Interaction between the partially insurmountable antagonist valsartan and human recombinant angiotensin II type 1 receptors.

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.

Inhibition of angiotensin II-induced inositol phosphate production by triacid nonpeptide antagonists in CHO cells expressing human AT1 receptors.

PURPOSE: The aim of the present work is to describe the inhibitory properties of LY301875 and LY303336, two polysubstituted 4-aminoimidazole AT1 receptor antagonists, on CHO cells expressing human recombinant AT1 receptors. METHODS: The binding of [3H]-angiotensin II to intact cells as well as to angiotensin II induced inositol phosphate accumulation is measured. RESULTS: Both antagonists inhibit specific [3H]-angiotensin II binding to AT1 receptors in these cells, with IC50 values of 5.9 and 5.2 nM, respectively. Preincubation of the cells with LY301875 results in a decline of up to 80% of the maximal angiotensin II-stimulated inositol phosphate (IP) production. A near complete decline of the maximal response is observed for LY303336. This insurmountable inhibition is attenuated for both antagonists when losartan is included during the preincubation of the cells. CONCLUSIONS: Functional recovery experiments, in which antagonist-preincubated cells are washed and exposed to fresh media, suggest that the insurmountable inhibition by LY301875 and LY303336 is related to their relatively slow dissociation from the AT1 receptors. As already described for losartan and the derived insurmountable AT1 antagonists candesartan. EXP3174, and irbesartan, coincubation experiments reveal that LY301875 and LY303336 interact with the AT1 receptor in a manner that is competitive with angiotensin II.

Effect of BIBP3226 on inositol phosphate accumulation and cytosolic calcium level in control and NPY Y1 receptor expressing CHO-K1 cells.

BIBP3226 was developed as a potent, selective and competitive antagonist for NPY Y1 receptors by mimicking the C-terminal part of NPY. In agreement with previous studies, NPY mediated a pertussis toxin sensitive elevation of intracellular calcium concentration in CHO-K1 cells that express recombinant human NPY Y1 receptors which can be inhibited by BIBP3226. Surprisingly micromolar concentrations of BIBP3226 were found to induce by itself a fast increase of intracellular calcium concentration followed by a sustained elevated level of this ion. These responses of BIBP3226 are not mediated by NPY receptor activation since (1) they are still present after NPY receptor activation and desensitization, (2) they are also evoked by the receptor inactive enantiomer BIBP3435, (3) they are not affected by pretreatment of the cells with pertussis toxin, (4) they also occur in non-transfected CHO-K1 cells. Preincubation of the cells with EGTA abolished only the sustained increase calcium concentration elicited by BIBP3226 suggesting that the fast increase of intracellular calcium concentration reflects the mobilization of intracellular calcium pools. The ability of thapsigargin to completely inhibit BIBP3226 mediated responses, in the presence or absence of extracellular calcium indeed indicated that BIBP3226 mobilizes intracellular Ins(1,2,3)P3 sensitive calcium stores. In agreement, BIBP3226 was found to activate phospholipase C since the responses were completely inhibited by U73122. Furthermore, when measured in the presence of 10 mM LiCl, BIBP3226 caused an increased accumulation of inositol phosphates. This effect of BIBP3226 is likely to be mediated by activation of an until now unknown receptor or cellular target that is endogeneously expressed in CHO-K1 cells.