Ca2+ uptake by the sarcoplasmic reticulum decreases the amplitude of depolarization-dependent [Ca2+]i transients in rat gastric myocytes

Abstract

Gastric myocytes loaded with fura-2 were voltage-clamped at -60 mV. Depolarizations to 0 mV evoked nifedipine-sensitive (5 microM) inward currents and Ca2+ transients. Cyclopiazonic acid (5 microM) elevated steady-state [Ca2+]i and reduced Ca current (ICa), but when divalent cations were omitted from the extracellular solution, cyclopiazonic acid had no effect on either the amplitude or the current-voltage relationship of the nifedipine-sensitive current. This suggests that the reduction in ICa was caused by the rise in steady-state [Ca2+]i. The relationship between the total Ca2+ influx carried by the Ca2+ current (sigmaI(Ca).dt) and the amplitude of the Ca2+ transient (delta[Ca2+]i) was analysed for experiments using physiological Ca2+ solutions by calculating the ratio delta[Ca2+]i/sigmaI(Ca).dt. Cyclopiazonic acid (5 microM) and ryanodine (10 microM) both increased this ratio, indicating a decrease in the buffering power of the cell. Mimicking the increase in steady-state [Ca2+]i produced by these agents by changing the holding potential to -40 mV, however, did not affect delta[Ca2+]i/sigmaI(Ca).dt. It was concluded that up-take by a ryanodine-sensitive store normally limits Ca2+ distribution to the bulk cytoplasm following entry to the cell through dihydropyridine-sensitive channels.

Ryanodine receptors in smooth muscle

“…If anything, it increases the elevation in [Ca 2+ ] i (82). Furthermore, the application of ryanodine to rat gastric myocytes increases the efficiency of VDCCs to elevate [Ca 2+ ] i (76). The fact that Ca 2+ influx through VDCCs is able to increase global [Ca 2+ ] i before triggering Ca 2+ sparks (59,81) and that ryanodine does not change the initial Ca 2+ buffering capacity (78) suggest that RyRs from smooth muscle are insensitive to Ca 2+ influx through VDCCs, even when the activation of VDCCs generates a strong increase in the subsarcolemmal [Ca 2+ ] (85).…”

“…However, these studies did not show the extent of CICR contribution to the depolarization-induced [Ca 2+ ] i transient. In addition, it appears that CICR is not universally present in smooth muscle cells (67,(74)(75)(76). To further complicate this picture, there are cases where CICR is evident only for the first depolarization pulse (77) or the first train of voltage pulses (68).…”

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes

“…Global [Ca 2ϩ ]c was measured by exciting fura 2 alternately at 340 and 380 nm and recording the ratio (R) of the emitted fluorescence (filtered at 510 nm) with a microfluorimeter (Photon Technology International). [Ca 2ϩ ]c was calculated from R by applying the equations of Grynkiewicz et al (11) with experimentally determined values of maximum R (R max), minimum R (Rmin), ␤ (ratio of maximum-to-minimum fluorescence excited at 380 nm) and a K d of 287 nM (35). Local changes in [Ca 2ϩ ] were imaged in fluo 4-loaded cells with a confocal scanning laser microscope (Bio-Rad, MR-A1) used in line-scan mode at a rate of 500 scans/s.…”

“…For experiments that required intracellular application of heparin or recording of spontaneous ionic currents, cells were voltage clamped with the whole cell patch-clamp technique as previously described in detail elsewhere (35). Changes in STOC activity were recorded at a holding potential of Ϫ40 mV with consecutive 1-s recording sweeps and a data sampling rate of 2 kHz.…”

Sarcoplasmic Reticulum Function in Smooth Muscle

“…Because of the difficulty of directly measuring [Ca] in microdomains, direct experimental support for them was lacking. However, with the advent of near-membrane Ca indicators (170, 809), evidence supporting or consistent with the superficial buffer barrier role of the SR in smooth muscles has been provided in a variety of preparations [vascular (587,713), gastric (754), bladder (789), uterus (631,793)]. The sites on the SR of Ca release and uptake during these processes may be functionally and spatially separate (635).…”