Portilla D, Mordhorst M, Bertrand W, Irwin C, Morrison A R
Department of Medicine, University of Arkansas College of Medicine, Little Rock.
J Lab Clin Med. 1992 Nov;120(5):752-61.
To evaluate the identity of the guanosine triphosphate--binding proteins coupling arginine vasopressin receptor occupancy with activation of phospholipase C, leading to Ca2+ mobilization, and activation of phospholipase A2, leading to arachidonate release and prostanoid formation, we used intact cells, saponin-permeabilized cells, and membranes of the rat mesangial cell. Arginine vasopressin 10(-7) mol/L produced a dose-dependent increase in cytosolic Ca2+ to maximal levels of 500 nmol/L with peak responses occurring within 10 seconds of addition of arginine vasopressin to cells in suspension. Arginine vasopressin 10(-7) mol/L elicited a maximal response. These increases were associated temporarily with a fourfold increase in tritiated D-myo-inositol 1,4,5-trisphosphate formation in prelabeled cells. Pertussis toxin (200 ng/ml) did not inhibit the Ca2+ increase nor did it inhibit the increase in tritiated D-myo-inositol 1,4,5-trisphosphate formation, suggesting a pertussis toxin--insensitive signaling pathway for phospholipase C hydrolysis in response to vasopressin. Membranes prepared from mesangial cells increased D-myo-inositol 1,4,5-trisphosphate formation in vitro in response to arginine vasopressin and guanosine-5'-0(3- thiotrisphosphate), and this stimulation was inhibited by guanosine-5'-0(2-thiodiphosphate), confirming the involvement of a guanosine triphosphate--binding protein. In contrast arginine vasopressin stimulated arachidonate release from intact mesangial cells, and this effect was blocked by pretreating cells with pertussis toxin. To demonstrate that this was through a pertussis toxin--sensitive guanosine triphosphate--binding protein, we permeabilized cells with saponin and determined that arginine vasopressin and guanosine-5'-0(3-thiotriphosphate) stimulated the release of arachidonic acid and the stimulation of guanosine-5'-0(3-thiotriphosphate) was inhibited by guanosine-5'-0(2-thiodiphosphate). Finally, pertussis toxin was able to stimulate adenosine diphosphate ribosylation in vivo of a substrate protein in mesangial cell membranes of 41 kd, and this ribosylation was inhibited by pretreating cells with pertussis toxin. These data suggest that the release of arachidonic acid by vasopressin in glomerular mesangial cells is linked to a pertussis toxin--sensitive guanosine triphosphate--binding protein and that this activation of phospholipase C in vasopressin is linked to a pertussis toxin--insensitive guanosine triphosphate--binding protein.
为评估将精氨酸加压素受体占据与磷脂酶C激活（导致Ca2+动员）以及磷脂酶A2激活（导致花生四烯酸释放和前列腺素形成）相偶联的鸟苷三磷酸结合蛋白的特性，我们使用了完整细胞、皂角苷通透细胞和大鼠系膜细胞的细胞膜。10(-7) mol/L的精氨酸加压素使细胞溶质Ca2+呈剂量依赖性增加至最大水平500 nmol/L，在向悬浮细胞中添加精氨酸加压素后10秒内出现峰值反应。10(-7) mol/L的精氨酸加压素引发最大反应。这些增加与预标记细胞中氚化D-肌醇1,4,5-三磷酸形成增加四倍暂时相关。百日咳毒素（200 ng/ml）既不抑制Ca2+增加，也不抑制氚化D-肌醇1,4,5-三磷酸形成增加，提示存在一条对百日咳毒素不敏感的磷脂酶C水解信号通路以响应加压素。从系膜细胞制备的细胞膜在体外对精氨酸加压素和鸟苷-5'-O(3-硫代三磷酸)反应增加D-肌醇1,4,5-三磷酸形成，且这种刺激被鸟苷-5'-O(2-硫代二磷酸)抑制，证实了鸟苷三磷酸结合蛋白的参与。相反，精氨酸加压素刺激完整系膜细胞释放花生四烯酸，且此效应被用百日咳毒素预处理细胞所阻断。为证明这是通过对百日咳毒素敏感的鸟苷三磷酸结合蛋白实现的，我们用皂角苷使细胞通透并确定精氨酸加压素和鸟苷-5'-O(3-硫代三磷酸)刺激花生四烯酸释放，且鸟苷-5'-O(3-硫代三磷酸)的刺激被鸟苷-5'-O(2-硫代二磷酸)抑制。最后，百日咳毒素能够在体内刺激系膜细胞膜中41 kd底物蛋白的二磷酸腺苷核糖基化，且这种核糖基化被用百日咳毒素预处理细胞所抑制。这些数据提示加压素在肾小球系膜细胞中释放花生四烯酸与对百日咳毒素敏感的鸟苷三磷酸结合蛋白相关，且加压素中磷脂酶C的这种激活与对百日咳毒素不敏感的鸟苷三磷酸结合蛋白相关。
