Phosphatidate metabolism in stimulated synaptosomes.

Acetylcholine increases the incorporation of 32P1 into phosphatidate of guinea-pig brain synaptosomes incubated in vitro (Durell & Sodd, 1966; Schacht & Agranoff, 1972; Yagihara & Hawthorne, 1972). Three pathways for the synthesis of phosphatidate operate in brain tissue: acylation of glycerol 3-phosphate, acylation of dihydroxyacetone phosphate (Pollock et al., 1975) and the phosphorylation of 1,2-diacylglycerol (Hokin & Hokin, 1959). It seems likely, though it has not yet been shown conclusively, that the increased synthesis caused by acetylcholine takes place by the thud of these pathways. The diacylglycerol kinase involved is more than ten times as active as the acylation enzymes (Lapetina & Hawthorne, 1971) and diacylglycerol is available from the hydrolysis of phosphatidylinositol. Further, Schacht & Agranoff (1974) have shown that acetylcholine does not increase the incorporation of 3H from [3H]glycerol or [3H]glucose into synaptosomal phosphatidate. This indicates that the synthesis of phosphatidate from glycerol 3-phosphate or dihydroxyacetone phosphate is unaffected by acetylcholine. Evidence from experiments with various tissues shows that the increased labelling of phosphatidate is associated with muscarinic rather than nicotinic cholinergic receptors (Michell, 1975) though electric organ of Torpedo marmorata is an exception (Bleasdale et al., 1976). If the major response of phosphatidate to acetylcholine is post-synaptic, it is difficult to understand why synaptosomal phosphatidate responds to this agonist. Synaptosomes are essentially pre-synaptic nerve terminals and though post-synaptic membranes are attached at the junctional complex they will be isolated from the metabolism of the synaptosome. There is also some confusion about the action of atropine, which blocks the action of acetylcholine on muscarinic receptors. Schacht & Agranoff (1 974) found that atropine blocked the increased labelling of phosphatidate due to acetylcholine when synaptosomes were incubated with 32PI. Yagihara et al. (1973) showed that the major increase in phosphatidate labelling was associated with the synaptic vesicle fraction, and more recent work (J. E. Bleasdale, unpublished observations) shows that atropine does not affect this increase. As pointed out previously (Hawthorne & Bleasdale, 1975) effects of acetylcholine in vitro on a mixed population of synaptosomes from brain cortex may not be