Garcia J G, Painter R G, Fenton J W, English D, Callahan K S
Department of Medicine, Indiana University School of Medicine, Indianapolis 46208.
J Cell Physiol. 1990 Jan;142(1):186-93. doi: 10.1002/jcp.1041420123.
The regulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGI2 was monitored by RIA of 6-keto PGF1 alpha and dose-dependent increases observed with human alpha- and gamma-thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6-keto PGF1 alpha approximating responses with 1 microM gamma-thrombin, 5 microM arachidonate, or 10 microM histamine. Diisopropyl phosphorofluoridate-inactivated alpha-thrombin did not stimulate PGI2 release, demonstrating that catalytic activity was required for thrombin-stimulated PGI2 release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGI2 synthesis in a dose-dependent and time-dependent manner (20 mM NaF, 4.4 +/- 0.5-fold increase at 10 min, 11.9 +/- 1.5-fold increase at 30 min). Neither alpha-thrombin nor NaF-stimulated PGI2 release was dependent upon the availability of extracellular Ca++). The hypothesis that G proteins are involved in agonist-stimulated PGI2 synthesis was further supported by studies using digitonin-permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5'-0-3-thiotrisphosphate (GTP gamma S), which effected significant dose-dependent increases in PGI2 synthesis compared with control levels of 6-keto PGF1 alpha. In contrast, the G-protein inhibitor GDP beta S, (guanosine 5'-0-2-thiodiphosphate), attenuated alpha-thrombin-mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 microgram/ml) did not inhibit the PGI2 synthesis stimulated by either alpha-thrombin, NaF, or histamine but catalyzed the ADP ribosylation of a 40 kDa membrane protein which cross-reacted with antisera against a synthetic peptide corresponding to an amino acid sequence common to the alpha-subunit of other G-proteins. Preincubation of HUVEC microsomal membranes with alpha-thrombin diminished pertussis toxin-catalyzed ADP ribosylation in a time-dependent manner. These data suggest that thrombin stimulation of PGI2 synthesis by HUVEC monolayers requires the catalytically functional enzyme and further suggests that the thrombin-occupied receptor is coupled to phospholipase activities by a pertussis toxin-insensitive guanine nucleotide regulatory protein in human endothelial cell membranes.
研究了培养的人脐静脉内皮细胞（HUVEC）对前列环素（PGI2）合成的调节作用。通过放射免疫分析法（RIA）检测6-酮-前列腺素F1α（6-keto PGF1α）来监测HUVEC单层细胞产生的PGI2，并观察到人类α-和γ-凝血酶、组胺或花生四烯酸可使其呈剂量依赖性增加。α-凝血酶（10 nM）产生的6-酮-前列腺素F1α水平与1 μM γ-凝血酶、5 μM花生四烯酸或10 μM组胺所引发的反应相近。经二异丙基氟磷酸酯灭活的α-凝血酶不会刺激PGI2释放，表明凝血酶刺激PGI2释放需要催化活性。已知能激活鸟嘌呤核苷酸调节蛋白（G蛋白）的氟化钠（NaF），以剂量依赖性和时间依赖性方式直接刺激HUVEC合成PGI2（20 mM NaF，10分钟时增加4.4±0.5倍，30分钟时增加11.9±1.5倍）。α-凝血酶和NaF刺激的PGI2释放均不依赖细胞外钙离子的存在。使用洋地黄皂苷通透的HUVEC单层细胞，并用另一种G蛋白激活剂鸟苷5'-O-3-硫代三磷酸（GTPγS）进行刺激的研究进一步支持了G蛋白参与激动剂刺激的PGI2合成这一假说，与6-酮-前列腺素F1α的对照水平相比，GTPγS可使PGI2合成显著增加且呈剂量依赖性。相反，G蛋白抑制剂GDPβS（鸟苷5'-O-2-硫代二磷酸）可减弱α-凝血酶介导的前列腺素生成。用百日咳毒素（1 μg/ml）处理HUVEC单层细胞，不会抑制α-凝血酶、NaF或组胺刺激的PGI2合成，但会催化一种40 kDa膜蛋白的ADP核糖基化，该蛋白与针对与其他G蛋白α亚基共同氨基酸序列的合成肽的抗血清发生交叉反应。用α-凝血酶对HUVEC微粒体膜进行预孵育，可使百日咳毒素催化的ADP核糖基化呈时间依赖性减少。这些数据表明，HUVEC单层细胞中凝血酶刺激PGI2合成需要具有催化功能的酶，进一步表明在人内皮细胞膜中，被凝血酶占据的受体通过一种对百日咳毒素不敏感的鸟嘌呤核苷酸调节蛋白与磷脂酶活性相偶联。
