Phospholipase C inhibitor, U73122, releases intracellular Ca2+, potentiates Ins(1,4,5)P3-mediated Ca2+ release and directly activates ion channels in mouse pancreatic acinar cells

Abstract

It is recognized in many cellular systems that the receptor/G-protein activation of phospholipase C and Ins(1,4,5)P3 production is the transduction pathway regulating the release of Ca2+ from internal stores. Ca2+ signals can now be monitored at the level of single cells but the biochemical detection of Ins(1,4,5)P3 cannot match this resolution. It is often difficult or impossible to directly attribute responses evoked in single cells by putative phospholipase C-coupled agonists to changes in Ins(1,4,5)P3 levels. U73122 is an aminosteroid that is reported to act as a specific inhibitor of phospholipase C and it has become an important tool in establishing the link between phospholipase C activation and cellular Ca2+ signalling. In the present study we use both patch-clamp electrophysiology and the imaging of fluorescent Ca2+ indicators to investigate the effect of U73122 in mouse pancreatic acinar cells. The study reveals that U73122 has effects other than the inhibition of phospholipase C. U73122 can directly activate ion channels. It can itself promote the release of Ca2+ from intracellular stores in permeabilized cells and in intact cells it triggers a release of Ca2+ that is initiated specifically at the secretory pole of these morphologically and functionally polarized cells. We also present evidence that U73122 can potentiate the response to Ins(1,4,5)P3; this is seen both in permeabilized cells and in patch-clamp protocols in which cells are internally dialysed with submaximal concentrations of Ins(1,4,5)P3. The effects of U73122 are therefore multiple and not specific for the inhibition of phospholipase C. Importantly, all the effects described influence Ca2+ signalling yet in many experimental protocols some of these effects can go unnoticed and might in error be attributed simply to the inhibition of Ins(1,4,5)P3 production.

Pancreatic protease activation by alcohol metabolite depends on Ca 2+ release via acid store IP 3 receptors

“…The most widely used inhibitor of phospholipase C (PLC) is the aminosteroid compound U73122 (29). U73122 is a powerful PLC inhibitor, but other effects-including release of Ca 2ϩ from IP 3 -sensitive stores-have been noted (29,30). We used U73122 at a concentration (10 M) that abolishes cytosolic Ca 2ϩ signal generation evoked by muscarinic receptor activation in many systems (29), including pancreatic acinar cells (31).…”

Inositol 1,4,5-Trisphosphate-independent Ca2+Mobilization Triggered by a Lipid Factor Isolated from Vitreous Body

“…This is in contrast with the data obtained after exposure of the cells to U73122, an inhibitor of PI-PLC. However, several reports have pointed out that the action of this drug is far from being specific, being able to also inhibit other PLCs, as well as phospholipase D (48,49). Taking this into account, the Ca 2ϩ mobilization induced by VLF could be explained by two alternate routes; either the PTX-sensitive G-protein is coupled to phospholipase D, inducing the intracellular accumulation of PA, or coupled to a different PLC, such as PC-PLC.…”

Agonist‐dependent cannabinoid receptor signalling in human trabecular meshwork cells

“…U73343, the inactive analogue of U73122, had no significant effect upon the release of Ca 2 þ (P40.05, n ¼ 18). A dose of 1 mM U73122 was not exceeded as this compound has been shown to release Ca 2 þ from internal stores at higher doses (Willems et al, 1994;Mogami et al, 1997;Jan et al, 1998 ( Figure 4a). These results suggest that the observed WIN55,212-2-mediated Ca 2 þ mobilization did not depend on CB 1 -G i/o coupling.…”