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Background and Purpose: Distinct vasopressin receptors are involved in different physiological and behavioural functions. Presently, no selective agonist is available to specifically elucidate the functional roles of the V1A receptor in the rat, one of the most widely used animal models. FE 201874 is a new derivative of the human selective V1A receptor agonist F180. In this study, we performed a multi-approach pharmacological and functional characterization of FE 201874 to determine whether it is selective for V1A receptors.Experimental Approach: We modified an available human selective V1A receptor agonist (F180) and determined its pharmacological properties in cell lines expressing vasopressin/oxytocin receptors (affinity and coupling to second messenger cascades), in an ex vivo model (aorta ring contraction) and in vivo in rats (proliferation of adrenal cortex glomerulosa cells and lactation).Key Results: FE 201874 exhibited nanomolar affinity for the rat V1A receptor; it was highly selective towards the rat V1B and V2 vasopressin receptors and behaved as a full V1A agonist in all the pharmacological tests performed. FE 201874 bound to the oxytocin receptor, but with moderate affinity, and behaved as an oxytocin antagonist in vitro, but not in vivo.Conclusions and Implications: On functional grounds, all the data demonstrate that FE 201874 is the first selective agonist of the rat V1A receptor isoform available. Hence, FE 201874 may have potential as a treatment for the vasodilator-induced hypotension occurring in conditions such as septic shock and could be the most suitable compound for discriminating between the behavioural effects of arginine vasopressin and oxytocin. [ABSTRACT FROM AUTHOR]

Background and Purpose Distinct vasopressin receptors are involved in different physiological and behavioural functions. Presently, no selective agonist is available to specifically elucidate the functional roles of the V1A receptor in the rat, one of the most widely used animal models. FE 201874 is a new derivative of the human selective V1A receptor agonist F180. In this study, we performed a multi-approach pharmacological and functional characterization of FE 201874 to determine whether it is selective for V1A receptors. Experimental Approach We modified an available human selective V1A receptor agonist ( F180) and determined its pharmacological properties in cell lines expressing vasopressin/oxytocin receptors (affinity and coupling to second messenger cascades), in an ex vivo model (aorta ring contraction) and in vivo in rats (proliferation of adrenal cortex glomerulosa cells and lactation). Key Results FE 201874 exhibited nanomolar affinity for the rat V1A receptor; it was highly selective towards the rat V1B and V2 vasopressin receptors and behaved as a full V1A agonist in all the pharmacological tests performed. FE 201874 bound to the oxytocin receptor, but with moderate affinity, and behaved as an oxytocin antagonist in vitro, but not in vivo. Conclusions and Implications On functional grounds, all the data demonstrate that FE 201874 is the first selective agonist of the rat V1A receptor isoform available. Hence, FE 201874 may have potential as a treatment for the vasodilator-induced hypotension occurring in conditions such as septic shock and could be the most suitable compound for discriminating between the behavioural effects of arginine vasopressin and oxytocin. [ABSTRACT FROM AUTHOR]

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1 The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments reveated that F-180 exhibited a high affinity for the human V[subla] receptor subtype (K[sub1]=II nM) and was selective for this receptor subtype. 2 Functional studies performed on CHO cells expressing human V[sub la] receptors indicate that similarly to AVP. F-180 can stimulate the accumulation of inositol phosphate. The activation constant (K[sub ac...]) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V[sub2] and V? receptor subtypes and an antagonist for the human OT receptor. 3 Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V[sub la] receptors, but weak affinity and non selective properties for rat V[sub la] receptors. 4 To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. 5 In conclusion, we demonstrate that F-180 is the first selective V[sub la], agonist described for human and bovine vasopressin receptors Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V[sub la] receptor subtypes. [ABSTRACT FROM AUTHOR]

The adenylate cyclase enzymes from pig kidney medulla, rat liver and rat brain membranes were solubilized with Triton X-100 into three different states : basal, NaF-activated and guanosine 5'-(β,γ-imino)triphosphate- [GuoPP(NH)P]-activated. The effects of 2-mercaptoethanol and other thiol-protecting reagents on the response of solubilized adenylate cyclase to NaF and GuoPP(NH)P and on its apparent sedimentation constants were examined. In the absence of 2-mercaptoethanol, GuoPP(NH)P produced very little stimulation of the adenylate cyclase activity in 200 000 x g supernatant fractions of detergent-treated membranes. 2-Mercaptoethanol markedly enhanced the response to GuoPP(NH)P and to a lesser extent to NaF. When membranes were solubilized and ultracentrifuged without 2-mercaptoethanol, the adenylate cyclases in all of the three states studied had the same apparent sedimentation coefficient (7.4 S, 7.6 S and 7.9 S for the enzymes from pig kidney, rat liver and rat brain respectively). In the presence of 2-mercaptoethanol the apparent sedimentation constant of the adenylate cyclase solubilized in the basal activity state fell by about 1 S, that of the GuoPP(NH)P-preactivated state remained unchanged and that of the NaF-preactivated state dropped with a concomitant loss in activity. The reduction in size and activity were abolished when NaF was added to the sucrose density gradients. Stimulation by GuoPP(NH)P or NaF of the light form of adenylate cyclase (basal state solubilized in the presence of 2-mercaptoethanol) raised the apparent sedimentation constant, and the values obtained were identical to those found for the preactivated forms of the enzyme. [ABSTRACT FROM AUTHOR]

Specific vasopressin receptors in rat liver membranes were recently characterized [Cantau et al., Journal of Receptors Research, in the press]. They differ from the receptors characterized earlier in kidney membranes as regards coupling with adenylate cyclase and specificity towards vasopressin structural analogues [Rajerison et al. (1974) J. Biol. Chem. 249, 6390–6400; Butlen et al. (1978) Mol. Pharmacol. 14, 1006–1017]. The object of the present work was to see whether these differences reflect a difference in the molecular size of liver and kidney vasopressin receptors. For this purpose, rat liver and kidney membranes were incubated with [³H]vasopressin and solubilized with Triton X-100 (0.3%). The properties of the macromolecular components of soluble extracts to which labelled vasopressin remained bound were observed to resemble those of the intact membrane receptors as regards binding reversibility at 30–37°C and sensitivity to guanyl nucleotides. The hydrodynamic parameters of the soluble hormone-receptor complexes were estimated from Ultrogel column filtration experiments and from sucrose density gradient ultracentrifugation experiments. The following values were obtained for liver and kidney receptors respectively: Stokes' radii: 5.4 and 5.6 nm; standard sedimentation coefficients: 3.7 and 3.7 S; partial specific volumes: 0.75 and 0.78 ml · g-1; molecular weight: 83000 and 80000. These results indicate that the marked functional differences between liver and kidney receptors are not accompanied by appreciable differences in molecular size. [ABSTRACT FROM AUTHOR]

The effects of 17 non-ionic detergents on pig kidney plasma membrane adenylate cyclase were tested (digitonin; detergents from the Triton series: X45, X100, X102, X114, X165, X305, X405, N101; the Brij series: 35, 56, 96 and the Tween series: 20, 40, 60, 80, 85). Membranes (2.66 mg protein/ml) were treated with increasing amounts of detergent for 150 min at 0 °C. The adenylate cyclase activities present in treated membranes and in their corresponding non-sedimentable fractions were measured at 30 °C. Only detergents having hydrophilic lipophilic balance values from 12 to 14 were effective solubilizing agents. For these detergents, the solubilization yield was dependent on their structure. When solubilized adenylate cyclase kept at 0 °C was incubated at 30 °C, the enzyme catalytic activity exponentially decreased towards an equilibrium value with a half-time of 5 min. This temperature-dependent adjustment of enzyme activity was reversible. Its magnitude was dependent on the detergent used. A more progressive but less pronounced effect was observed with non-solubilizing detergents. The micellar form of detergent in the incubation medium was found to be responsible for the appearance of microheterogeneity in the population of solubilized enzyme molecules differing by the substrate accessibility. This effect was suppressed when reducing the detergent concentration in the incubation medium. [ABSTRACT FROM AUTHOR]

We have synthesized and fully characterized by fast-atom-bombardment-mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-ArgTyr(3I)-NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V1a receptor [dissociation constant (Kd) = 54 ± 20 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841-1849] and for both the rat vasopressin V1a, receptor expressed in Spodoptera frugiperda 9 cells (Sf9 cells, Kd = 688 ± 35 pM) and in COS-7 cells (Kd = 320 ± 20 pM). This probe labels specifically the V1a receptors in an ultraviolet-dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands. (63 kDa and 93.6 kDa) for receptor-probe associations obtained in Sf9 and COS-7 cells respectively. These molecular masses are consistent with non-glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V1a receptor and for mapping its antagonist-binding site. [ABSTRACT FROM AUTHOR]

Agonist-induced intracellular calcium signals may propagate as intercellular Ca2+ waves in multicellular systems as well as in intact organs. The mechanisms initiating intercellular Ca2+ waves in one cell and determining their direction are unknown. We investigated these mechanisms directly on fura2-loaded multicellular systems of rat hepatocytes and on cell populations issued from peripheral (periportal) and central (perivenous) parts of the hepatic lobule. There was a gradient in vasopressin sensitivity along connected cells as demonstrated by low vasopressin concentration challenge. Interestingly, the intercellular sensitivity gradient was abolished either when D-myoinositol 1,4,5-trisphosphate (InsP3) receptor was directly stimulated after flash photolysis of caged InsP3 or when G proteins were directly stimulated with AlF4­. The gradient in vasopressin sensitivity in multiplets was correlated with a heterogeneity of vasopressin sensitivity in the hepatic lobule. There were more vasopressin-binding sites, vasopressin-induced InsP3 production and V1a vasopressin receptor mRNAs in perivenous than in periportal cells. Therefore, we propose that hormone receptor density determines the cellular sensitivity gradient from the peripheral to the central zones of the liver cell plate, thus the starting cell and the direction of intercellular Ca2+ waves, leading to directional activation of Ca2+-dependent processes. [ABSTRACT FROM AUTHOR]

After partial hepatectomy, liver mass is restored through a complex regulatory network of cytokines, growth factors, and hormones. In this study, we show that an interplay between hypothalamic vasopressin secretion and a remodeling of Ca&sup2+; signals in the liver contributes significantly to rat liver compensatory growth and bile flow. [ABSTRACT FROM AUTHOR]