Rovati G E, Oliva D, Sautebin L, Folco G C, Welton A F, Nicosia S
Biochem Pharmacol. 1985 Aug 15;34(16):2831-7. doi: 10.1016/0006-2952(85)90003-6.
Leukotriene C4 (LTC4), one of the major components of the slow-reacting substance of anaphylaxis (SRS-A), is a potent constrictor of bronchial smooth muscle in many species including humans. Here we report the identification and characterization of specific binding sites for LTC4 in membranes from human lung parenchyma. At 4 degrees, 3H-LTC4 binding is specific, saturable (Bmax = 32-41 pmoles/mg prot.), rapid (equilibrium being attained within 15 min), reversible and of high affinity (Kd = 3.6-7 X 10(-8) M). The binding sites are sensitive to heat and probably possess a protein moiety, being inactivated upon trypsinization. CaCl2 affects both the association and the dissociation rate and dose-dependently enhances the binding of 3H-LTC4 at equilibrium; maximal enhancement (4-fold) occurred at 10(-2)M CaCl2. Unlabelled LTC4 is able to complete with 3H-LTC4 for its binding sites with an IC50 of 7.8 X 10(-8) M. The addition of 10(-2) M CaCl2 increases the potency of LTC4 in inhibiting the binding (2.2-fold); both the competition curves are monophasic, indicating the existence of a homogeneous class of binding sites. In the presence of CaCl2, LTD4, LTE4 and the SRS-A antagonist FPL 55712 can inhibit 3H-LTC4 specific binding, being, however, less potent than LTC4 (IC50 S = 2.2 X 10(-6), 2.4 X 10(-5) M, for LTD4, LTE4 and FPL 55712, respectively). FPL 55712 displayed a competitive mechanism; its affinity, however, was lower if absorption to glass was not prevented. The present studies indicate that specific binding sites for 3H-LTC4 exist in human lung parenchyma, and that a receptor-mediated process might be involved in the bronchoconstriction induced by LTC4.
白三烯C4（LTC4）是过敏反应慢反应物质（SRS - A）的主要成分之一，在包括人类在内的许多物种中是支气管平滑肌的强效收缩剂。在此，我们报告人肺实质细胞膜中LTC4特异性结合位点的鉴定和特性。在4℃时，3H - LTC4结合具有特异性、可饱和性（Bmax = 32 - 41皮摩尔/毫克蛋白）、快速（15分钟内达到平衡）、可逆且具有高亲和力（Kd = 3.6 - 7×10⁻⁸M）。这些结合位点对热敏感，可能含有蛋白质部分，经胰蛋白酶处理后失活。CaCl2影响结合和解离速率，并剂量依赖性地增强平衡时3H - LTC4的结合；在10⁻²M CaCl2时出现最大增强（4倍）。未标记的LTC4能够与3H - LTC4竞争其结合位点，IC50为7.8×10⁻⁸M。添加10⁻²M CaCl2可增加LTC4抑制结合的效力（2.2倍）；两条竞争曲线均为单相，表明存在一类同质的结合位点。在CaCl2存在下，LTD4、LTE4和SRS - A拮抗剂FPL 55712可抑制3H - LTC4特异性结合，但其效力低于LTC4（LTD4、LTE4和FPL 55712的IC50分别为2.2×10⁻⁶、2.4×10⁻⁵M）。FPL 55712表现出竞争机制；然而，如果不防止其吸附到玻璃上，其亲和力较低。目前的研究表明，人肺实质中存在3H - LTC4的特异性结合位点，并且受体介导的过程可能参与LTC4诱导的支气管收缩。
