Assay of receptor-stimulated phosphoinositide turnover

The stimulation of phosphoinositide turnover is one of the key means by which receptors evoke responses in target cells and tissues. This is true for both G protein-coupled receptors and receptors that couple via tyrosine kinase activity. The protocols in this unit allow for pharmacological analysis of receptors coupled to phosphoinositide turnover. In general, the [(3)H]myo-inositol prelabeling methodology (described for both tissue slices and cultured cells) is the more widely applicable, since it requires fewer experimental steps and typically gives rise to a better signal-to-noise ratio. Individual inositol phosphates can also be determined as described by chromatographic separation on ion-exchange columns. In some circumstances (for example, when rapid responses to receptor stimulation are to be investigated or when the absolute levels of the active inositol phosphate are to be examined), it is preferable to use the mass assay described here for inositol (1,4,5)-trisphosphate from either tissue slices and cultured cells. This unit also provides support protocols for the preparation of [(3)H]myo-inositol, chromatography columns, tissue slices, and the IP(3)-binding protein.