A SIMPLE ASSAY FOR ECTO-5'-NUCLEOTIDASE USING INTACT PULMONARY-ARTERYENDOTHELIAL-CELLS - EFFECT OF ENDOTOXIN-INDUCED CELL INJURY

Adenosine may be protective in acute vascular injury by inhibiting pla telet aggregation and neutrophil oxidant release. In contrast, adenine nucleotides, which may be released with acute vascular injury, stimul ate platelet aggregation and neutrophil oxidant release. Ectonucleotid ases, membrane enzymes that catabolize extracellular nucleotides, are the primary mechanism for degrading circulating nucleotides to adenosi ne. Ecto-5'-nucleotidase converts extracellular AMP to adenosine. We h ypothesized that endothelial cell injury alters ecto-5'-nucleotidase a ctivity. Using a novel assay first reported by Jamal et al. (Biochem J 250: 369-373, 1988) with rat adipocytes, we studied the properties of ecto-5'-nucleotidase in intact monolayers of cultured bovine pulmonar y artery endothelial cells (BPAEC) and examined the effect of endotoxi n on enzyme activity. The assay uses a fluorescent analog of AMP, 1,N6 -etheno-AMP (E-AMP), as the substrate for ecto-5'-nucleotidase, and me asures etheno-adenosine (E-Ado) formation. Etheno-AMP in Hepes buffer, pH 7.4, at 22-degrees, was added to confluent monolayers of BPAEC; sa mples of supernatant were collected after various intervals, and E-AMP and E-Ado were quantitated by HPLC. Using these methods we found a K( m) of 15 +/- 6 muM, a pH optimum of 7.48, minimal effect of MgCl2 or C aCl2 at physiologic pH, and inhibition by alpha,beta-methylene ADP, a known 5'-nucleotidase inhibitor. We established that the monolayer ass ay was indeed measuring cell surface associated 5'-nucleotidase. To de termine the effect of endotoxin, we incubated confluent monolayers wit h endotoxin in Minimal Essential Medium plus 10% fetal bovine serum fo r 24 hr, washed them, and assessed the conversion of E-AMP to E-Ado by the endotoxin-injured cells. Endotoxin stimulated endothelial ecto-5' -nucleotidase activity. This increase in 5'-nucleotidase activity in r esponse to endotoxin injury may represent an important clearance mecha nism for circulating adenine nucleotides and may be protective in acut e vascular injury by increasing adenosine production.