BRAIN PHOSPHOINOSITIDES AND BARBITURATES

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The mode of action of anesthetics and anticonvulsants is not yet clarified although the most useful of anticonvulsant drugs, phenobarbital, has been in use for 75 years. Among lipophilic drugs, the barbiturates are the nearest in structure to inositol; both contain a six-membered ring with several hydrogen bonding sites (O, OH, or NH groups). Since the enzymes metabolizing phosphoinositides undoubtedly recognize the inositol moiety, it may be suspected that barbiturates will inhibit polyphosphoinositide metabolism. We find that phenobarbital is an effective inhibitor of the kinases, especially the phosphatidylinositol-4-phosphate (PIP) kinase. It inhibits the phosphohydrolases, especially phosphatidyl-4,5-bisphosphate (PIP)2 phosphodiesterase (phospholipase C), only weakly. Therefore, the pharmacological action of phenobarbital may be connected to a depression of PIP2 synthesis. This lipid, on cell stimulation, yields the second messenger, inositol-1,4,5-trisphosphate (IP3), which in turn mobilizes intracellular calcium; thus, the inositide-calcium response may be dampened. We plan to investigate this possibility and related issues. Experiments will be carried out with synaptosomal and synaptoneurosomal brain fractions - which contain all relevant enzymes - and, if appropriate, platelets. The ED50 of phenobarbital anaesthesia will be determined (tadpole righting reflex) to confirm that PIP-kinase inhibition in vitro coincides with in vivo (clinical) concentration. Other barbiturates (non- anticonvulsant) will be tested, as well as barbiturate inhibition of other enzymes (e.g. CD P-diacylglycerol-inositoltransferase, inositol phosphate(s) hydrolases, phospholipase A2, diglyceride kinase); the results will indicate if the most sensitive response to barbiturates is indeed that of PIP-kinase. The mechanism of PIP-kinase inhibition by phenobarbital will be determined (is it competitive?). The depression of PIP2 (and also PIP) synthesis by phenobarbital should lead to a decrease in these lipids and an increase in phosphatidylinositol (PI); this will be tested by quantitative analysis of the inositides after incubation (synaptosomes, synaptoneurosomes, and platelets) with and without the drug. The expected dampening effect of phenobarbital on the mobilization of calcium resulting from stimulation of synaptosomes or synaptoneurosomes by cholinergic muscarinic agonists, and of platelets by thrombin will be measured as diminished release of Ca from the intracellular membranes, followed fluorimetrically as Ca-fura-2 formation.