Ca²⁺ regulation in guinea‐pig colonic smooth muscle: the role of the Na⁺‐Ca²⁺ exchanger and the sarcoplasmic reticulum

Abstract

To study the contribution of the Na+‐Ca2+ exchanger to Ca2+ regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca2+]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 InsP3 (InsP3). Depletion of the SR of Ca2+ (verified by the absence of a response to caffeine and InsP3) by either ryanodine (50 μm), to open the ryanodine receptors (RyRs), or thapsigargin (500 nm) or cyclopiazonic acid (CPA, 10 μm), to inhibit the SR Ca2+ pumps, reduced neither the magnitude of the Ca2+ transient nor the relationship between the influx of and the rise in [Ca2+]c evoked by depolarisation. This suggested that Ca2+‐induced Ca2+ release (CICR) from the SR did not contribute significantly to the depolarisation‐evoked rise in [Ca2+]c. However, although Ca2+ 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 Ca2+ transient. Indeed, the SR Ca2+ content increased following depolarisation as assessed by the increased magnitude of the [Ca2+]c levels evoked each by InsP3 and caffeine, relative to controls. The increased SR Ca2+ content following depolarisation returned to control values in approximately 12 min via Na+‐Ca2+ exchanger activity. Thus inhibition of the Na+‐Ca2+ exchanger by removal of external Na+ (by either lithium or choline substitution) prevented the increased SR Ca2+ content from returning to control levels. On the other hand, the Na+‐Ca2+ exchanger did not appear to regulate bulk average Ca2+ directly since the rates of decline in [Ca2+]c, following either depolarisation or the release of Ca2+ from the SR (by either InsP3 or caffeine), were neither voltage nor Na+ dependent. Thus, no evidence for short term (seconds) control of [Ca2+]c by the Na+‐Ca2+ exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca2+ from the cytosol after its elevation by depolarisation. This Ca2+ is then removed from the SR to outside the cell by the Na+‐Ca2+ exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca2+]c following transient elevations in the ion produced either by depolarisation or by release from the store.

Ca2+ entry following P2X receptor activation induces IP3 receptor‐mediated Ca2+ release in myocytes from small renal arteries

“…Bradley et al, 2002;2004) demonstrating that complete depletion of the SR of Ca 2+ (including IP3-sensitive store) does not reduce [Ca 2+ ]i transients induced by step-like depolarization of the cell membrane. The latter results suggest that CICR is not recruited.…”

“…The ability of Ca 2+ entering the SMC via VGCCs to induce RyR-mediated Ca 2+ release has also been demonstrated in voltage-clamp experiments performed on SMCs from pregnant myometrium (Shmigol et al, 1998), ileum (Kohda et al, 1997), mesenteric artery (Bolton and Gordienko, 1998), cerebral arteries (Kamishima and McCarron, 1997) and portal vein (Coussin et al, 2000). However, other research groups working on different or even the same SMC type failed to detect any evidence of CICR (Fleischmann et al, 1996;Kamishima and McCarron, 1996;Bradley et al, 2002). The ability of Ca 2+ liberated via IP3Rs to trigger RyR-mediated Ca 2+ release also varies among different types of smooth muscles and depends on receptor distribution and isoform expression (reviewed in Wray and Burdyga, 2010 Two major sympathetic cotransmitters, noradrenaline and ATP, mediate vasoconstriction of small arteries (Wier et al, 2009) by acting on metabotropic a1-adrenoceptors and ionotropic P2X receptors respectively.…”

The Sarcoplasmic Reticulum, Ca2+Trapping, and Wave Mechanisms in Smooth Muscle

“…Each of these findings demonstrates that, in response to sarcolemma depolarization, Ca 2+ influx is essential for a rise in [Ca 2+ ] c and that channel activity or membrane potential changes themselves failed to alter [Ca 2+ ] c in the absence of influx. Moreover, Ca 2+ influx produced by depolarization of the sarcolemma is quantitatively adequate to account for the entire [Ca 2+ ] c increase, without SR involvement (3). At least 100 times more Ca 2+ enters the cell than appears as free Ca 2+ in the cytosol (3).…”

“…Not surprisingly, therefore, depletion of the SR Ca 2+ stores using the Ca 2+ pump inhibitors thapsigargin or cyclopiazonic acid or by ryanodine and caffeine to open RyR on the SR (Fig. 1B) does not reduce depolarization-evoked [Ca 2+ ] c increases (3). These results suggest that the SR does not contribute to the rise in [Ca 2+ ] c evoked by Ca 2+ influx via CICR, although controversy persists as to the precise contribution of CICR in smooth muscle.…”

“…Moreover, Ca 2+ influx produced by depolarization of the sarcolemma is quantitatively adequate to account for the entire [Ca 2+ ] c increase, without SR involvement (3). At least 100 times more Ca 2+ enters the cell than appears as free Ca 2+ in the cytosol (3). Not surprisingly, therefore, depletion of the SR Ca 2+ stores using the Ca 2+ pump inhibitors thapsigargin or cyclopiazonic acid or by ryanodine and caffeine to open RyR on the SR (Fig.…”