Spurney R F, Onorato J J, Albers F J, Coffman T M
Department of Medicine, Duke University Medical Center, Durham, North Carolina.
Am J Physiol. 1993 Feb;264(2 Pt 2):F292-9. doi: 10.1152/ajprenal.1993.264.2.F292.
Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (1R-[1 alpha(Z),2 beta,3 beta,5 alpha])-(+)-7-(5-[(1,1'-biphenyl)- 4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)
血栓素A2（TxA2）刺激肾小球系膜细胞收缩。然而，系膜细胞TxA2受体此前尚未得到鉴定。因此，我们使用特异性血栓素受体激动剂（1S-[1α,2β(5Z),3α-(1E,3S)4α]）-7-(3-[3-羟基-4-(对碘苯氧基)-1-丁烯基]7-氧杂双环[2.2.1]庚-2-基)-5-庚烯酸（IBOP），研究了大鼠肾小球系膜细胞中TxA2的结合及与TxA2相关的信号转导途径。在这些细胞中，[125I]BOP结合具有饱和性、可置换性且亲和力高。Scatchard分析显示存在一类结合位点，解离常数（Kd）为293 pM，最大结合位点密度（Bmax）为33 fmol/mg蛋白。血栓素激动剂（15S）-羟基-11α,9α-(环氧亚甲基)前列腺-5Z,13E-二烯酸（U-46619）[抑制剂解离常数（Ki）= 297 nM]以及TxA2受体拮抗剂SQ 29548（Ki = 1 nM）和（1R-[1α(Z),2β,3β,5α]）-(+)-7-(5-[(1,1'-联苯)-4-基甲氧基]-3-羟基-2-(1-哌啶基)环戊基]-4-庚烯酸（GR 32191）（Ki = 92 nM）可抑制特异性结合。结合对血栓素也具有高度特异性，因为前列腺素E2（Ki = 16 μM）和无活性的血栓素代谢产物TxB2（Ki = 41 μM）抑制结合的效力约低1000倍。IBOP刺激磷脂酰肌醇水解，产生最大反应50%时的药物有效浓度为229 pM，这与平衡Kd密切相关，并增强了蛋白激酶C的酸性80 kDa蛋白底物的磷酸化。（摘要截短于250字）
