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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.

Stimulation of rat pancreatic acinar cells with low concentrations of phosphatidylinositol (PI)-linked secretagogues induces [Ca2+]i oscillations, without measurable changes in the formation of inositol 1,4,5-trisphosphate. Therefore, we tested U73122 a new phospholipase C inhibitor to determine if PI turnover is necessary for the generation of [Ca2+]i oscillations. In acini prelabeled with [3H]inositol, PI hydrolysis on stimulation with either cholecystokinin or carbachol was inhibited dose-dependently by U73122, with a maximal effect seen at 10 microM; the formation of inositol 1,4,5-trisphosphate, measured using a radioreceptor assay, was also similarly inhibited. By contrast secretin- or vasoactive intestinal peptide-stimulated production of cAMP was unaffected by 10 microM U73122. These studies indicate that U73122 is a relatively specific inhibitor of G-protein-mediated phospholipase C activation in pancreatic acini. In fura-2-loaded acini, U73122 inhibited the increases in [Ca2+]i stimulated by these high concentrations of secretagogues which can be demonstrated to elicit PI turnover. The [Ca2+]i signal generated by directly stimulating G-proteins with sodium fluoride was also inhibited by U73122; however, the [Ca2+]i rise induced by thapsigargin was unaffected. These data indicate that the mechanism of inhibition was distal to the occupation of cell surface receptors but did not involve an interference of Ca2+ metabolism in general. When [Ca2+]i oscillations were elicited by low concentrations of cholecystokinin or carbachol, U73122 rapidly inhibited the oscillating [Ca2+]i signal. In contrast, oscillations induced by an analogue of cholecystokinin, JMV-180, which does not stimulate changes in PI metabolism at any concentration, were unaffected. This indicates that cholecystokinin- and carbachol-induced oscillations are probably initiated by small, localized changes in PI metabolism, which are not readily detectable. However, the inability of U73122 to inhibit JMV-180-induced oscillations indicates that PI metabolism may not necessarily be a prerequisite for the generation of [Ca2+]i oscillations.[1]


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