Fig. 3
From: Identification of a Novel V1-type AVP Receptor Based on the Molecular Recognition Theory
Functional characterization of the V6 cDNA encoded AVP receptor expressed in Cos1pMAM-V6 cells. (A) AVP-induced 45Ca2+ mobilization in intact Cos1pMAM-V6 cells. Cells were exposed to 0.1 µM AVP (●) in comparison to basal control conditions (i.e., no hormone added [○]) and 45Ca2+-efflux measured as the % 45Ca2+-retained at 10, 20 and 30 sec. (B) Peptide hormone response profile of 45Ca2+ mobilization in Cos1pMAM-V6 cells. The % 45Ca2+ efflux activity was determined in response to various peptide ligands (1 nM): AVP, angiotensin II (AngII), bradykinin (bradyk), and endothelin-1 (Et-1). Specificity for AVP stimulation was tested with the concurrent incubation of AVP (1 nM) with a V1 antagonist, [dCH2)5, Tyr(Me)]-AVP (24) (V1 antag) at 100 nM. 45Ca2+ efflux activity was defined as the difference between cells exposed to PB containing the different ligands and cells exposed to PB alone; incubation time was 10 sec. Results are expressed as percent of 45Ca2+ efflux activity relative to the activity obtained in Cos1pMAM-V6 cultures exposed to 1 nM AVP. (C) Concentration dependence of AVP-induced Ca2+ mobilization in Cos1pMAM-V6 cells. 45Ca2+ efflux was measured in response to varying concentrations of AVP (from 10−13−10−7 M) at the 10-sec time point. Results are expressed as percent 45Ca2+ efflux activity relative to Cos1pMAM-V6 cells exposed to 100 nM AVP for 10 sec. (D) Characterization of AVP-induced IP3 formation in Cos1pMAM-V6 cells. IP3 formation (pmols 10 −6 cells) was measured in Cos1pMAM-V6 cells exposed to 0.1 µM AVP (●) or to control buffer (○) at 10, 20, and 30 sec. (E) Concentration dependence of the AVP-induced IP3 formation in Cos1pMAM-V6 cells measured at 10 sec. (F) Pharmacologic properties of the V6 cDNA encoded AVP receptor. Competition for 3H-AVP specific binding on intact Cos1pMAM-V6 cells by various AVP analogs is presented in comparison to dissociation by unlabeled AVP per se (●). (◆), V1 antagonist, [dCH2)5, Tyr(Me)]-AVP (24); (◇)V1/V2 antagonist, [d(CH2)5, D-Ile2, Ile4]-AVP (25); and (○), V2 agonist, DVDAVP (26). Values for respective affinities (Kd and Ki) are presented in Table 1. Results shown in (A), (B), (C), (D), and (E) are from experiments performed in triplicate with corresponding standard deviations (bars). Results shown in (F) are from experiments performed in triplicate with an average standard deviation of 8.7%.