Functionally separate intracellular Ca2+ stores in smooth muscle

In smooth muscle, release via the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) controls oscillatory and steady-state cytosolic Ca2+ concentrations ([Ca2+](c)). The interplay between the two receptors, itself determined by their organization on the SR, establishes the time course and spatial arrangement of the Ca2+ signal. Whether or not the receptors are co-localized or distanced from each other on the same store or whether they exist on separate stores will significantly affect the Ca2+ signal produced by the SR. To date these matters remain unresolved. The functional arrangement of the RyR and Ins(1,4,5)P3R on the SR has now been examined in isolated single voltage-clamped colonic myocytes. Depletion of the ryanodine-sensitive store, by repeated application of caffeine, in the presence of ryanodine, abolished the response to Ins(1,4,5)P-3, suggesting that Ins(1,4,5)P3R and RyR share a common Ca2+ store. Ca2+ release from the Ins(1,4,5)P3R did not activate Ca2+-induced Ca2+ release at the RyR. Depletion of the Ins(1,4,5)P-3-sensitive store, by the removal of external Ca2+, on the other hand, caused only a small decrease (similar to 26%) in caffeine-evoked Ca2+ transients, suggesting that not all RyR exist on the common store shared with Ins(1,4,5)P3R. Dependence of the stores on external Ca2+ for replenishment also differed; removal of external Ca2+ depleted the Ins(1,4,5)P-3-sensitive store but caused only a slight reduction in caffeine-evoked transients mediated at RyR. Different mechanisms are presumably responsible for the refilling of each store. Refilling of both Ins(1,4,5)P-3-sensitive and caffeine-sensitive Ca2+ stores was inhibited by each of the SR Ca2+ ATPase inhibitors thapsigargin and cyclopiazonic acid. These results may be explained by the existence of two functionally distinct Ca2+ stores; the first expressing only RyR and refilled from [Ca2+](c), the second expressing both Ins(1,4,5)P3R and RyR and dependent upon external Ca2+ for refilling.