PHOSPHATIDYLCHOLINE HYDROLYSIS AND DNA-SYNTHESIS IN CULTURED RETINAL CAPILLARY PERICYTES

In order to verify the role of activation of phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) in the initiation of mitogenic pro cess of retinal capillary pericytes, platelet-derived growth factor (P DGF), a known PC hydrolysis stimulator, and exogenous PLD have been us ed to stimulate pericytes. Exogenous PLD (Streptomyces chromofuscus PL D) or PDGF BE homodimer (PDGF) was added to a medium of quiescent peri cytes prelabeled with [P-32]orthophosphate. In the presence of ethanol (300 mM), phosphatidic acid (PA) and its stable transphosphatidylated product, phosphatidylethanol (PEt), were determined. In parallel, [H- 3]thymidine incorporation was measured. Downregulation of PKC was achi eved by long-term treatment with a phorbol ester. The addition of exog enous PLD or PDGF stimulated both [H-3]thymidine incorporation and [P- 32]PEt formation in a similar kinetic fashion, suggesting that PC hydr olysis is involved in PDGF-mitogenic signaling pathway. PDGF-stimulate d [H-3]PA formation was significantly higher in the presence than in t he absence of PA phosphohydrolase (PAP) inhibitor, indicating the acti vation of PLD/PAP pathway. In the presence of ethanol, a substantial l evel of PA at the steady state can be abolished by an inhibitor of dia cylglycerol (DAG) kinase. This phenomenon indicates the existence of P C-phospholipase C (PLC)/DAG kinase pathway in PC hydrolysis. Insulin p otentiated both PLD- and PDGF-induced DNA synthesis. Though similariti es occur in the induction of DNA synthesis and PC hydrolysis by exogen ous PLD and PDGF, the maximum extent of DNA synthesis of exogenous PLD was only approximately 43% of that induced by PDGF. Moreover, exogeno us PLD-induced DNA synthesis was not blunted, while PDGF-elicited DNA synthesis was markedly reduced, by PKC downregulation. In addition, PD GF-induced PC hydrolysis was attenuated by a tyrosine kinase inhibitor , whereas exogenous PLD-induced PC hydrolysis was unchanged. Taken tog ether, exogenous PLD may mimic PDGF action and partially account for t he efficacy on DNA synthesis elicited by PDGF. The signal transduction initiated by exogenous PLD is able to bypass the PKC- and PTK-depende nt activation of endogenous PLD. These findings provide evidence for t he importance of PLD-mediated PC hydrolysis in pericyte DNA synthesis stimulated by PDGF. (C) 1995 Academic Press, Inc.