Modulation of ecto-5'-nucleotidase by phospholipids in human umbilical vein endothelial cells (HUVEC).

Ecto-5'-nucleotidase, the major enzyme controlling extracellular adenosine production, can be activated by phospholipids, e.g. lysophosphatidylcholine (LPC). This study examined the structural requirements of phospholipids to evoke this enzyme activation and figured out two new activators of ecto-5'-nucleotidase: platelet activating factor (PAF) and sphingosylphosphorylcholine (SPC). Potential signal transduction pathways including an involvement of protein kinase C and PAF-receptor were evaluated on the model of human umbilical vein endothelial cells (HUVEC). Cells were pre-incubated with 10 microM of various phospholipids including lysophosphatidylcholine, beta-arachidonyl-gamma-palmityl-alpha-phosphatidylcholine, beta,gamma-dipalmityl-alpha-phosphatidyl-choline, beta,gamma-dipalmityl-alpha-phosphatidylethanolamine, beta,gamma-dipalmityl-alpha-phosphatidylserine, gamma-acyl-beta-lyso-alpha-phosphatidylethanolamine, beta-acetyl-gamma-O-hexadecyl-alpha-phosphatidylcholine (platelet activating factor), lysophosphatidylic acid, sphingosine-1-phosphate and sphingosylphosphorylcholine. In the cell supernatant the extracellular dephosphorylation rate of the fluorescent AMP-analogue 1,N6-etheno-5'AMP to 1,N6-etheno-adenosine was measured by HPLC. Out of these ten structurally related phospholipids only lysophosphatidylcholine, sphingosylphosphatidylcholine and platelet activating factor dose-dependently increased the activity of ecto-5'-nucleotidase. Pharmacological blocking experiments revealed that neither the activation of PAF-receptor nor of protein kinase C were important for mediating the activation of ecto-5'-nucleotidase. Thus, using information on the known molecular structures of tested phospholipids, a phosphatidylcholine residue in alpha-position and a short chain length fatty acid esterified in beta-position seem essential for activation of ecto-5'-nucleotidase by glycerophospholipids. Since all tested phospholipids have similar fatty acid chain lengths and residues in alpha-position, they should act similarly on membrane fluidity. It is concluded that the observed effects are not based on changes in membrane fluidity by the added phospholipids, but rather involve a yet to be determined phospholipid-receptor.