Characterization of glycerophosphocholine phosphodiesterase activity and phosphatidylcholine biosynthesis in cultured retinal microcapillary pericytes. Effect of adenosine and endothelin-1.
In pericytes from bovine retina, the enzyme glycerophosphocholine phosphodiesterase, catalyzing the hydrolysis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, Km, inhibitors, and subcellular localization. In these cells, the natural substrate sn-glycero-3-phosphocholine 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 culture (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 lysophosphatidylcholine from the bloodstream to nervous tissue. In addition, capillary pericytes in culture were able to incorporate 3H-choline rapidly into choline-containing soluble phosphorylated intermediates and into phosphatidylcholine. To find a positive 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 3H-choline incorporation compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phosphatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabolism in pericytes.
Anfuso
Carmelina D
CD
Department of Biochemistry, Faculty of Medicine, University of Catania, 95125 Catania, Italy.
Sipione
Simonetta
S
Lupo
Gabriella
G
Ragusa
Nicolò
N
Alberghina
Mario
M
D000241:Adenosine / Q000494:pharmacology*; D000818:Animals; D001813:Blood-Retinal Barrier; D002196:Capillaries / Q000166:cytology; D002412:Cations / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002794:Choline / Q000096:biosynthesis; D019332:Endothelin-1 / Q000494:pharmacology*; D020286:Pericytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D010743:Phospholipids / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology*
D002412:Cations; D019332:Endothelin-1; D010713:Phosphatidylcholines; D010743:Phospholipids; D010727:Phosphoric Diester Hydrolases; C020677:glycerophosphocholine phosphodiesterase; D000241:Adenosine; D002794:Choline