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Polarized Ca2+ release in saponin-permeabilized parotid acinar cells evoked by flash photolysis of 'caged' inositol 1,4,5-trisphosphate.

ABSTRACT: In exocrine acinar cells, agonist stimulation results in a polarized Ca2+ signal, termed the 'Ca2+ wave', that propagates from the apical pole towards the basolateral region. We attempted to detect the inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ wave in saponin-permeabilized rat parotid acinar cells using a digital imaging system. The permeabilized acinar cells were labelled with the membrane-bound Ca2+ indicator Calcium Green C18 to detect changes in Ca2+ concentration adjacent to the membrane of intracellular organelles. Application of InsP3 was made by the photolysis of InsP3 P4(5)-1-(2-nitrophenyl)ethyl ester (caged InsP3) to expose simultaneously all regions of the permeabilized acinar cells to InsP3. The increase in fluorescence ratio following the photolysis of 0.5 microM caged InsP3 started at the apical region of the acinar cells within 0.1 s and spread towards the basolateral region, indicating that Ca2+ release from intracellular Ca2+ stores was initially evoked at the apical region. Pretreatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pumps, failed to prevent the InsP3-induced Ca2+ wave, suggesting that the generation of the Ca2+ wave is not attributed to the polarized distribution of the Ca2+ pumps. The photolysis of a high concentration (10 microM) of caged InsP3 caused a homogeneous increase in the fluorescence ratio throughout the cells, indicating that all regions of intracellular Ca2+ stores similarly responded to the high concentration of InsP3. The present study is the first demonstration of the InsP3-induced Ca2+ wave in permeabilized exocrine acinar cells. The result provides fresh evidence that the apical region contains elements of intracellular Ca2+ stores particularly sensitive to InsP3 and that the Ca2+ wave results from a polarized distribution of InsP3-sensitive Ca2+ stores.

SUBMITTER: Tanimura A

PROVIDER: S-EPMC1219539 | biostudies-other | 1998 Jun

REPOSITORIES: biostudies-other

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Project description:In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.

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Polarized Ca2+ release in saponin-permeabilized parotid acinar cells evoked by flash photolysis of 'caged' inositol 1,4,5-trisphosphate.

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