Comparative study of high-glucose effect on phosphatidylcholine hydrolysis of cultured retinal capillary pericytes and endothelial cells

Abstract

Recent studies have suggested the importance of phosphatidylcholine (PC) hydrolysis in the control of cell growth. Because of the different replicative behavior between retinal pericytes and endothelial cells in early diabetic retinopathy, we have compared PC metabolism of these two types of cells under high-glucose conditions. Both types of cells growing in controlled glucose conditions were prelabeled with [ 32P]P i for testing phosphatidylethanol (PEt), a unique product of PC-specific phospholipase D, with [ 14C]glycerol for determining 1,2-diacylglycerol (DAG) de novo synthesis and labeled with selective fatty acids for PC-derived and phosphoinositide (PI)-derived DAG. The prelabeled cells were stimulated with ATP, a purinergic agonist. Analyses of products of PI and PC hydrolysis were carried out by TLC and/or HPLC. In both types of cells, ATP activated PI and PC hydrolysis. The dose and time dependencies of IP 3 and PI-derived DAG formation with respect to ATP were essentially identical. When the level of IP 3 and PI-derived DAG returned toward the baseline, a second increase in DAG mainly derived from PC occurred, lasting at least 1 h. High glucose altered DAG kinetics: in pericytes it suppressed both PI- and PC-derived DAG formation; in endothelial cells it stimulated the PC-derived DAG increase, but kept the PI-derived DAG unchanged. DAG de novo synthesis was stimulated in endothelial cells growing in high glucose, but was left unchanged in pericytes. Contrast to endothelial cells, in pericytes, subsequent mitogenic process may be negatively regulated by the inhibitory effects of high glucose on PI and PC hydrolysis, resulting in reduced levels of PC-derived DAG.