Ca2+-Induced Ca2+ Release through Localized Ca2+ Uncaging in Smooth Muscle

Abstract

Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca2+ release or Ca2+ sparks and, in some spiking tissues, as Ca2+ release that is triggered by the activation of sarcolemmal Ca2+ channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca2+ (DMNP-EDTA) in Fluo-4–loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca2+ activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca2+ release in the form of Ca2+ sparks and Ca2+ waves that were distinguishable from increases in Ca2+ associated with Ca2+ uncaging, unequivocally demonstrating that Ca2+ release occurs subsequent to a localized rise in [Ca2+]i. TPFP-triggered Ca2+ release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca2+ sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca2+ release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca2+]i through inositol trisphosphate (InsP3) receptors (InsP3Rs). We conclude that CICR activated by localized Ca2+ release bears essential similarities to those observed by the activation of ICa (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca2+ release through InsP3R can occur at high local [Ca2+]i.