Hydrolysis of Endogenous Phospholipids by Rat Brain Microsomes

Abstract

Phosphatidylcholine of rat brain microsomes was labeled in vivo by intracerebral injection of either [3H]oleic acid or [methyl-3H]choline chloride. These labeled microsomes served both as the enzyme source as well as a source of endogenously labeled substrate. Phospholipase D (PLD) activity was detected with these particles only in the presence of exogenous oleate, its activator. Ca2+ and the ionophore A 23187 inhibit PLD activity of oleate-labeled microsomes. In oleate-labeled particles, besides phosphatidic acid the product of PLD action radioactivity was also detected in diglyceride as a result of resident phosphatidate phosphohydrolase, which hydrolyzed the phosphatidic acid. The phosphatidate phosphohydrolase could not be completely inhibited by KF and propranolol. The release of endogenous fatty acids from labeled phospholipid by a mellitin-stimulated phospholipase A2 also present in these particulates produced minimal stimulation of endogenous PLD. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are hydrolyzed by 50% in the presence of mellitin and 90% of the radioactivity was found in the lyso-compounds. Mellitin and oleate together reduced the radioactivity found in lyso-PC and increased that in lyso-PE.

The phosphatidylcholine pathway of diacylglycerol formation stimulated by phorbol diesters occurs via phospholipase D activation

“…Most of what is known of mammalian phospholipase D comes from the work of Witter and Kanfer [19] [9,18]. It is interesting to note that agonist-induced PC hydrolysis is severely depressed or totally absent in several transformed derivatives of the parent REF52 cell line [9].…”

Cytosolic phospholipase A2 is induced in reactive glia following different forms of neurodegeneration

“…The brain is rich in arachidonic acid-containing phospholipids (Mead et al, 1977) and contains significant PLA 2 activity (Witter and Kanfer, 1985;Farooqui et al, 1997). The central nervous system (CNS) appears to contain multiple PLA 2 types, including calcium-dependent and calcium-independent forms of various molecular masses and substrate specificities (Gray and Strickland, 1982;Ross et al, 1995;Thomson and Clark, 1995;Hirashima et al, 1992;Yang et al, 1996).…”

Specificity of Phospholipase D Activation by Cholecystokinin and Phorbol Myristate Acetate but Not by Carbamylcholine and A23187 in Rat Pancreatic Acini

“…There is also some evidence for the involve ment of phospholipase A2 (PLA?) in hormonal signal transduction and enzyme release from pancreatic acinar cells [6], Recently, we have demonstrated [7] that caerulein can activate phospholipase D (PLD) in pancreatic acini by a mechanism which seems independent of PK-C and PLC activation; this finding thus provides an alternate path way in the generation of second messengers derived from membrane phospholipids [8,9]. PLD catalyzes the hydro lysis of the terminal diester bond of phosphatidylcholine (PC) and possibly of other glycerophosphatides with the formation of phosphatidic acid (PA) and choline [8].…”

“…PA can then serve as a substrate for DAG biosynthesis through the action of PA phosphohydrolase. Propranolol, a (3-adrenergic receptor antagonist, has been found to inhibit the conversion of PA to DAG [9], On the other hand, in the presence of a primary alcohol, PLD catalyzes a transphosphatidylation reaction which produces phosphatidylalcohol. Being PLD-specific, this reaction be comes the marker of choice for the enzyme's measure ment [10],…”