AGONIST‐STIMULATED BREAKDOWN OF MYO‐[2‐3H] INOSITOL‐LABELLED PHOSPHATIDYLINOSITOL IN MOUSE PANCREAS

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Abstract
Agonist-stimulated hydrolysis of phosphatidylinositol (PI) in cell membranes has been proposed to lead to an increase of cytosol calcium concentration and activation of the cellular response in certain smooth muscles and glands. A method is described which allows the rapid, reproducible measurement of hydrolysis of phosphatidylinositol in mouse pancreas. The technique involves the in vivo labelling of the pancreas with myo-[2-3H] inositol. The majority of the label incorporated into phospholipids is in the form of PI. with only a small proportion of label in di- and tri phosphoinositides. Tissue pieces of the labelled pancreata are incubated in vitro in the presence or absence of secretagogues, and the PI in homogenates of these pieces is precipitated with trichloroacetic acid. No PI remains in the acid-soluble supernatant. This technique does not require the time-consuming extraction and chromatographic separation of lipids which has been necessary in other assays of PI hydrolysis. Using this method, we have confirmed that PI breakdown is stimulated by carbachol and cholecystokinin-octapeptide in the presence or absence of extracellular Ca2+. Agonist-Stimulated hydrolysis of PI was potentiated by extracellular Ca2+, and was not dependent on agonist-activated Na+ influx. This technique will facilitate the investigation of the importance of PI breakdown in stimulus-response coupling.

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