High Throughput 96-Well Plate Assay for Receptor-Mediated Phosphatidylinositol Turnover

Abstract

The G-protein coupled receptor family represents a large number of neurotransmitter receptors. Among the diverse signal transduction pathways mediated via G-proteins, phospholipase C mediated phosphatidylinositol hydrolysis represents one of the best characterized signal transduction mechanisms. Accordingly, the measurement of agonist-induced phosphatidylinositol turnover has been used as a convenient functional assay for receptor activation. Assays currently used for this purpose, however, are not suitable for high throughput screening. In this article, an improved technique using 96-well microtiter plate format for measuring phosphatidylinositol turnover is introduced. Anion exchange columns were prepared on fiber glass 96-well multiscreen filter plate. Separation and detection of released inositol phosphates were conducted in a 96-well format. Cells expressing certain neurotransmitter receptors were challenged with agonists and the receptor-mediated PI turnover was measured by the new technique and the results obtained were compared to that obtained from traditional assays. The results indicate that the 96-well assay is 10 to 20 times more efficient than the traditional method and is, furthermore, suitable for high throughput drug screening. Our data also indicate that this method is particularly useful for characterizing multiple antagonists by Schild analysis.