Verheijen I, Fierens F L, Debacker J P, Vauquelin G, Vanderheyden P M
Department of Molecular and Biochemical Pharmacology, Free University of Brussels, Belgium.
Fundam Clin Pharmacol. 2000 Nov-Dec;14(6):577-85. doi: 10.1111/j.1472-8206.2000.tb00443.x.
The interaction between the AT1 receptor-selective antagonist valsartan, and its human receptor, was investigated by direct radioligand binding as well as by its inhibition of angiotensin II induced inositol phosphate accumulation in CHO cells expressing human recombinant AT1 receptors. Specific binding of [3H]-valsartan rapidly reached equilibrium at 37 degrees C. It was saturable and occurred to a homogeneous class of sites with a K(D) of 0.88+/-0.076. It was inhibited by other AT1 receptor antagonists with the same potency order as previously described for the binding of [3H]-angiotensin II and [3H]-candesartan to human AT1 receptors (i.e. candesartan > or = EXP3174 > valsartan = irbesartan = angiotensin II > losartan). When valsartan and angiotensin II were applied simultaneously to the CHO-AT1 cells. the antagonist caused a rightward shift of the angiotensin II concentration response curve. Hence, valsartan interacts with the AT1 receptor in a manner that is competitive with angiotensin II. Pre-incubation of the cells with 0.5, 5 and 50 nM valsartan caused an additional, concentration-dependent, up to 55% decline of the maximal response. The partial nature of this insurmountable inhibition by valsartan was confirmed by biphasic antagonist concentration-inhibition curves. These data reflect the co-existence of a fast reversible/surmountable as well as a tight binding/insurmountable valsartan receptor complex. In agreement, pre-incubation of the CHO-AT1 cells with 5 and 50 nM valsartan produced a partial inhibition of the angiotensin II induced increase of the free intracellular calcium concentration. [3H]-Valsartan dissociated from its receptors with a half-life of 17 min. In functional recovery experiments with valsartan-pre-treated cells, the angiotensin II-mediated response was half-maximally restored within approximately 30 min. These kinetic data suggest that the insurmountable inhibition by valsartan is related to its relatively slow dissociation from the human AT1 receptors.
通过直接放射性配体结合以及其对表达人重组AT1受体的CHO细胞中血管紧张素II诱导的肌醇磷酸积累的抑制作用，研究了AT1受体选择性拮抗剂缬沙坦与其人受体之间的相互作用。[3H] - 缬沙坦的特异性结合在37℃下迅速达到平衡。它是可饱和的，并且发生在一类均匀的位点上，K(D)为0.88±0.076。它被其他AT1受体拮抗剂抑制，其效力顺序与先前描述的[3H] - 血管紧张素II和[3H] - 坎地沙坦与人AT1受体结合的顺序相同（即坎地沙坦≥EXP3174＞缬沙坦 = 厄贝沙坦 = 血管紧张素II＞氯沙坦）。当缬沙坦和血管紧张素II同时应用于CHO - AT1细胞时，拮抗剂导致血管紧张素II浓度反应曲线向右移动。因此，缬沙坦以与血管紧张素II竞争的方式与AT1受体相互作用。用0.5、5和50 nM缬沙坦对细胞进行预孵育导致最大反应额外的、浓度依赖性的下降，高达55%。缬沙坦这种不可克服抑制作用的部分性质通过双相拮抗剂浓度 - 抑制曲线得到证实。这些数据反映了快速可逆/可克服以及紧密结合/不可克服的缬沙坦受体复合物的共存。一致的是，用5和50 nM缬沙坦对CHO - AT1细胞进行预孵育对血管紧张素II诱导的细胞内游离钙浓度升高产生部分抑制作用。[3H] - 缬沙坦从其受体解离的半衰期为17分钟。在用缬沙坦预处理的细胞进行的功能恢复实验中，血管紧张素II介导的反应在大约30分钟内恢复到最大反应的一半。这些动力学数据表明，缬沙坦的不可克服抑制作用与其从人AT1受体相对缓慢的解离有关。
