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

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Abstract
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‐ (t1/2, 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 μM) 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 pre‐treatments 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‐thiotri‐phosphate (GTP‐γ‐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‐γ‐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. Phorbol ester pretreatment was also effective in reversing the increase in [3H]IP accumulation and 45Ca2+ mobilisation evoked by noradrenaline. However, following preincubation with carbachol there was no loss of nor‐adrenaline‐stimulated phosphoinositide breakdown although its ability to mobilise 45Ca2+ was blocked.