine and InsP(3)) by either ryanodine (50 microM), to open the ryanodine receptors (RyRs), or thapsigargin (500 nM) or cyclopiazonic acid (CPA, 10 microM), to inhibit the SR Ca(2+) pumps, reduced neither the magnitude of the Ca(2+) transient nor the relationship between the influx of and the rise in [Ca(2+)](c) evoked by depolarisation. This suggested that Ca(2+)-induced Ca(2+) release (CICR) from the SR did not contribute significantly to the depolarisation-evoked rise in [Ca(2+)](c). However, although Ca(2+) was not released from it, the SR accumulated the ion following depolarisation since ryanodine and thapsigargin each slowed the rate of decline of the depolarisation-evoked Ca(2+) transient. Indeed, the SR Ca(2+) content increased following depolarisation as assessed by the increased magnitude of the [Ca(2+)](c) levels evoked each by InsP(3) and caffeine, relative to controls. The increased SR Ca(2+) content following depolarisation returned to control values in approximately 12 min via Na(+)-Ca(2+) exchanger activity. Thus inhibition of the Na(+)-Ca(2+) exchanger by removal of external Na(+) (by either lithium or choline substitution) prevented the increased SR Ca(2+) content from returning to control levels. On the other hand, the Na(+)-Ca(2+) exchanger did not appear to regulate bulk average Ca(2+) directly since the rates of decline in [Ca(2+)](c), following either depolarisation or the release of Ca(2+) from the SR (by either InsP(3) or caffeine), were neither voltage nor Na(+) dependent. Thus, no evidence for short term (seconds) control of [Ca(2+)](c) by the Na(+)-Ca(2+) exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca(2+) from the cytosol after its elevation by depolarisation. This Ca(2+) is then removed from the SR to outside the cell by the Na(+)-Ca(2+) exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca(2+)](c) following transient elevations in the ion produced either by depolarisation or by release from the store.

Figure 1. Ryanodine-induced SR store Ca²⁺ depletion did not reduce the magnitude of depolarisation-evoked Ca²⁺ transients

Depolarisation (C) triggered an inward Ca²⁺ current (I_Ca; D) and a Ca²⁺ transient (A). Caffeine (10 mm, B; CAF) and InsP₃ (A, ↑) each produced Ca²⁺ transients (A). In the presence of ryanodine (50 μm), the responses to both caffeine (B) and InsP₃ (A) were abolished (A); the depolarisation-evoked Ca²⁺ transient (C) was not reduced (A). At the break in the record (∼3 min; A-C) several depolarisation-evoked Ca²⁺ transients in response to different voltages were investigated to match peak I_Ca amplitude in ryanodine to that of control (D and Ea and b). In Ea (control) and in Eb (in ryanodine, 50 μm) the time course of the ‘measured’ [Ca²⁺]_c increase (dashed line) followed that of the ‘calculated’ increase in [Ca²⁺]_c (dotted line^.; see Methods) over the period of the depolarisation. Depolarisation activated I_Ca (Ea, continuous line, control, −60 to +30 mV, and Eb, continuous line, in ryanodine, −70 mV to +15 mV; C and D). The amount of Ca²⁺ entering the cell, i.e. the ‘calculated’ increase in [Ca²⁺]_c, in both control (Ea, dotted line) and in ryanodine (Eb, dotted line) was sufficient (by 200-fold) to account for the ‘measured’ increase in [Ca²⁺]_c (control Ea, dotted line, and in ryanodine Eb, dashed line). Ryanodine had no significant effect on the ‘measured’ [Ca²⁺]_c (Eb, dashed line) but reduced the ‘calculated’ increase in [Ca²⁺]_c (Eb, dotted line). These results show that CICR did not contribute to the depolarisation-evoked increase in [Ca²⁺]_c. A-E are components of the same experiment. Note the expanded time axis in D.

Figure 2. Thapsigargin-induced SR store Ca²⁺ depletion did not reduce the magnitude of depolarisation-evoked Ca²⁺ transients

Caffeine (10 mm, B; CAF) and InsP₃ (A, ↑) each evoked Ca²⁺ transients (A). Depolarisation (−70 mV to 0 mV, C) triggered I_Ca (D, note the expanded time axis) and a Ca²⁺ transient (A). Thapsigargin (500 nm), an inhibitor of SERCA pumps on the SR, abolished Ca²⁺ transients evoked by InsP₃ (A) and caffeine (B, A) but not that evoked by depolarisation (which in some cells was increased) (A). In Ea (controls) and in Eb (thapsigargin) the time course of the ‘measured’ [Ca²⁺]_c increase (dashed line) closely followed that of the ‘calculated’ increase in [Ca²⁺]_c (dotted line) over the duration of the depolarisation. I_Ca (control, Ea, continuous line; in thapsigargin, Eb, continuous line) was triggered by a depolarisation (−70 mV to 0 mV; C and D). The ‘calculated’ increase in [Ca²⁺]_c was determined (during the first 200 ms of the depolarising pulse) from an integral of I_Ca. The amount of Ca²⁺ entering the cell, i.e. the ‘calculated’ increase in [Ca²⁺]_c in control (Ea, dotted line) and in thapsigargin (Eb, dotted line) was more than adequate (by 200-fold) to account for the ‘measured’ rise in [Ca²⁺]_c (control, Ea, dotted line; and in thapsigargin, Eb, dashed line measured at 10 Hz). These results confirm that CICR does not contribute to the depolarisation-evoked increase in [Ca²⁺]_c. A-E are components of the same experiment.

Figure 3. Ryanodine and thapsigargin each slowed the rate of decline of depolarisation-evoked Ca²⁺ transients

Ryanodine (50 μm) slowed the rate of decline of depolarisation-evoked Ca²⁺ transients at the 95 % level (* P < 0.01) without significantly decreasing the amplitude (Aa). The mean percentage decay rates from 0 to 95 % for [Ca²⁺]_c in the absence and presence of ryanodine are shown in Ab (n = 21). Inhibition of the SR store uptake by thapsigargin (500 nm) also slowed the decline of the depolarisation-evoked Ca²⁺ transient without decreasing its amplitude significantly (Ba). Thapsigargin was more effective than ryanodine especially at higher [Ca²⁺]_c. The mean perecntage decay rates from 0 to 95 % for [Ca²⁺]_c in the absence and presence of thapsigargin are shown in Bb (n = 5). The decline was slowed significantly from the 70 to the 95 % levels by thapsigargin (* P < 0.05). These results suggest that the SR removes Ca²⁺ from the cytosol following a depolarisation-evoked increase, particularly at lower Ca²⁺ concentrations. •, control; ○, drug treated.

Figure 4. Caffeine-evoked (A) and InsP₃-evoked (B) Ca²⁺ transient amplitudes were each enhanced by prior depolarisation

Caffeine (10 mm, CAF, Ad) and InsP₃ (Bb, ↑) each evoked approximately reproducible Ca²⁺ transients (Ab and Bb; Ca²⁺ measurements were made at 10 Hz.). Depolarisation (3 s, −70 mV to 0 mV, Ac, or 3 s, −60 to +10 mV, Bc) increased [Ca²⁺]_c (Ab and Bb). Following a depolarisation-evoked rise in [Ca²⁺]_c the caffeine-evoked Ca²⁺ transient was increased to 174 % of that prior to depolarisation (Ab) and the InsP₃-evoked Ca²⁺ transient to 145 % of that before depolarisation (Bb). Aa is a summary of the significant increase (* P < 0.01) in [Ca²⁺]_c evoked by caffeine before and after depolarisation (n = 24) and (Ba) of that (* P < 0.05) evoked by InsP₃ before and after depolarisation (n = 6). ‘\\’ indicates the time during which depolarisation-induced increases in [Ca²⁺]_c occurred in both cases. The fluorescence ratio reached 2.9 ± 0.2 F/F₀ units above baseline in Aa and 2.7 ± 0.6 F/F₀ units above baseline in Ba and have been omitted from the figure to enable the caffeine and InsP₃ responses to be followed more easily.

Figure 5. Restoration of caffeine-evoked Ca²⁺ transients, following depolarisation, was dependent on the Na⁺-Ca²⁺ exchanger

Caffeine (10 mm; Ac, Bc and Cc, CAF) evoked approximately reproducible Ca²⁺ transients (Ab, Bb and Cb; Ca²⁺ measurements were made at 10 Hz). Depolarisation (−70 mV to +10 mV; Ad, Bd and Cd) also increased [Ca²⁺]_c (Ab, Bb and Cb). Following depolarisation (45 s), the SR store had accumulated Ca²⁺, as indicated by the increased amplitude of the caffeine-evoked Ca²⁺ transient (Ab) and the latter had returned close to control levels 12 min after the depolarisation (Bb). However, when external Na⁺ was replaced by Li⁺, so inhibiting forward mode activity of the Na⁺-Ca²⁺ exchanger, the caffeine-evoked Ca²⁺ transient did not return to control levels within the 12 min period (Cb). Aa, Ba and Ca show summarised results of the changes in the fluorescence ratio (F/F₀) above baseline produced by caffeine before and after depolarisation (perpendicular dotted line; all experiments were paired, n = 9; * P < 0.05). Clearly the SR Ca²⁺ content, as indicated by the Ca²⁺ transients evoked by caffeine, remains increased following depolarisation only when the Na⁺-Ca²⁺ exchanger is inhibited, thus preventing the removal of Ca²⁺ from the cell suggesting that the Na⁺-Ca²⁺ exchanger and the SR are involved in Ca²⁺ removal. Ab-d, Bb-d and Cb-d show results from three different cells.

Figure 6. The decline in [Ca²⁺]_c following depolarisation was independent of both membrane voltage and extracellular Na⁺

A, depolarisation from −70 mV to 0 mV (Ab) initially increased [Ca²⁺]_c (measured using fura-2); this returned to resting levels on repolarisation (Aa). Clamping close to E_Ca (+130 mV, Ab) to inhibit exchanger forward mode did not change the rate of decline of the Ca²⁺ transient (Aa). The absence of a Ca²⁺ transient during a control pulse to +130 mV (included in each experiment) confirmed that E_Ca had been reached (Ab). Summarised data (Ac) showed no significant differences between the rates of decline of controls and when membrane voltage was clamped at +130 mV (n = 5) demonstrating that Ca²⁺ removal was voltage independent. ▪, control; ○, at +130 mV. Ba shows records of Ca²⁺ transients, evoked by depolarisation (−70 mV to 0 mV, Bb). Inhibition of exchanger forward mode activity by replacing external Na⁺ with Li⁺ (Na⁺ free) failed to alter the rate of decline of [Ca²⁺]_c (Ba). The mean rates of decline (n = 5, Bc; ▪, control; ○, Na⁺ free) of [Ca²⁺]_c were unchanged following substitution, indicating that the removal of Ca²⁺ was Na⁺ independent. C, depolarisation (−70 mV to 0 mV, C) raised [Ca²⁺]_c (Ca) (fluorescence trace has been smoothed using a sliding boxcar average of three data points). Comparison of the measured current (Cb) with the Na⁺-Ca²⁺ exchanger current (I_Na-Ca) predicted for stoichiometric exchange of 3 Na⁺ ions for 1 Ca²⁺ ion (Cb, dotted line), indicated the absence of the latter which confirmed that the exchanger was not involved in removal of Ca²⁺ from the cytosol following depolarisation. The change in the level of noise in the current recording occurred because of a change in sampling from 1.5 kHz to 500 Hz.

Figure 7. The rate of decline of the Ca²⁺ transient following Ca²⁺ release from the SR store was Na⁺ independent

The rate of decline of the Ca²⁺ transients (measured using fluo-3; at 10 Hz) evoked by InsP₃ (Aa, ↑) or by caffeine (10 mm, •, Ba) were unaffected by replacement of external Na⁺ by Li⁺ (Na⁺ free). The data for InsP₃ and caffeine in the presence and absence of Na⁺ are summarised in Ac, n = 10 and Bc, n = 4–6 respectively. The rate of decline of Ca²⁺ transients evoked by depolarisation (Cb, −70 mV to 0 mV (Ca) and the corresponding summarised data (Cc, n = 6) also were unaffected by external Na⁺ substitution. ▴, control; ▵, Na⁺ free.

Figure 8. The rate of decline of Ca²⁺ following release from the SR was voltage independent

The rates of decline of the Ca²⁺ transients (measured using fluo-3; at 10 Hz) evoked by InsP₃ (A, ↑) or depolarisation (from −70 mV to 0 mV; C) were unaffected when membrane voltage was clamped at +130 mV. B and D show summarised data (n = 4 and n = 3, respectively, control and at +130 mV). Inhibition of the exchanger by clamping at +130 mV did not affect the rate of removal of [Ca²⁺]_c following Ca²⁺ influx or release from the SR. •, control (−70 mV); ○, +130 mV.