Role of the human V1 vasopressin receptor COOH terminus in internalization and mitogenic signal transduction

We studied the role played by the intracellular COOH-terminal region of the human arginine vasopressin (AVP) V1-vascular receptor (V1R) in ligand binding, trafficking, and mitogenic signal transduction in Chinese hamster ovary cells stably transfected with the human AVP receptor cDNA clones that we had isolated previously. Truncations, mutations, or chimeric alterations of the V1R COOH terminus did not alter ligand binding, but agonist-induced V1R internalization and recycling were reduced in the absence of the proximal region of the V(1)R COOH terminus. Coupling to phospholipase C was altered as a function of the COOH-terminal length. Deletion of the proximal portion of the V1R COOH terminus or its replacement by the V2-renal receptor COOH terminus prevented AVP stimulation of DNA synthesis and progression through the cell cycle. Mutation of a kinase consensus motif in the proximal region of the V1R COOH terminus also abolished the mitogenic response. Thus the V1R cytoplasmic COOH terminus is not involved in ligand specificity but is instrumental in receptor trafficking and facilitates the interaction between the intracellular loops of the receptor, G protein, and phospholipase C. It is absolutely required for transmission of the mitogenic action of AVP, probably via a specific kinase phosphorylation site.     

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.     

Induction of c-fos protein by activation of vasopressin receptors in smooth muscle cells

Stimulation of vasopressin (V1) receptors of rat aortic smooth muscle cells (A-10, ATCC CRL 1476) results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) with the mobilization of intracellular calcium. When A-10 cells are exposed to arginine vasopressin (AVP), there is an increase in the level of c-fos oncoprotein. The extent of induction of c-fos oncoprotein depends on both the time of exposure of the cells to AVP, reaching a maximum at 60 min after which there is a slow decline, and the concentration of AVP used, with an approximate EC50 of 1 nM which corresponds well with the Kd of vasopressin binding to these receptors. This vasopressin-mediated increase in c-fos protein level is inhibited by a V1/V2 antagonist (SKF 101498) suggesting that this is a receptor-mediated event. In addition dDAVP, a V2 selective agonist, is much less effective than AVP in inducing c-fos protein suggesting that AVP mediates its effect via V1 receptors. Desensitization of vasopressin receptors by prolonged exposure to AVP resulted in no additional induction of c-fos protein level in response to second challenge of AVP. In addition to AVP, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), also stimulates the accumulation of c-fos protein although to a lesser extent than AVP. The above data suggest that c-fos protein levels in smooth muscle cells are regulated by AVP and the hormonal effect may be mediated through PI turnover and DAG, IP3 and Ca2+ signals.     

Arginine-vasopressin modulates intracellular pH via V1 and V2 receptors in renal collecting duct cells.

Arginine-vasopressin (AVP) has been proposed to be involved in the modulation of acid-base transporters; however, the nature of the mechanisms underlying AVP direct action on intracellular pH (pH(i)) in the cortical collecting duct (CCD) is not yet clearly defined. The aim of the present study was to elucidate which are the proteins implicated in AVP modulation of pH(i), as well as the receptors involved in these responses using a CCD cell line (RCCD(1)); pH(i) was monitored with the fluorescent dye BCECF in basal conditions and after stimulation with basolateral 10(-8) M AVP. Specific V1- or V2-receptor antagonists were also used. RT-PCR studies demonstrated that RCCD(1) cells express V1a and V2 receptors. Functional studies showed that while V2-receptor activation induced a biphasic response (alkalinization-acidification), V1-receptor activation resulted in an intracellular acidification. The V2-mediated alkalinization phase involves the activation of basolateral NHE-1 isoform of the Na(+)/H(+) exchanger while in the acidification phase CFTR is probably implicated. On the other hand, V1-mediated acidification was due to activation of a Cl(-)/HCO(3)(-) exchanger. We conclude that in RCCD(1) cells AVP selectively activates, via a complex of V1 and V2 receptor-mediated actions, different ion transporters linked to pH(i) regulation which might have physiological implications.     

Palmitoylation of the vasopressin V1a receptor reveals different conformational requirements for signaling, agonist-induced receptor phosphorylation, and sequestration

In this study, we establish that the V1a vasopressin receptor (V1aR) is palmitoylated, and we show that this modification has an important functional role. Palmitoylation of the V1aR occurs within the Cys371/Cys372 couplet located in the proximal C-terminal tail domain. Substitution of these residues in a [C371G/C372G]V1aR construct effectively disrupted receptor palmitoylation. Our data also indicate an additional palmitoylation site at another locus in the receptor, as yet undefined. [3H]Palmitate incorporation was agonist-sensitive and increased following exposure to [Arg8]vasopressin (AVP). Given the hydrophobic nature of the acyl chain, palmitoylation of the C terminus of G-protein-coupled receptors has been proposed to form an additional intracellular loop. Consequently, palmitoylation/depalmitoylation will have a profound effect on the local conformation of this domain. The V1aR palmitoylation status regulated both phosphorylation and sequestration of the receptor, and furthermore, palmitoylation, phosphorylation, and sequestration were all regulated by AVP. The palmitoylation-defective construct [C371G/C372G]V1aR exhibited decreased phosphorylation compared to wild-type V1aR, under both basal and AVP-stimulated conditions, and was sequestered at a faster rate. In contrast, the binding of four different classes of ligand and intracellular signaling were not affected by palmitoylation. This study therefore establishes that there are different conformational requirements for signaling, agonist-induced phosphorylation, and sequestration of the V1aR.     

Vascular vasopressin receptors mediate phosphatidylinositol turnover and calcium efflux in an established smooth muscle cell line

Vasopressin-induced phosphatidylinositol turnover and mobilization of intracellular Ca2+ was studied using an established smooth muscle cell line (A-10). The cells were subcloned to ensure a monoclonal cell population. The accumulation of inositol mono-, di-, and tris-phosphates (IP1, IP2, and IP3, respectively), and the mobilization of intracellular Ca2+ were dependent on the time of incubation and the concentration of arginine vasopressin (AVP). IP1, IP2, and IP3 were significantly elevated after 15 sec and remained elevated for up to 2 hr. The concentrations of AVP required for half-maximal stimulation of IP1, IP2, and IP3 formation were 2, 12, and 4 nM, respectively. LiCl was required to observe the accumulation of inositol phosphates in response to AVP. Significant 45Ca2+ efflux was observed within 15 sec after exposure to AVP. By employing the vasopressin receptor subtype selective antagonists [d(CH2)5Tyr(Me)AVP, V1; d(CH2)5D-Tyr(Et)VAVP,V1/V2; d(CH2) 5D-IleVAVP,V2] and agonists [AVP, V1/V2; dDAVP, V2; dVDAVP, V2], we found that the vasopressin-induced stimulation of phosphatidylinositol turnover and 45Ca2+ efflux were mediated by receptors of the vascular V1 subtype. Pertussis toxin pretreatment partially inhibited vasopressin-induced phosphatidylinositol turnover. These data demonstrate that activation of V1 receptors of vascular smooth muscle cells resulted in enhanced phosphatidylinositol turnover and mobilization of intracellular Ca2+.     

Dynamic interaction of human vasopressin/oxytocin receptor subtypes with G protein-coupled receptor kinases and protein kinase C after agonist stimulation

Examination of the structure of [Arg(8)]-vasopressin receptors (AVPRs) and oxytocin receptors (OTRs) suggests that G protein-coupled receptor kinases (GRKs) and protein kinase C (PKC) are involved in their signal transduction. To explore the physical association of AVPRs and OTRs with GRKs and PKC, wild types and mutated forms of these receptor subtypes were stably expressed as green fluorescent protein fusion proteins and analyzed by fluorescence, immunoprecipitation, and immunoblotting. Addition of a C-terminal GFP tag did not interfere with ligand binding, internalization, and signal transduction. After agonist stimulation, PKC dissociated from the V(1)R, did not associate with the V(2)R, but associated with the V(3)R and the OTR. After AVP stimulation, only GRK5 briefly associated with AVPRs following a time course that varied with the receptor subtype. No GRK associated with the OTR. Exchanging the V(1)R and V(2)R C termini altered the time course of PKC and GRK5 association. Deletion of the V(1)R C terminus resulted in no PKC association and a ligand-independent sustained association of GRK5 with the receptor. Deletion of the GRK motif prevented association and reduced receptor phosphorylation. Thus, agonist stimulation of AVP/OT receptors leads to receptor subtype-specific interactions with GRK and PKC through specific motifs present in the C termini of the receptors.     

Solubilization of human platelet vasopressin receptors

The human platelet membrane receptor for vasopressin (AVP) has been solubilized with the cholic acid derivative detergent 3-( [3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. Rapid and simple separation of free tritiated AVP ( [3H]AVP) from the solubilized receptor-hormone complex was done by filtration through polyethylenimine-treated filters. [3H]AVP binds to this soluble receptor with an equilibrium dissociation constant of 11.03 +/- 1.86 nM and a maximal number of binding sites = 288 +/- 66 fmol/mg protein while the corresponding values of the membrane-bound receptor are 1.62 +/- 0.21 nM and 237 +/- 38 fmol/mg of protein, respectively. The Ki value for native AVP derived from competition experiments is 11.02 +/- 2.05 nM for the soluble receptor. Competition experiments with specific vascular and renal antagonists confirm that the solubilized receptor belongs to the V1-vascular subtype.     

Pancreatic vasopressin V1b receptors: characterization in In-R1-G9 cells and localization in human pancreas

Vasopressin (AVP) receptors present in In-R1-G9 cells, a hamster glucagon-secreting alpha-pancreatic cell line, were characterized using SSR-149415, a selective nonpeptide V1b receptor antagonist, and reference AVP compounds. Binding experiments, using [3H]AVP as a ligand, identified a single population of high-affinity binding sites. SSR-149415 competitively inhibited this binding and exhibited nanomolar and stereospecific affinity for these sites. The affinity of various AVP/oxytocin ligands confirmed a V1b binding profile. In functional studies, AVP was a potent stimulant in inducing intracellular Ca2+ increase, glucagon secretion, and cell proliferation. These effects were fully antagonized by SSR-149415 with a nanomolar potency, whereas its diasteroisomer as well as two selective V1a and V2 receptor antagonists were much less potent. Additionally, the order of potency of AVP agonists and antagonists was in agreement with V1b-mediated effects. By RT-PCR, we confirmed the presence of V1b receptor mRNA in both In-R1-G9 cells and in human pancreas. The distribution pattern of V1b receptors investigated in human pancreas by immunohistochemistry showed strong labeling in islets of Langerhans, and colocalization studies indicated that this receptor was expressed in alpha-glucagon, beta-insulin, and somatostatin pancreatic cells. Thus, in In-R1-G9 cells, AVP mediates intracellular Ca2+ increase, glucagon secretion, and cell proliferation by activating V1b receptors, and these effects are potently antagonized by SSR-149415. Moreover, the presence of V1b receptors also found in human Langerhans islets could suggest hormonal control of AVP in human pancreas.     

Intracellular pathways of V(1) and V(2) receptors activated by arginine vasopressin in rat hippocampal neurons.

To explore the intracellular pathways activated by vasopressin receptors, the effects of arginine vasopressin (AVP) and its analogues mediating glycine (Gly)-induced Cl(-) currents (I(Gly)) were examined in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch recording technique. AVP and its analogues inhibited I(Gly) in a concentration-dependent manner. The inhibitory actions of AVP(4-9) (AVP metabolite) and NC-1900 (AVP(4-9) analogue) were reversed by a V(1) receptor antagonist, or pretreatment with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N', N'-tetraacetic acid. In contrast, these blocking procedures had no effect on the 1-desamino-8-D-AVP (DDAVP; V(2) agonist) action. A V(2) receptor antagonist did not block the inhibitory action of AVP(4-9) or NC-1900, but blocked that of DDAVP. The inhibitory action of AVP was completely blocked by the co-application of the V(1) and V(2) antagonists. The inhibitory action of NC-1900 was not affected by perfusion with a Ca(2+)-free external solution, but was strongly blocked by thapsigargin. The intracellular application of heparin or anti-inositol 1,4,5-triphosphate (IP(3)) also blocked the NC-1900 action. Furthermore, Ca(2+)/calmodulin (CaM) inhibitors blocked the NC-1900 action, while a CaM-dependent kinase II inhibitor and PKC modulators had no effect. 2',5'-Dideoxyadenosine (an adenylate cyclase inhibitor), H-89, and Rp-cAMPS blocked the inhibitory actions of NC-1900 and DDAVP. These results suggest that the activation of the V(1) receptor in the hippocampal neurons induces the production of IP(3), which releases Ca(2+) from the IP(3)-sensitive Ca(2+) storage sites. The Ca(2+) binds to CaM, resulting in the activation of Ca(2+/)CaM-sensitive adenylate cyclases. The activation of protein kinase A through the adenylate cyclase inhibits I(Gly).     

Vasopressin elevation of Na+/H+ exchange is inhibited by genistein in human blood platelets.

The regulation of intracellular Na+ and pHi in human blood platelets is known to be controlled by the function of the Na+/H+ exchanger. The phosphorylation state of the Na+/H+ exchanger which determines the exchanger activity in human blood platelets is regulated by the activities of protein kinases and protein phosphatases. Observations in this study indicate that arginine vasopressin (AVP) that interacts with a V1 receptor, activates the Na+/H+ exchange in human blood platelets through a genistein-inhibited mechanism. The AVP-activated Na+/H+ exchange is probably not regulated by protein kinase C (PKC), since this activation is not inhibited by staurosporine. The multiple ways in which platelet Na+/H+ exchange can be modulated may indicate the critical role played by this exchanger in the homeostasis control of pHi in human blood platelets.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Desensitization of the human V2 vasopressin receptor. Homologous effects in the absence of heterologous desensitization.

Three main pathways have been implicated in desensitization of receptors that stimulate adenylylcyclase (AC): cAMP-mediated phosphorylation; cAMP-independent phosphorylation, and receptor internalization. Cell lines derived from the murine Ltk- cell were found useful in exploring the contribution of cAMP-dependent phosphorylation in V2 vasopressin receptor desensitization. The HTB-2 cell expresses the human V2 vasopressin receptor, introduced by transfection of human genomic DNA, and the prostaglandin E1 (PGE1) receptor, endogenous to the Ltk- cell. The A7 cell expresses the hamster beta 2-adrenoceptor, which undergoes the above-mentioned desensitization processes. Treatment of HTB-2 cells with arginine-vasopressin (AVP) had no effect on AC responsiveness to PGE1, but promoted desensitization of the AVP response. This was seen as a 5-6-fold right shift in the dose-response curves for AVP action (cAMP accumulation in intact cells and AC stimulation in homogenates and isolated membranes) and in a decrease in the maximum effect of AVP on these parameters. AVP treatment caused a decrease in cell surface receptors to approximately 75% of control without changes in KD, as determined by Scatchard analysis. When cAMP was increased by treatment with 10 microM PGE1 and isobutylmethylxanthine, desensitization of the PGE1 receptor was observed but not of the AVP receptor. In A7 cells the same treatment caused, as expected, a 3-fold right shift in the dose-response curve for AC stimulation by isoproterenol, indicating that L cells can mediate heterologous desensitization. These data demonstrate that the V2 vasopressin and the PGE1 receptors undergo homologous desensitization in the absence of cAMP-mediated phosphorylation and that this component is not required for vasopressin receptor internalization.     

Fibroblast growth factor receptors have different signaling and mitogenic potentials.

Fibroblast growth factor (FGF) receptors (FGFRs) are structurally related receptor protein tyrosine kinases encoded by four distinct genes. Activation of FGFR-1, -2, and -3 by FGFs induces mitogenic responses in various cell types, but the mitogenic potential of FGFR-4 has not been previously explored. We have compared the properties of BaF3 murine lymphoid cells and L6 rat myoblast cells engineered to express FGFR-1 or FGFR-4. Acidic FGF binds with high affinity to and elicits tyrosine phosphorylation of FGFR-1 or FGFR-4 receptors displayed on BaF3 cells, but only FGFR-1 activation leads to cell survival and growth. FGFR-4 activation also fails to elicit detectable signals characteristic of the FGFR-1 response: tyrosine phosphorylation of SHC and extracellular signal-related kinase (ERK) proteins and induction of fos and tis11 RNA expression. The only detected response to FGFR-4 activation was weak phosphorylation of phospholipase C gamma. A chimeric receptor containing the extracellular domain of FGFR-4 and the intracellular domain of FGFR-1 confers FGF-dependent growth upon transfected BaF3 cells, demonstrating that the intracellular domains of the receptors dictate their functional capacity. Activation of FGFR-1 in transfected L6 myoblasts induced far stronger phosphorylation of phospholipase C gamma, SHC, and ERK proteins than could activation of FGFR-4 in L6 cells, and only FGFR-1 activation induced tyrosine phosphorylation of a characteristic 80-kD protein. Hence, the signaling and biological responses elicited by different FGF receptors substantially differ.     

Phosphorylation and recycling kinetics of G protein-coupled receptors

The rate of ligand-induced phosphorylation of the V2 and V1a vasopressin receptors was characterized in HEK 293 cells. Both receptors were phosphorylated predominantly by GRKs, and the V1a receptor was also phosphorylated by protein kinase C regardless of the presence or absence of ligand. Phosphorylation of the V1aR catalyzed by GRKs reached maximal values at the shortest measured time: 15 seconds, and decayed rapidly with a t1/2 of 6 min in the continuous presence of AVP. In agreement with the hypothesis that dephosphorylation must precede receptor recycling to the cell surface, the V1aR returned rapidly to the cell surface after removal of the hormone from the medium. Phosphate incorporation into the V2R proceeded at a slower pace, and the internalized phosphorylated receptor failed to recycle to the cell surface and retained its phosphate for a long time in the presence or absence of ligand. A single mutation in the carboxy terminus of the V2R accelerated de-phosphorylation of the protein and conferred recycling properties to the V2R. These experiments provided molecular evidence for the hypothesis that internalization is required for de-phosphorylation and recycling of reactivated G protein coupled receptors to the cell surface.     

Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.     

Vasopressin produces inhibition on phrenic nerve activity and apnea through V(1A) receptors in the area postrema in rats

The area postrema (AP) is the most caudal circumventricular organ in the central nervous system and contains arginine vasopressin (AVP) receptors. To investigate that AVP receptors in the AP might participate in the modulation of respiration, the adult rat was anesthetized with urethane (1.2 g/kg, i.p.), paralyzed, ventilated artificially, and maintained at normocapnia in hyperoxia. The phrenic nerve was separated at C4 level. Phrenic burst was amplified, filtered, integrated, and then stored in the hard disc via the PowerLab system. Three doses of AVP and an AVP V(1A) receptor antagonist, [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,-O-Me-Tyr2,Arg8]-vasopressin, were microinjected into the AP through a pair of microelectrodes. The moderate and high doses of AVP reduced the PNA to 72% and 45% of the control (P < 0.05), extended the mean TE from 1.4 s before AVP to 4.0 s and 7.6 s, (P < 0.05), and decrease in BP by 26 and 37 mmHg (P < 0.05), respectively. These significant reductions in PNA and BP and elongation of TE were totally abolished by the pre-treatment of the AVP V(1A) receptor antagonist and by application of lidocaine or CoCl2 at the nucleus tractus solitarius (NTS). Moreover, pulmonary inhibition caused by AVP was significantly attenuated by hypercapnia. These results strongly suggest that AVP V(1A) receptors in the AP may participate in the modulation of cardiopulmonary functions through the activation of V(1A) receptors and the pathway connected to the NTS. They may also indicate that a putative vasopressinergic pathway has a projection to the AP to alter the excitability of neurons having AVP V(1A) receptors and results in an inhibition of cardiopulmonary functions via the connection between the AP and NTS.     

Contribution of the vasopressin V1 receptor to its hydrosmotic response

Toad urinary bladder epithelial cells grown in culture (primary) show a significant increase in water-soluble inositol phosphates when treated with 10(-8) M vasopressin (AVP), but not with (1-deamino-8-D-arginine)vasopressin (dDAVP), a V2-agonist. The increase in inositol phosphates was blocked by the V1-antagonist, d(CH2)5Tyr(Me)AVP, suggesting a V1-coupled phosphoinositide breakdown. The V1-antagonist had no effect on basal adenylate cyclase activity nor on that stimulated by AVP. However, the V1-antagonist was found to attenuate the hydrosmotic response of AVP, suggesting some role of the V1-receptor cascade in the water flow response. Mezerein (MZ), a non-phorbol activator of protein kinase C (PKC) increased osmotic water flow when added to the mucosal surface. The response was less in magnitude and occurred over a longer period (90 min) than that observed with AVP. In an attempt to emulate the V1-response, activation of PKC, and an increase in intracellular calcium, toad bladders were incubated with MZ and the calcium ionophore A23187 (IP). It was found that IP enhanced the water flow response to MZ at all times measured. Mz and IP were also found to enhance cAMP-mediated water flow, suggesting that apical membrane permeability may be regulated in part through V1-receptor stimulation and its respective second messengers. Collectively, these observations suggest that the V1 receptor may play a role not only as part of a negative feedback system, but also as an integral component of the enhanced water permeability that occurs at the apical membrane.     

Cell cycle-dependent coupling of the vasopressin V1a receptor to different G proteins

Arginine vasopressin (AVP) regulates biological processes by binding to G protein-coupled receptors. In Swiss 3T3 fibroblasts, expressing the V(1a) subtype of vasopressin receptors, AVP mobilizes calcium from intracellular stores. In proliferating cells, the AVP-induced increase in intracellular calcium concentration ([Ca(2+)](i)) was mediated by G proteins of the G(q) family, which are insensitive to pertussis toxin (PTX) pretreatment of the cells. In quiescent cells, the AVP-induced increase in [Ca(2+)](i) was partially PTX-sensitive, suggesting an involvement of G(i) proteins. We confirmed this by photoaffinity labeling of G proteins in Swiss 3T3 cell membranes activated by AVP. In Swiss 3T3 cells arrested in the G(0)/G(1) phase of the cell cycle, the AVP-induced increase in [Ca(2+)](i) was also partially PTX-sensitive but was PTX-insensitive in cells arrested in other phases of the cell cycles. The blocking effect of PTX pretreatment in G(0)/G(1) cells was mimicked by microinjection of antisense oligonucleotides suppressing the expression of the Galpha(i3) subunits. These results were confirmed by microinjection of antibodies directed against the C terminus of G protein alpha-subunits. The data presented indicate that in Swiss 3T3 fibroblasts synchronized in the G(0)/G(1) phase of the cell cycle the V(1a) receptor couples to G(q/11) and G(i3) to activate the phospholipase C-beta, leading to release of intracellular calcium.