Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand

[(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V(1b) receptor (V(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V(1b)R was performed using SSR-149415/[(3)H]SSR-149415 in binding and functional studies in vitro. [(3)H]SSR-149415 binding was time-dependent, reversible, and saturable. Scatchard plot analysis gave a single class of high-affinity binding sites with apparent equilibrium dissociation constant (K(d)) approximately 1 nM and maximum binding density (B(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V(1b) rank order of affinity: SSR-149415 = AVP > dCha > dPen > dPal > dDavp > SSR-126768A > SR-49059 > SSR-149424 > OT > SR-121463B. Species differences between human, rat, mouse, and hamster V(1b)R were observed. Autoradiography studies with [(3)H]SSR-149415 on rat and human pituitary showed intense specific labeling confined to corticotroph cells and absence of labeling in the other tissues examined. SSR-149415 potently and stereospecifically antagonized the AVP-induced inositol phosphate production and intracellular Ca(2+) increase (EC(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V(1b)R. AVP (10(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V(1b)R induced their rapid internalization. Preincubation with 10(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10(-6) M SSR-149415 treatment. Thus SSR-149415/[(3)H]SSR-149415 are unique tools for studying animal and human V(1b)R.     

Mapping the binding site of arginine vasopressin to V1a and V1b vasopressin receptors

Starting from the 2.8-A resolution x-ray structure of bovine rhodopsin, three-dimensional molecular models of the complexes between arginine vasopressin and two receptor subtypes (V1a, V1b) have been built. Amino acid sequence alignment and docking studies suggest that four key residues (1.35, 2.65, 4.61, and 5.35) fine tune the binding of vasopressin and related peptide agonists to both receptor subtypes. To validate these predictions, a series of single or double mutants were engineered at V1a and V1b receptor subtypes and tested for their binding and functional properties. Two negatively charged amino acids at positions 1.35 and 2.65 are key anchoring residues to the Arg8 residue of arginine vasopressin. Moreover, two amino acids (V(4.61) and P(5.35)) delineating a hydrophobic subsite at the human V1b receptor are responsible for the recognition of V1b selective peptide agonists. Last, one of the latter positions (5.35) is hypothesized to explain the pharmacological species differences between rat and human vasopressin receptors for a V1b peptide agonist. Altogether these refined three-dimensional models of V1a and V1b human receptors should enable the identification of further new selective V1a and V1b agonists as pharmacological but also therapeutic tools.     

Glucagonlike peptide-1(7-36)amide suppresses glucagon secretion and decreases cyclic AMP concentration in cultured In-R1-G9 cells.

We previously reported that GLP-1(7-36)amide had glucagonostatic action as well as insulinotropic action in the perfused rat pancreas. In this study, we examined the effect of GLP-1(7-36)amide on glucagon secretion and cAMP concentration in glucagon-secreting cell line, In-R1-G9. GLP-1(7-36)amide (1nM) significantly suppressed glucagon secretion and decreased cAMP concentration in the cells. GLP-1(1-37) did not affect glucagon secretion. It is suggested that inhibitory effect of GLP-1(7-36)amide on glucagon secretion is at least partly mediated by adenylate cyclase system.     

A vasopressin analog that binds but does not activate V1 or V2 vasopressin receptors is not internalized into cells that express V1 or V2 receptors.

To assess whether receptor binding is sufficient to initiate vasopressin receptor endocytosis in cells expressing the vasopressin V1 or V2 receptors, we synthesized a novel fluorescent-labeled vasopressin analog, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)-D-tyrosine, 4-valine, 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-CLVP), that binds to vasopressin receptors but does not activate intracellular events such as the mobilization of intracellular calcium or the activation of adenylate cyclase. We compared the manner in which this analog was endocytosed in cells expressing V1 (A-10, rat smooth muscle cells) or V2 (LLC-PK1, porcine kidney cells) receptors with that of a full agonist, [1-(beta-mercaptopropionic acid), 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) [Lutz et al. (1990) J. Biol. Chem. 265, 4657-4663; Lutz et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,6507-6511]. We showed that R-CLVP bound to both types of receptors with good affinity. It failed to increase cyclic AMP concentrations in LLC-PK1 cells and did not increase the mobilization of intracellular calcium in A-10 cells. It bound to the surface of both these cell types in a diffuse manner and it did not undergo receptor endocytosis in either cell type. In contrast, R-MLVP, an agonist that bound to both receptor subtypes and elicited changes in intracellular cyclic AMP and calcium, bound to the surface of these cells in a diffuse manner at early times after exposure, and rapidly underwent endocytosis. We conclude that binding of vasopressin to its receptors alone is insufficient to cause receptor endocytosis, and other events distal to the receptor are required to initiate endocytosis. R-CLVP should be a useful analog in determining the factors responsible for initiating receptor endocytosis.     

Oxytocin and V1 vasopressin receptors in rabbit endometrium during pregnancy

Neurohypophysial hormone receptors were identified and characterized in rabbit endometrium and decidua by radioligand binding methods. The results strongly support the presence of a heterogeneity of sites in the decidua of parturient rabbits. The oxytocin site (R1) binds oxytocin and oxytocin analogues ([Thr4, Gly7]oxytocin and OTA) with high affinity, whereas the AVP site (R2) was selective for the V1 AVP analogues, [Phe2, Orn8]VT and d(CH2)5TyrMeAVP. The concentration of oxytocin receptors was low (50-100 fmol/mg protein) at oestrus (Day 0) and on Day 29 of pregnancy, but increased significantly (about 8-fold, P less than 0.05) during parturition. Conversely, V1 AVP receptors were more concentrated than the oxytocin sites at the end of pregnancy (150 fmol/mg protein) but did not change during parturition. These results indicate that neurohypophysial hormones have specific receptors not only in the myometrium but also in the uterine mucosa and we suggest that these receptors may participate in the regulation of uterine activity during pregnancy.     

Lack of V1 vasopressin receptors in rabbit hepatocytes.

Vasopressin does not induce glycogenolysis in rabbit hepatocytes; glucagon, angiotensin, phenylephrine and ATP are as potent as with rat hepatocytes, whereas isoprenaline is nearly 10000 times more potent in the rabbit. Binding studies of [3H]vasopressin reveal the complete absence of specific vasopressin receptors on rabbit liver plasma membranes. We verified that vasopressin acts as an antidiuretic and vasopressor agent in the rabbit. We conclude that there is a selective lack of V1 vasopressin receptors in rabbit liver.     

Mediators of the mitogenic action of human V(1) vascular vasopressin receptors

Arginine vasopressin (AVP) activation of V(1) vascular receptors (V(1)Rs) stimulates cell growth and proliferation in different tissues via cellular signaling pathways that remain to be identified. To explore the intracellular mediators of the mitogenic action of V(1)R, Chinese hamster ovary (CHO) cells were stably transfected with the human V(1)R cDNA clone we isolated previously. We assessed AVP effects on kinase activation (immunoblotting with phosphospecific antibodies), DNA synthesis (tritiated thymidine uptake), cell cycle progression (flow cytometry analysis after nuclear labeling with propidium iodide), and cell proliferation (conversion of the colorimetric reagent MTS) in the presence or absence of various pathway inhibitors. AVP stimulation of V(1)Rs leads to the phosphorylation of several kinases, an increase in DNA synthesis, a progression through the S and G(2)-M phases of the cell cycle, and an increase in cell proliferation. The mediators of the mitogenic action of V(1)R activation included calcium mobilization, coupling to a G(q) protein, and the simultaneous and parallel activation of several kinases, mainly calcium/calmodulin-dependent kinase II, phosphatidylinositol 3 kinase, protein kinase C, and p42/p44 mitogen-activated protein kinase.     

Arginine vasopressin inhibits apoptosis of rat glomerular mesangial cells via V1a receptors

Arginine vasopressin (AVP) promotes proliferation of glomerular mesangial cells. We examined whether AVP modulates an apoptosis of cultured rat glomerular mesangial cells at 3-17th passages. The agarose gel electrophoresis demonstrated that AVP attenuated a ladder formation stimulated by the serum deprivation. The quantitation of oligonucleosomes by ELISA also showed that AVP suppressed the serum deprivation-induced apoptosis. Such an antiapoptotic effect of AVP was dose-dependent. An AVP V1a receptor antagonist, d(CH2)5Tyr(Me)AVP, abolished the antiapoptotic effect of AVP. The inhibitory effect of AVP on the apoptosis was reduced by staurosporine and mimicked by phorbol-12-myristate-13-acetate. These results suggest that AVP inhibits serum deprivation-induced apoptosis of glomerular mesangial cells via V1a receptor-protein kinase C pathway.     

Immunocytochemical localization of elastase 1 in human pancreas

By light and electron microscopic immunocytochemistry the distribution is described of human pancreatic elastase 1 (E1) during ontogenesis, in adults, in cases of acute and chronic panceatitis, acute pancreatic ischaemia as well as pancreatic tumours. E1-positive cells were first detected in ductal sprouts in the 14th gestational week. Complete acini expressing E1 could be found from the 17th to the 20th week of gestation onwards. Scattered distinct E1-positive epithelia could be found in the ducts of fetal and adult pancreas. By immunoelectron microscopy, E1 was localized in rough endoplasmic reticulum, condensing vacuoles, zymogen granules of acinar epithelia and in acinar lumina. E1 appeared to be distributed homogeneously in zymogen granules. As specific markers of acinar cells, both monoclonal antibodies under study identified heterotopic pancreatic acini in peribiliar glands of the liver and also helped to visualize different damage patterns in pancreatitis. The acinar epithelia surrounding acute lipolytic necroses initially reacted more intensely with the E1-antibodies than undamaged pancreatic tissue. In acute ischaemia, acinar cells which are dissociated from intercalated ducts lost their immunocytochemical reactivity for E1. Pancreatic parenchyma involved in advanced acute pancreatitis as well as in chronic inflammation was detected only weakly by both E1-antibodies. However, atrophic lobules in post-inflammatory scars were stained more intensely by the E1-antibodies than normal parenchyma. Pancreatic tumours (adenomas, adenocarcinomas, solid-cystic tumours and islet cell tumours) were not labelled by these antibodies.     

Central vasopressin V(1a) and V(1b) receptors modulate the cardiovascular response to air-jet stress in conscious rats.

This study investigates the contribution of central vasopressin receptors in the modulation of systolic arterial pressure (SAP) and heart rate (HR) response to air-jet stress in conscious Wistar rats equipped with a femoral arterial catheter and intracerebroventricular cannula using novel non-peptide and selective vasopressin V(1a) (SR49059) and V(1b) (SSR149415) antagonists. The effects of stress on SAP and HR were evaluated by measuring the maximal response to stress, the latency of the maximal response, the duration of the recovery period, and the increase in the low frequency (LF) short-term variability component. Stress induced a parallel and almost immediate increase in both SAP and HR, followed by enhanced LF SAP variability in the recovery period. Pretreatment of rats with V(1a) antagonist did not affect the maximal increase or the latency of SAP and HR response to acute stress, but shortened the recovery period of SAP and HR and prevented the increase in LF SAP. The V(1b) antagonist reduced the maximal increase in SAP without affecting HR and their latencies, shortened the recovery period of SAP and inhibited the increase in LF SAP variability. These results indicate that both central V(1a) and V(1b) receptors mediate cardiovascular changes induced by air-jet stress in conscious rats.     

Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis

G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from several studies suggesting that GPCRs can form both homo- and heterodimers. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) approaches, we established that the vasopressin V1a, V2, and the oxytocin receptors exist as homo- and hetero-dimers in transfected human embryonic kidney 293T cells. Each receptor protomer had a similar propensity to form homo- and heterodimers, indicating that their relative expression levels may determine the homo-/heterodimer ratio. The finding that immature forms of the receptor can be immunoprecipitated as homo- and heterodimers and the detection by BRET of such oligomer in endoplasmic reticulum-enriched fractions suggest that the oligomerization processes take place early during biosynthesis. Treatment with agonists or antagonists did not modify the BRET among any of the vasopressin and oxytocin receptor pairs studied, indicating that the dimerization state of the receptors is not regulated by ligand binding once they have reached the cell surface. Taken together, these results strongly support the notion that GPCR dimerization is a constitutive process.     

Subcellular localization of leukotriene receptors in human endothelial cells

Leukotriene B₄ (LTB₄), a potent chemotactic and immune-modulating mediator, signals via two receptors, BLT₁ and BLT₂. Recently, we reported that BLT₁ is the predominating BLT expressed on human umbilical vein endothelial cells (HUVEC), and that BLT₁ mediated functions are enhanced by LTB₄ and lipopolysaccharide (LPS), but not by TNFα. Here, we demonstrate that BLT₁ is found on the outer cell membrane of HUVECs but also in intracellular granules, co-localized with monocyte chemotactic protein-1 and P-selectin, but not with interleukin-8 and von Willebrand factor. Upon stimulation with LTB₄ or LPS, more BLT₁ protein is found, now evenly distributed over the cytoplasm and in the cell nucleus, but less on the cell surface. An MAP kinase inhibitor prevented this enhancement and translocation, suggesting this signaling pathway to be crucial. Thus, BLT₁, a G-protein-coupled 7-transmembrane receptor, is located in various subcellular compartments in endothelial cells, which may have implications for cellular LT dependent responses and target accessibility for BLT₁ antagonists.     

Attenuated stress-induced catecholamine release in mice lacking the vasopressin V1b receptor

Vasopressin V(1b) receptor is specifically expressed in the pituitary and mediates adrenocorticotropin release, thereby regulating stress responses via its corticotropin releasing factor-like action. In the present study we examined catecholamine release in response to two types of stress in mice lacking the V(1b) receptor gene (V(1b)R(-/-) mice) vs. wild-type mice. There were no significant differences in the basal plasma levels of catecholamines between the two genotypes. In response to stress induced by forced swimming, norepinephrine (NE), but not epinephrine (E) or dopamine (DA), was increased in wild-type mice, whereas the increases in NE and DA were not observed in V(1b)R(-/-) mice. In wild-type mice, E, but not NE or DA, was increased in response to social isolation stress, whereas the increase in E was not observed in V(1b)R(-/-) mice. These results suggest that the V(1b) receptor regulates stress-induced catecholamine release. Because it has been suggested that arginine-vasopressin (AVP) is related to the development of depression, we also evaluated immobility time in the forced swimming test, and we found no significant change in V(1b)R(-/-) mice. Taken together, these findings suggest that, in addition to the previously elucidated effect on the hypothalamic-pituitary-adrenal axis, vasopressin activity via V(1b) receptors regulates stress-induced catecholamine release.     

V1b and CRHR1 receptor heterodimerization mediates synergistic biological actions of vasopressin and CRH

Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.     

Purification and characterization of the rat liver vasopressin (V1) receptor

Utilizing a proteoliposome reconstitution system, we have purified the rat liver V1 vasopressin receptor to near homogeneity. The receptor was purified approximately 21,000-fold from rat liver membranes, using differential detergent solubilization, size exclusion gel filtration, lectin affinity, and ion-exchange chromatography. The purified receptor exhibits a Kd of 6 nM, when, prior to solubilization, the membranes were exposed to 1 microM vasopressin. This resulted in the association of a pertussis toxin-insensitive guanine nucleotide-binding protein with the receptor during most of the purification procedure. In the absence of this association, the receptor had a Kd of approximately 30 nM. Association of the receptor with a G-protein was confirmed by the ability of vasopressin to stimulate the hydrolysis of [gamma-32P]GTP. The specific activity of the vasopressin-stimulated hydrolysis was 25 nmol/min/mg, approximately 8,000-fold higher than values obtained with crude reconstituted receptor preparations. Cross-linking of 125I-vasopressin to a partially purified preparation of receptor demonstrated that the receptor had a molecular weight of approximately 68,000 under reducing conditions, and 58,000 under nonreducing conditions. The purification procedure may prove useful in purifying a number of small peptide hormone receptors (e.g. bradykinin, angiotensin II) and perhaps their associated G-proteins as well.     

Cytokine-mediated downregulation of vasopressin V(1A) receptors during acute endotoxemia in rats

The reduced pressure response to vasopressin during acute sepsis has directed our interest to the regulation of vasopressin V(1A) receptors. Rats were injected with lipopolysaccharide for induction of experimental gram-negative sepsis. V(1A) receptor gene expression was downregulated in the liver, lung, kidney, and heart during endotoxemia. Inasmuch as the concentrations of proinflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were highly increased during sepsis, the influence of these cytokines on V(1A) receptor expression was investigated in primary cultures of hepatocytes and in the aortic vascular smooth muscle cell line A7r5. V(1A) receptor expression was downregulated by the cytokines in a nitric oxide-independent manner. Blood pressure dose-response studies after injection of endotoxin showed a diminished responsiveness to the selective V(1) receptor agonist Phe(2),Ile(3),Orn(8)-vasopressin. Our data show that sepsis causes a downregulation of V(1A) receptors and suggest that this effect is likely mediated by proinflammatory cytokines. We propose that this downregulation of V(1A) receptors contributes to the attenuated responsiveness of blood pressure in response to vasopressin and, therefore, contributes to the circulatory failure in septic shock.     

V1b vasopressin receptor trafficking and signaling: Role of arrestins,G proteins and Src kinase

The signaling pathway of G protein‐coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V_1b subtype (V_1bR) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of β‐arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V_1bR‐mediated MAP kinase pathway. Using MEF cells Knocked‐out for β‐arrestins 1 and 2, we demonstrated that both β‐arrestins 1 and 2 play a fundamental role in internalization and recycling of V_1bR with a rapid and transient V_1bR‐β‐arrestin interaction in contrast to a slow and long‐lasting β‐arrestin recruitment of the V₂ vasopressin receptor subtype (V₂R). Using V_1bR‐V₂R chimeras and V_1bR C‐terminus truncations, we demonstrated the critical role of the V_1bR C‐terminus in its interaction with β‐arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation‐independent manner. In parallel, V_1bR MAP kinase activation was dependent on arrestins and Src‐kinase but independent on G proteins. Interestingly, Src interacted with hV_1bR at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V_1bR involving both arrestins and Src kinase family.      

Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells

Background

Sigma-1 receptors are involved in regulation of neuronal activities presumably through regulation of the activity of ion channels. Sigma-1 receptors also play a role in growth and metastasis of cancerous cells. Intracellular distribution of sigma-1 receptors have been linked to sphingolipid-enriched domains.

Results

We report that in CHO-K1 cells sigma-1 receptors target to focal adhesion contacts (FAC) where they colocalize with Talin and Kv1.4 potassium channels. The levels of sigma-1 receptors in the FAC were significantly increased by application of sigma-1 receptor ligands and by filamentous actin (F-actin) polymerization with phalloidin. The total length of FAC (measured by the focal adhesion marker, talin) was concomitantly increased in the presence of sigma-1 receptors upon phalloidin treatment. Only sigma-1 receptor ligands, however, resulted in an increase of sigma-1 receptors in the FAC, independent of talin. Additionally, a novel approach was utilized to allow an assessment of the half life of endogenous sigma-1 receptors in CHO-K1 cells, which was measured to be at least 72 hours.

Conclusion

Ligand activated sigma-1 receptors translocate into FAC from a pool of receptors stored in ER lipid rafts presumably for inhibition of Kv1.4 channels. Stabilization of actin filaments is likely to be important for targeting sigma-1 receptors to Focal Adhesion Contacts in CHO-K1 cells.