Bonitati A E, Agarwal K C, Rounds S
Providence Department, Veterans Affairs Medical Center, RI 02908.
Biochem Pharmacol. 1993 Oct 19;46(8):1467-73. doi: 10.1016/0006-2952(93)90113-b.
Adenosine may be protective in acute vascular injury by inhibiting platelet aggregation and neutrophil oxidant release. In contrast, adenine nucleotides, which may be released with acute vascular injury, stimulate platelet aggregation and neutrophil oxidant release. Ectonucleotidases, membrane enzymes that catabolize extracellular nucleotides, are the primary mechanism for degrading circulating nucleotides to adenosine. Ecto-5'-nucleotidase converts extracellular AMP to adenosine. We hypothesized that endothelial cell injury alters ecto-5'-nucleotidase activity. 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 pulmonary artery endothelial cells (BPAEC) and examined the effect of endotoxin 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 measures ethenoadenosine (E-Ado) formation. Etheno-AMP in Hepes buffer, pH 7.4, at 22 degrees, was added to confluent monolayers of BPAEC; samples of supernatant were collected after various intervals, and E-AMP and E-Ado were quantitated by HPLC. Using these methods we found a Km of 15 +/- 6 microM, a pH optimum of 7.48, minimal effect of MgCl2 or CaCl2 at physiologic pH, and inhibition by alpha,beta-methylene ADP, a known 5'-nucleotidase inhibitor. We established that the monolayer assay was indeed measuring cell surface associated 5'-nucleotidase. To determine the effect of endotoxin, we incubated confluent monolayers with endotoxin in Minimal Essential Medium plus 10% fetal bovine serum for 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 response to endotoxin injury may represent an important clearance mechanism for circulating adenine nucleotides and may be protective in acute vascular injury by increasing adenosine production.
腺苷可能通过抑制血小板聚集和中性粒细胞氧化剂释放，对急性血管损伤起到保护作用。相比之下，可能伴随急性血管损伤而释放的腺嘌呤核苷酸会刺激血小板聚集和中性粒细胞氧化剂释放。外核苷酸酶是一类分解细胞外核苷酸的膜酶，是将循环核苷酸降解为腺苷的主要机制。外5'-核苷酸酶可将细胞外的AMP转化为腺苷。我们推测内皮细胞损伤会改变外5'-核苷酸酶的活性。利用Jamal等人（《生物化学杂志》250: 369 - 373, 1988）首次报道的一种用于大鼠脂肪细胞的新型检测方法，我们研究了培养的牛肺动脉内皮细胞（BPAEC）完整单层中外5'-核苷酸酶的特性，并检测了内毒素对酶活性的影响。该检测方法使用AMP的荧光类似物1,N6-乙烯基-AMP（E-AMP）作为外5'-核苷酸酶的底物，并测量乙烯基腺苷（E-Ado）的生成。将pH 7.4、22℃的Hepes缓冲液中的E-AMP添加到汇合的BPAEC单层中；在不同时间间隔后收集上清液样本，通过高效液相色谱法对E-AMP和E-Ado进行定量。使用这些方法，我们发现其Km为15±6微摩尔，最适pH为7.48，在生理pH下MgCl2或CaCl2的影响最小，且受到已知的5'-核苷酸酶抑制剂α,β-亚甲基ADP的抑制。我们确定单层检测确实测量的是细胞表面相关的5'-核苷酸酶。为了确定内毒素的影响，我们将汇合的单层细胞与内毒素在最低限度基本培养基加10%胎牛血清中孵育24小时，洗涤后，评估内毒素损伤细胞将E-AMP转化为E-Ado的情况。内毒素刺激内皮外5'-核苷酸酶活性。这种对内毒素损伤的5'-核苷酸酶活性增加可能代表了循环腺嘌呤核苷酸的一种重要清除机制，并且可能通过增加腺苷生成对急性血管损伤起到保护作用。
