Stimulation of inositol phospholipid breakdown in rat cortical and hippocampal miniprisms by noradrenaline, 5-hydroxytryptamine and carbachol: some methodological aspects.

After incubation of miniprisms from rat cerebral cortex or hippocampus with 3H-myo-inositol, labelling of the phospholipid ("Lipid") and inositol phosphate ("InsP") fractions was found. Inositol phospholipid hydrolysis ("PI breakdown") was stimulated by noradrenaline, 5-hydroxytryptamine and carbachol. Expressing data as InsP/(Lipid + InsP) was found to be a superior measure of the rate of PI breakdown compared with the more commonly used InsP d.p.m. unit, since the former was found to be independent of the volume of the miniprism aliquot used and the degree of labelling of inositol phospholipids. The PI breakdown responses to noradrenaline, 5-hydroxytryptamine and particularly carbachol were found to be enhanced by increasing the assay [K+] from 5.88 mM to 18.2 mM. Storage of hippocampal samples at -70 degrees by the "slow freeze-fast thaw" method of Hardy et al. (1983) resulted in a decreased degree of labelling of the Lipid and InsP fractions and a loss of the PI response to noradrenaline when assayed at a [K+] of 5.88 mM, but a reasonable response was seen in these samples at an assay [K+] of 18.2 mM. The temperature of the Krebs-Henseleit buffer used in the preparation of the miniprisms was found to be important for the PI breakdown response.