Characterization and Localization of Phospholipase A2Activity in Sympathetic Ganglia

Abstract:Superior cervical ganglion phospholipase A2activity was characterized using l‐palmitoyl‐2‐[l‐14C]arachidonoyl‐sn‐glycero‐3‐phosphocholine as a substrate. The enzyme activity exhibited linearity with interval of incubation and tissue concentration; there appeared to be two pH optima of the enzyme, at pH 6.0 and 9.0. A Lineweaver‐Burk plot of the reciprocal of activity versus substrate concentration yielded an apparent Kmof 0.53 mMand aVmaxof 5.3 nmol/h/mg of protein. The enzyme exhibited a partial Ca2+dependence; in the absence of Ca2+and presence of EGTA, activity was reduced by 40%. The phospholipase A2activity was heat sensitive and was completely inactivated after treatment at 100°C for 30 min. For determination of whether the enzyme had a preference for hydrolysis of specific fatty acid substituents in the 2 position of phosphatidylcholine, several different substrates were tested. The order of preference for hydrolysis by the ganglionic enzyme was l‐palmitoyl‐2‐[l‐14C]arachidonoyl‐sn‐glycero‐3‐phosphocholine = l‐palmitoyl‐2‐[1‐14C]linoleoyl‐sn‐glycero‐3‐phosphocholine > l‐palmitoyl‐2‐[l‐14C]palmitoyl‐sn‐gly‐cero‐3‐phosphocholine. For determination of the localization of the phospholipase A2enzyme in sympathetic ganglia, two approaches were used. Guanethidine, which results in destruction of adrenergic cell bodies in sympathetic ganglia, was administered to rats; an ˜50% decline in phospholipase A2activity was observed after this treatment. In other experiments, the preganglionic nerve to the ganglion was sectioned in rats; after 2 weeks of denervation, no significant change in ganglionic phospholipase A2activity was seen, although after 4 weeks of denervation, a small but non‐significant decrease in enzyme activity was observed. These results suggest that the enzyme has a predominantly post‐synaptic localization. Extrinsic phospholipase A2enzymes have previously been shown to affect postsynaptic function in rat sympathetic ganglia. The presence of a phospholipase A2in the tissue itself could suggest that endogenous phospholipase A2enzymes can regulate postsynaptic neuronal function.