ABSTRACT: In pericytes from bovine retina, the enzyme glyc- erophosphocholine phosphodiesterase, catalyzing the hydroly- sis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, K m , inhibitors, and subcellular localiza- tion. In these cells, the natural substrate sn-glycero-3-phospho- choline was present at relatively high concentration (6.4 ± 1.2 nmol/mg protein), and the EDTA-sensitive phosphodiesterase activity was also found to be markedly high (9.80 ± 1.5 nmol/min/mg protein) compared to that estimated in liver and brain (1–3 nmol/min/mg protein) or in renal epithelial cell cul- ture (0.27 nmol/min/mg protein). The reaction conditions were in general agreement with those found earlier in brain and other tissues. The majority of the enzyme specific activity was located in the plasma membrane, whereas a minor part was present in the microsomal fraction. The physiological significance of the high catabolic phosphodiesterase activity in these cells may be related to the transfer, followed by deacylation, of lysophos- phatidylcholine from the bloodstream to nervous tissue. In ad- dition, capillary pericytes in culture were able to incorporate 3 H-choline rapidly into choline-containing soluble phosphory- lated intermediates and into phosphatidylcholine. To find a pos- itive and negative effector on phosphatidylcholine formation, adenosine, an important intercellular mediator in the retina in response to alterations in oxygen delivery, and endothelin-1, a potent paracrine mediator present at the blood–brain and blood–retina barrier, were tested. The cells cultured for 1 or 24 h in a medium containing adenosine at concentrations of 10 -6 and 10 -4 M showed significant reduction in 3 H-choline incorpo- ration compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phos- phatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabo- lism in pericytes. Paper no. L9094 in Lipids 38, 45–52 (January 2003). Our previous studies concerned the transport across the blood–retina and blood–brain barrier of plasma lysophos- phatidylcholine (lyso-PtdCho), a transport form to tissues, and especially the brain, of both choline and unsaturated FA (1). We were stimulated to investigate whether endothelial cells (EC) and pericytes, which organize together the physical bar- rier between blood and tissues in microvessels, may deacylate lyso-PtdCho to sn-glycero-3-phosphocholine (GroPCho), and how high the potential is for these cells to catabolize this last phosphorylated intermediate to liberate free choline. We first demonstrated a marked presence of glycerophospho- choline phosphodiesterase (sn-glycero-3-phosphocholine glyc- erophosphohydrolase, GroPChoPDE, EC 3.1.4.2) in bovine brain microvessel preparations (2). This suggested investigating the cellular origin, whether endothelial or pericytal, of this en- zyme activity related to the last step of phosphatidylcholine (Ptd- Cho) degradation. Therefore, in the present study, experiments were performed to measure the activity in cultured pericytes and endothelial cells, both isolated from bovine retina microcapillar- ies. We found that the enzyme activity tested was high in peri- cytes (9.80 + 1.5 nmol/min/mg protein) and was present to a much lesser extent in EC (0.39 + 0.08 nmol/min/mg protein). In view of this result, the elevated GroPChoPDE activity we had previously measured in brain microvessels (2) could be attrib- uted to the pericyte component. The consequence of this finding was to promote our interest in expanding the study of such highly expressed GroPChoPDE in this cell type; here we further char- acterize its enzymatic parameters and subcellular localization. GroPChoPDE activity is enriched in rat kidney (40 nmol/min/mg protein) (3), and it is present at lower levels of activity in renal epithelial cell culture (0.27 nmol/min/mg protein) (4) and in many organs including brain (1–3 nmol/min/mg protein) (3–7), but not in myelin (8). Among brain subcellular fractions, microsomes contain the greatest amount (6). A glycerylphosphorylcholine diesterase, secreted by epithelial cells in the rat uterus during preovulatory estro- gen surge, has been purified and characterized (9); native gly- coprotein is a trimer of 105 kDa. At the same time, we tested for the presence of some en- zymes involved in PtdCho biosynthesis, degradation, and re- modeling in pericytes, for which no studies have been done. Indeed, their physiological function and biochemical charac- teristics are not yet clearly identified. Because they contain contractile elements (muscle actin) similar to those present in the vascular smooth muscle cells, a role as modulators of mi- crocapillary dilation has been proposed (10). Copyright © 2003 by AOCS Press 45 Lipids, Vol. 38, no. 1 (2003) *To whom correspondence should be addressed at Department of Biochem- istry, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy. E-mail: malber@unict.it Abbreviations: CDP-Cho, cytidine diphosphocholine; CT, CTP:phospho- choline cytidylyltransferase; EC, endothelial cells; ET-1, endothelin 1; γ- GPT, γ-glutamyltranspeptidase; GroPCho, sn-glycero-3-phosphocholine; GroPChoPDE, glycerophosphocholine phosphodiesterase; lyso-PtdCho, lysophosphatidylcholine; PCho, phosphorylcholine; PLA 2 , phospholipase A 2 ; PtdCho, phosphatidylcholine; SN, supernatant. Characterization of Glycerophosphocholine Phosphodiesterase Activity and Phosphatidylcholine Biosynthesis in Cultured Retinal Microcapillary Pericytes. Effect of Adenosine and Endothelin-1 Carmelina D. Anfuso, Simonetta Sipione, Gabriella Lupo, Nicolò Ragusa, and Mario Alberghina* Department of Biochemistry, Faculty of Medicine, University of Catania, 95125 Catania, Italy