Characteristics of Phorbol Ester- and Agonist-Induced Down-Regulation of Astrocyte Receptors Coupled to Inositol Phospholipid Metabolism

We have examined some of the characteristics of phorbol ester- and agonist-induced down-regulation of astrocyte receptors coupled to phosphoinositide metabolism. Our results show that preincubation of 3H]inositol-labelled astrocyte cultures with phorbol 12-myristate 13-acetate (PMA) resulted in a time- (t 1/2, 1-2 min) and concentration-dependent (IC50, 1 nM) decrease in the accumulation of 3H]inositol phosphates (IP) evoked by muscarinic receptor stimulation. Much longer (30-40 min) preincubation periods with higher concentrations (IC50, 600 microM) were required to elicit the same effect with the receptor agonist carbachol. Following preincubation, agonist-stimulated 3H]IP accumulation recovered with time; in both cases pretreatment levels of inositol lipid metabolism were attained within 2 days. Both phorbol ester and agonist pretreatments were also effective in reversing the carbachol-evoked mobilisation of 45Ca2+ in these cells. However, their effects on phosphoinositide metabolism were found not to be additive. Although neither pretreatment affected the incorporation of 3H]inositol into phosphoinositides, both resulted in a loss of membrane muscarinic receptors as assessed by 3H]N-methylscopolamine binding. In washed membranes prepared from 3H]inositol-labelled cultures, the guanine nucleotide analogue, guanosine 5'-O-thiotriphosphate (GTP-gamma-S), caused a dose-dependent increase in 3H]IP formation. This response was enhanced when carbachol was also included in the incubation medium, although the agonist alone was without effect. Pretreatment with either PMA or carbachol had no effect on GTP-gamma-S-stimulated 3H]IP accumulation but did reduce the ability of carbachol to augment this response. Similar findings were obtained when membranes were exposed directly to PMA.(ABSTRACT TRUNCATED AT 250 WORDS)