Effects of YM471, a nonpeptide AVP V(1A) and V(2) receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells

Abstract

YM471, (Z)-4'-[4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl]-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [(3)H]-AVP binding to V(1A) and V(2) receptors with K(i) values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V(1B) and oxytocin receptors with K(i) values of 16.4 microM and 31.6 nM, respectively. In CHO cells expressing V(1A) receptors, YM471 potently inhibited AVP-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, exhibiting an IC(50) value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca(2+)](i) increase (IC(50)=193 nM), and did not affect AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC(50) value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V(1A) and V(2) receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP.

Figure 1

Chemical structure of YM471, (Z)-4′-{4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-phenylbenzanilide monohydrochloride.

Figure 2

Inhibition of specific [³H]-AVP or [³H]-oxytocin bound to plasma membranes prepared from CHO cells expressing human (a) V_1A, (b) V₂, (c)V_1B, and (d) oxytocin receptors by AVP receptor antagonists. Results are representative data from three to five independent experiments performed in duplicate. The combined results of all experiments are summarized in Table 1.

Figure 3

Scatchard plots of [³H]-AVP binding to plasma membranes prepared from CHO cells expressing human (a) V_1A and (b) V₂ receptors in the absence or presence of YM471. Results are representative data from four independent experiments performed in duplicate.

Figure 4

(a) Effect of AVP on [Ca²⁺]_i increase and (b) effect of AVP receptor antagonists on 10 nM AVP-induced [Ca²⁺]_i increases in CHO cells expressing human V_1A receptors. Values are mean±s.e.mean from three to eight independent determinations.

Figure 5

(a) Effect of AVP on [Ca²⁺]_i increase and (b) effect of AVP receptor antagonists on 10 nM AVP-induced [Ca²⁺]_i increases in CHO cells expressing human V_1B receptors. Values are mean±s.e.mean from three to eight independent determinations.

Figure 6

(a) Effect of oxytocin on [Ca²⁺]_i increase and (b) effect of AVP receptor antagonists on 100 nM oxytocin-induced [Ca²⁺]_i increases in human USMC. Values are mean±s.e.mean from three to eight independent determinations.

Figure 7

(a) Effect of AVP on production of cellular cyclic AMP and (b) effect of AVP receptor antagonists on 10 nM AVP-induced cellular cyclic AMP production in CHO cells expressing human V₂ receptors. Values are mean±s.e.mean from eight independent determinations.