Benzodiazepine modulation of recombinant alpha1beta3gamma2 GABA(A) receptor function efficacy determination using the Cytosensor microphysiometer

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.     

Activation of neurotensin receptors and purinoceptors in human colonic adenocarcinoma cells detected with the microphysiometer

Activation of endogenous neurotensin (NT) receptors and P2-purinoceptors expressed by human colonic adenocarcinoma HT-29 cells increased extracellular acidification rates that were detected in the microphysiometer. NT (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), NT[8-13] (Arg-Arg-Pro-Tyr-Ile-Leu), NT[9-13] (Arg-Pro-Tyr-Ile-Leu), and NT1 (N alpha methyl-Arg-Lys-Pro-Trp-Tle-Leu [Tle = tert-leucine]) were full agonists, whereas XL 775 (N-[N-[2-[3-[[6-amino-1-oxo-2-[[(phenylmethoxy)carbonyl]-amino]hex yl]amino]phenyl]-3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-L-isoleucyl]-L-le ucine) was a partial agonist for activating NT receptors expressed by HT-29 cells. Desensitization induced by NT was rapid and monophasic with 85% of the initial response lost by a 30-s exposure. Once initiated, the rate and extent of desensitization were similar for different concentrations of a given agonist, for agonists of different potencies, and for agonists of different efficacies, which suggests that desensitization may be independent of receptor occupancy or agonist efficacy. Resensitization was a much slower process, requiring 60 min before the full agonist response to NT was recovered. ATP, via P2-purinoceptors, also activated cellular acidification rates in a concentration-dependent manner. ATP induced a biphasic desensitization of purinoceptors with a loss of ca. 50% of the initial stimulation detectable between 30 and 90 s of exposure to the agonist. Desensitization of NT receptors did not influence the activation of P2-purinoceptors by ATP, suggesting there was no heterologous desensitization between the two types of receptors. Superfusion with NT receptor agonists for 15 min at concentrations that did not elicit changes in extracellular acidification rates blocked, in a concentration-dependent manner, the agonist response induced by 100 nM NT. This may reflect sequestration of the receptor. These results suggest that the high agonist affinity state of NT receptors may modulate receptor sequestration, whereas activation of the low agonist affinity state may be linked to cellular metabolism. Comparison of our results with published data found differences as well as similarities of NT responses among three lines of HT-29 cells.     

Binding of human single chain urokinase to Chinese Hamster Ovary cells and cloning of hamster u-PAR

In the present study, we assessed oxidative stress in patients with dilated cardiomyopathy of ischemic or idiopathic etiology. For this reason we measured whole blood reduced glutathione, erythrocyte superoxide dismutase, susceptibility of erythrocyte membranes and erythrocytes to peroxidation, and SH content of erythrocyte membranes in 12 patients (8 men and 4 women, ages 31 to 66 years) with idiopathic dilated cardiomyopathy, in 11 patients (8 men and 3 women, ages 32 to 65 years) with ischemic dilated cardiomyopathy, and in 21 healthy volunteers (12 men and 9 women, ages 25 to 67 years). There was no statistically significant difference between the two patient groups for the indicators studied (P >0.05). Blood glutathione, erythrocyte superoxide dismutase, and membrane SH content of both groups of patients was decreased compared with controls (P <0.05), whereas erythrocyte and membrane susceptibility to peroxidation were increased (P <0.05). We conclude that patients with idiopathic or ischemic dilated cardiomyopathy exhibit abnormalities of a range of markers of increased oxidative stress. These abnormalities may contribute to contractile dysfunction, increased incidence of fatal arrhythmias, and sudden death.     

Activation of phospholipase A2 by the nociceptin receptor expressed in Chinese hamster ovary cells

To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca2+-dependent cytosolic PLA2 (cPLA2) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA2, suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX-sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor on human thymocytes

Interleukin-7 (IL-7) is a glycoprotein that regulates lymphocyte precursor growth and differentiation. However, the exact mechanism whereby the IL-7 receptor (IL-7R) mediates these cell growth signals remains unknown. One of the earliest metabolic events linked to mitogenic responses in other growth factor receptor systems is the activation of phosphatidylinositol-3 kinase (PI-3 kinase). We demonstrate here that ligation of the IL-7R results in dose- and time-dependent increases in PI-3 kinase activity. These results suggest that PI-3 kinase is involved in signal transduction via the IL-7R in human thymocytes.     

Activation of dopaminergic and cholinergic neurotransmission by tachykinin NK3 receptor stimulation: an in vivo microdialysis approach in guinea pig

The regulation of dopaminergic and cholinergic function by neurokinin-3 (NK3) receptor activation was examined in vivo in urethane-anaesthetized guinea pigs with microdialysis probes. The local application of the NK3 tachykinin receptor agonist senktide in the region of dopamine cell bodies (pars compacta of the substantia nigra and ventral tegmental area) and in the area of cholinergic cell bodies (septal area) markedly enhanced the extracellular dopamine (DA) and acetylcholine (ACh) concentration throughout their respective target areas, i.e. striatum, nucleus accumbens, prefrontal cortex for dopaminergic systems and hippocampus for cholinergic neurons. The enhancing effect of senktide on neurotransmitter release was dose dependently blocked by the selective non-peptide NK3 receptor antagonist SR142801 (0.1-1 mg/kg, i.p.), whereas its inactive S-enantiomer SR142806 (0.3-1 mg/kg, i.p.) did not exert any antagonistic activity on the effect of intranigral or intraseptal application of senktide. These results demonstrate that NK3 receptors can modulate the activity of central DA and ACh systems.     

Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two different sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Specifically, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highly-metastatic cells. EGFR was found to associate with c-Src in colon cancer cells and specific inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identified in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.     

Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells

The novel hematopoietic growth factor FLT3 ligand (FL) is the cognate ligand for the FLT3, tyrosine kinase receptor (R), also referred to as FLK-2 and STK-1. The FLT3R belongs to a family of receptor tyrosine kinases involved in hematopoiesis that also includes KIT, the receptor for SCF (stem cell factor), and FMS. the receptor for M-CSF (macrophage colony- stimulating factor). Restricted FLT3R expression was seen on human and murine hematopoietic progenitor cells. In functional assays recombinant FL stimulated the proliferation and colony formation of human hematopoietic progenitor cells, i.e. CD34+ cord and peripheral blood, bone marrow and fetal liver cells. Synergy was reported for co-stimulation with G-CSF (granulocyte-CSF). GM-CSF (granulocyte-macrophage CSF), M-CSF, interleukin-3 (IL-3), PIXY-321 (an IL-3/GM-CSF fusion protein) and SCF. In the mouse, FL potently enhanced growth of various types of progenitor/precursor cells in synergy with G-CSF, GM-CSF, M-CSF, IL-3, IL-6, IL-7, IL-11, IL-12 and SCF. The well-documented involvement of this ligand-receptor pair in physiological hematopoiesis brought forth the question whether FLT3R and FL might also have a role in the pathobiology of leukemia. At the mRNA level FLT3R was expressed by most (80-100%) cases of AML (acute myeloid leukemia) throughout the different morphological subtypes (MO-M7), of ALL(acute lymphoblastic leukemia) of the immunological subtypes T-ALL and BCP-ALL (B cell precursor ALL including pre-pre B-ALL, cALL and pre B-ALL), of AMLL (acute mixed-lineage leukemia), and of CML (chronic myeloid leukemia) in lymphoid or mixed blast crisis. Analysis of cell surface expression of FLT3R by flow cytometry confirmed these observations for AML (66% positivity when the data from all studies are combined), BCP-ALL (64%) and CML lymphoid blast crisis (86%) whereas less than 30% of T-ALL were FLT3R+. The myeloid, monocytic and pre B cell type categories also contained the highest proportions of FLT3R+ leukemia cell lines . In contrast to the selective expression of the receptor, FL expression was detected in 90-100% of the various cell types of leukemia cell lines from all hematopoietic cell lineages. The potential of FL to induce proliferation of leukemia cells in vitro was also examined in primary and continuously cultured leukemia cells. The data on FL-stimulated leukemia cell growth underline the extensive heterogeneity of primary AML and ALL samples in terms of cytokine-inducible DNA synthesis that has been seen with other effective cytokines. While the majority of T-ALL (0-33% of the cases responded proliferatively; mean 11%) and BCP-ALL (0-30%; mean 20%) failed to proliferate in the presence of FL despite strong expression of surface FLT3R, FL caused a proliferative response in a significantly higher percentage of AML cases (22-90%; mean 53%). In the panel of leukemia cell lines examined only myeloid and monocytic growth factor- dependent cell lines increased their proliferation upon incubation with FL, whereas all growth factor-independent cell lines were refractory to stimulation. Combinations of FL with G-CSF, GM-CSF, M-CSF, IL-3, PIXY- 321 or SCF and FL with IL-3 or IL-7 had synergistic or additive mitogenic effects on primary AML and ALL cells, respectively. The potent stimulation of the myelomonocytic cell lines was further augmented by addition of bFGF (basic fibroblast growth factor), GM-CSF, IL-3 or SCF. The inhibitory effects of TGF-beta 1 (transforming growth factor-beta 1) on FL- supported proliferation were abrogated by bFGF. Taken together, these results demonstrate the expression of functional FLT3R capable of mediating FL- dependent mitogenic signaling in a subset of AML and ALL cases further underline the heterogeneity of AML and ALL samples in their proliferative response to cytokine.     

Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin

Integrins mediate cell adhesion and can induce different cellular responses, including changes in intracellular pH, changes and oscillation in intracellular free calcium, and protein phosphorylation on tyrosine. During bone resorption, the integrin alphav beta3 regulates adhesion of osteoclasts to bone extracellular matrix proteins, such us osteopontin (Opn). Adhesion via alphav beta3 is followed by osteoclast polarization onto the bone surface and by the onset of bone resorption. To characterize these events at the molecular level, we investigated the state of activation of alphav beta3 on the human osteoclast-like cell line GCT23 using the monoclonal antibody AP5 which binds to and can induce, under low calcium conditions, activated alphav beta3. By flow cytometry, approximately 50% of alphav beta3 on the surface of the osteoclast-like cell line GCT23 was reactive with AP5 and was therefore in the activated state. Incubation with AP5 in the presence of low calcium concentrations increased activated alphav beta3 to 90-100%. Activation of alphav beta3 increased the efficiency of GCT23 adhesion to Opn by 2-fold. Furthermore, haptotactic migration on Opn was also enhanced about 40% compared to control. We propose that changes in the activation state of alphav beta3 may be a regulation point for osteoclasts during bone resorption.     

Evolutionary variants of the human immunodeficiency virus type 1 V3 region characterized by using a heteroduplex tracking assay

Syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) are evolutionary variants that are associated with rapid CD4+ cell loss and rapid disease progression. The heteroduplex tracking assay (HTA) was used to detect evolutionary V3 variants by amplifying the V3 sequences from viral RNA derived from 50 samples of patient plasma. For this V3-specific HTA (V3-HTA), heteroduplexes were formed between the patient V3 sequences and a probe with the subtype B consensus V3 sequence. Evolution was then measured by divergence from the consensus. The presence of evolutionary variants was correlated with SI detection data on the same samples from the MT-2 cell culture assay. Evolutionary variants were correlated with the SI phenotype in 88% of the samples, and 96% of the SI samples contained evolutionary variants. In most cases the evolutionary V3 variants represented discrete clonal outgrowths of virus. Sequence analysis of the six discordant samples that did not show this correlation indicated that three non-syncytium-inducing (NSI) samples had V3 sequences that had evolved away from the consensus sequence but not toward an SI genotype. A fourth sample showed little evolution away from the consensus but was SI, which indicates that not all SI variants require basic substitutions in V3. The other two samples had SI-like genotypes and NSI phenotypes, suggesting that V3-HTA was able to detect SI emergence in these samples in the absence of their detection in vitro. V3-HTA was also used to confirm SI variant selection in MT-2 cells and to examine the possibility of variant selection during virus culture in peripheral blood cells.     

Production of IL-13 by human lung mast cells in response to Fcepsilon receptor cross-linkage

BACKGROUND: Cross-linkage of the high affinity Fcepsilon receptors (FcepsilonRI) on the surface of the mast cell by the allergen-IgE complex is a central event in the induction of allergic inflammatory reactions. However, the precise roles of human mast cells in the perpetuation of allergic inflammation is not well known. IL-13 plays an important role in the regulation of allergic inflammation, especially being involved in the induction of IgE synthesis. OBJECTIVE: We investigated whether human lung mast cells have the capacity to produce IL-13 by cross-linking of the FcepsilonRI. METHODS: Lung mast cells were purified by affinity magnetic selection with monoclonal antibody YB5.B8 against c-kit to achieve a final mast cell purity of more than 93%. Purified mast cells were precultured with human myeloma IgE (3 microg/mL) for 16 h before challenge with stem cell factor (SCF) (50 ng/mL) and anti-IgE (1 microg/mL). By RT-PCR, ELISA and immunocytochemistry, we evaluated the capacity of human lung mast cells to express and produce IL-13. RESULTS: IgE-dependent activation of human lung mast cells caused an increase in IL-13 mRNA expression which persisted for up to 12 h. Immunoreactive IL-13 was detectable 24 h after activation of sensitized lung mast cells with SCF and anti-IgE in 6 of 13 non-asthmatic donors and a million of mast cells secreted 106.7 +/- 42.65 (mean +/- SE) pg of IL-13 into the culture supernatants. SCF alone induced 61.63 +/- 31.12 pg of IL-13 from 106 mast cells. This difference was statistically significant (P = 0.028, n = 13). Furthermore, we confirmed by immunocytochemistry that immunological activation induced an increase of intracellular IL-13. CONCLUSION: These findings demonstrate the capacity of human lung mast cells to transcribe IL-13 after IgE-dependent activation and to synthesize and release IL-13.     

Activation of the cloned human kappa opioid receptor by agonists enhances [35S]GTPgammaS binding to membranes: determination of potencies and efficacies of ligands

Activation of kappa receptors inhibits adenylate cyclase, enhances K+ conductance and reduces Ca++ conductance via pertussis toxin-sensitive G proteins. We recently cloned a human kappa opioid receptor and stably expressed it in Chinese hamster ovary (CHO) cells. In this study, the effects of activation of the human kappa receptor by agonists on [35S]GTPgammaS binding to CHO cell membranes were examined. The presence of GDP and Mg++ was essential for the kappa agonist (-)-U50,488H-induced increase in [35S]GTPgammaS binding to be observed and the optimal concentration was 3 microM and 5 mM, respectively. The presence of 100 mM Na+ was necessary to produce the maximal signal-to-background ratio. (-)U50,488H-induced increase in [35S]GTPgammaS binding was time- and tissue concentration-dependent. (-)U50,488H increased [35S]GTPgammaS binding in a dose-dependent manner with an EC50 of 3.1 nM. (+)-U50,488H had no effect, which indicates that this effect is stereospecific. Naloxone (1 microM) or norbinaltorphimine (10 nM) shifted the dose-response curve of (-)-U50,488H to the right by 100-fold. These results indicate that enhancement of [35S]GTPgammaS binding by (-)-U50,488H is a kappa receptor-mediated event. Pretreatment of the cells with pertussis toxin, but not cholera toxin, abolished the (-)-U50,488H-induced increase in [35S]GTPgammaS binding, which indicates the involvement of Gi and/or Go proteins. [35S]GTPgammaS binding induced by (-)-U50,488H had a Kd value of 0.34 +/- 0.08 nM and a Bmax value of 431 +/- 29 fmol/mg protein. The rank order of potencies of opioid ligands tested in stimulating [35S]GTPgammaS binding was dynorphin A 1-17 > (+/-)-ethylketocyclazocine > beta-funaltrexamine, (-)-U50,488H, tifluadom > nalorphine > pentazocine, nalbuphine > buprenorphine. Dynorphin A 1-17, (+/-)-ethylketocyclazocine, (-)-U50,488H, tifluadom and beta-funaltrexamine were full agonists, but nalorphine and pentazocine were partial agonists producing maximal responses of 68% and 23% of those of full agonists, respectively. Nalbuphine and buprenorphine had low levels of agonist activities. Norbinaltorphimine and naloxone were antagonists devoid of activities. Enhancement of [35S]GTPgammaS binding by kappa agonists provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of opioid ligands at the kappa receptor.     

Activation of the alternative pathway of human complement by apoptotic human umbilical vein endothelial cells

Complement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the alpha chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major alpha chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the alpha 1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its alpha' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.     

Antiapoptotic signaling by the insulin receptor in Chinese hamster ovary cells

We have sought to determine whether insulin can promote cell survival and protect Chinese hamster ovary (CHO) cells from apoptosis induced by serum starvation. Low concentrations of insulin were antiapoptotic for cells overexpressing wild-type insulin receptors but not in cells transfected with kinase-defective insulin receptor mutants that lacked a functional ATP binding site. However, treatment with orthovanadate (50 microM), a widely used tyrosine phosphatase inhibitor, led a dramatic reduction in internucleosomal DNA fragmentation in both cell lines. Cells transfected with truncated receptor mutants in either the juxtamembrane or C-terminal domain were as responsive as cells overexpressing wild-type receptors in mediating insulin antiapoptotic protection. The mechanisms underlying insulin antiapoptotic protection were investigated using a variety of pharmacological tools known to inhibit distinct signaling pathways. The phosphatidylinositol-3' kinase inhibitors wortmannin and LY294002 had only a modest influence whereas blocking protein farnesylation with manumycin severely disrupted the antiapoptotic capacity of the insulin receptor. Of interest, cells gained antiapoptotic potential following inhibition of extracellular signal-regulated kinase activation with the pharmacological agent PD98059. Insulin induced MKK3/MKK6 phosphorylation and activation of p38 MAP kinase whose activity was inhibited with SB203580. However, the inhibition of p38 MAP kinase had no effect on the protection offered by insulin. We conclude that the antiapoptotic function of the insulin receptor requires intact receptor kinase activity and implicates a farnesylation-dependent pathway. Increase in cellular phosphotyrosine content, however, triggers antiapoptotic signal that may converge downstream of the insulin receptor.     

Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells

Three subtypes of human (h) arginine vasopressin (AVP) receptors, hV1A, hV1B and hV2, were stably expressed in Chinese hamster ovary (CHO) cells and characterized by [3H]-AVP binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. Scatchard analysis of saturation isotherms for the specific binding of [3H]-AVP to membranes, prepared from CHO cells transfected with hV1A, hV1B and hV2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.39, 0.25 and 1.21 nM and a maximum receptor density (Bmax) of 1580 fmol mg(-1) protein, 5230 fmol mg(-1) protein and 7020 fmol mg(-1) protein, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Pharmacological characterization of the transfected human AVP receptors was undertaken by measuring the relative ability of nonpeptide AVP receptor antagonists, YM087, OPC-21268, OPC-31260, SR 49059 and SR 121463A, to inhibit binding of [3H]-AVP. At hV1A receptors, the relative order of potency was SR49059>YM087>OPC-31260>SR 121463A> >OPC-21268 and at hV2 receptors, YM087=SR 121463A>OPC-31260>SR 49059> >OPC-21268. In contrast, the relative order of potency, at hV1B receptors, was SR 49059> >SR 121463A=YM087=OPC-31260=OPC-21268. In CHO cells expressing either hV1A or hV1B receptors, AVP caused a concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 1.13 nM and 0.90 nM, respectively. In contrast, stimulation of CHO cells expressing hV2 receptors resulted in an accumulation of cyclic AMP with an EC50 value of 2.22 nM. The potency order of antagonists in inhibiting AVP-induced [Ca2+]i or cyclic AMP response was similar to that observed in radioligand binding assays. In conclusion, we have characterized the pharmacology of human cloned V1A, V1B and V2 receptors and used these to determine the affinity, selectivity and potency of nonpeptide AVP receptor antagonists. Thus they may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of AVP.