The origins and metabolism of diacylglycerol and phosphatidate formed upon stimulation of neural receptors.

Abstract

1,2-diacyl-sn-glycerol (DAG), besides being a biosynthetic precursor of glycerolipids, has recently been shown to act as a second messenger activating protein kinase C (PKC). The sources and metabolism of both this second messenger DAG and the phosphatidate (PA) formed upon inositide-linked receptor stimulation were studied. DAG isolated from biological samples was benzoylated and the molecular species of the benzoylated DAG's were separated from each other by reverse-phase high-performance liquid chromatography. The total mass and the masses of individual molecular species were quantified by using an internal standard. Molecular species of all the phosphoglycerides were also analyzed after being hydrolyzed into DAG. About thirty different molecular species were identified in resting rat brains (45 nmol of total DAG/g brain). Studies on postmortem changes of brain DAG showed that the initial ($<$5 min) rapid increase of the DAG's was mainly due to the breakdown of PI's, while at later time points the DAG's also originated from phosphatidylcholine (PC). In human SK-N-SH neuroblastoma cells, muscarinic agonists, such as carbachol (CCh), elicited a rapid and sustained increase (60%) in DAG. Within 10 sec of stimulation the molecular species of newly-formed DAG were similar to those of PI's, however, at later time points ($>$1 min) they originated both from PI's (30-15%) and PC (70-85%). The molecular species profile of the incremental PA (200% over control) was very similar to that of PI's. When cells were preincubated with staurosporine, a PKC inhibitor, CCh produced DAG only from PI's. In contrast, 1-oleoyl-2-acetyl glycerol, a PKC activator, generated DAG solely from hydrolysis of PC. From these studies in neuroblastoma cells, it was concluded that DAG generated from PI's activates PKC and is recycled back to PI's via PA. The activated PKC stimulates PC-specific phospholipase C causing the breakdown of PC to DAG, which in turn may activate other kinds of PKC resulting in long-term effects. PC-specific phospholipase D is not stimulated after receptor activation and DAG produced from PC is not phosphorylated to PA.