ryanodine and inositol-1-4-5-trisphosphate-1-(2-nitrophenyl)ethyl-ester

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Endoplasmic Reticulum; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Mice; Mitochondria; Pancreas; Receptors, Cytoplasmic and Nuclear; Ryanodine; Ryanodine Receptor Calcium Release Channel; Uncoupling Agents

Ca2+ signalling events were analyzed in single myocytes from rat portal vein by using a laser confocal microscope combined with the patch-clamp technique. Increase in inositol 1,4,5-trisphosphate (InsP3) concentration was obtained by photorelease from a caged precursor or intracellular dialysis of 3F-InsP3. Low InsP3 concentrations activated either small elevations of [Ca2+]i or localized Ca2+ transients whereas high InsP3 concentrations activated either homogeneous Ca2+ responses or propagated Ca2+ waves. The InsP3-evoked localized Ca2+ transients had spatio-temporal properties characteristic of Ca2+ sparks. In addition, compounds that blocked Ca2+ sparks and Ca2+ responses activated by Ca2+ jumps reduced the global InsP3-activated Ca2+ responses and suppressed the Ca2+ transients. In contrast, Ca2+ responses evoked by flash-photolytic Ca2+ jumps or caffeine were not affected by heparin (an InsP3 receptor antagonist). These results suggest that the absence of elementary Ca2+ events evoked by InsP3 may be related to the lack of clustered InsP3 receptor units in these cells, as confirmed by immunocytochemistry. Cooperativity between InsP3- and ryanodine-sensitive Ca2+ channels may represent a novel mechanism to amplify Ca2+ release from the same intracellular store and give rise to propagated Ca2+ waves.

Topics: Acetates; Animals; Calcium Channels; Cells, Cultured; Ethylenediamines; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ion Channel Gating; Microinjections; Microscopy, Confocal; Patch-Clamp Techniques; Photolysis; Portal Vein; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Ryanodine; Ryanodine Receptor Calcium Release Channel; Signal Transduction