The synthesis and biological activity of four novel fluorescent vasopressin analogs

We synthesized and tested the biological properties of four fluorescent vasopressin analogs: [1-(2-mercapto)propionic acid]-8-lysine-N6-5-dimethylamino-naphthalene-1-sulfonyl vasopressin (D-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxyfluorescein vasopressin (F-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-2-N-methylanthranilamide vasopressin (MA-MLVP), and [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxytetramethylrhodamine vasopressin (R-MLVP). All fluorescent analogs were prepared by coupling the appropriate fluorochrome to the 6-amino group of the lysine residue in [1-(2-mercapto)propionic acid]-8-lysine vasopressin (MLVP) which was synthesized by the Merrifield solid-phase method. The structures of high performance liquid chromatography-purified MLVP and the fluorescent analogs were confirmed by fast atom bombardment mass spectrometry. F-MLVP, MA-MLVP, and R-MLVP effectively competed for 8-arginine vasopressin (AVP)-binding sites in canine renal plasma membranes and on the surface of porcine kidney cells (LLC-PK1, ATCC CL101). Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 32, 8.8, and 26 nM, respectively, were calculated from the results of competition binding assays conducted with membranes. D-MLVP did not bind to plasma membranes. Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 390, 38, and 160 nM, respectively, were calculated from the results of competition binding assays conducted with cells. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased adenylate cyclase activity in canine renal plasma membranes to values 2.4, 2.9, and 2.6 times that of basal activity, respectively. A maximally active concentration of AVP (1 microM) increased adenylate cyclase activity in canine renal plasma membranes to a value 2.7 times that of basal activity. D-MLVP did not stimulate adenylate cyclase activity. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased the cAMP content of porcine kidney cells from a basal level of 43 to 267, 160, and 469 pmol/mg of cell protein, respectively. Specific binding of these fluorescent analogs to receptors on the surface of LLC-PK1 cells was observed by fluorescence microscopy. These observations indicate that F-MLVP, MA-MLVP, and R-MLVP are biologically active fluorescent vasopressin analogs which are well-suited to the study of renal vasopressin receptors by fluorescence microscopy.     

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function.

A novel mutant of the LLC-PK1 renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V2 receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK1 cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5'-[beta, gamma-imino] triphosphate, 8-bromo adenosine-3',5'-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK1 and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V2 receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V2 receptor gene using DNA-transfer.     

125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP: iodination and binding characteristics of a vasopressin receptor ligand

A radioiodinated vasopressin antagonist, d(CH2)5[Tyr(NH2)9]AVP has been prepared. Iodination was carried out at the phenyl moiety of the tyrosylamide residue at position 9, followed by HPLC purification. Non-radiolabelled monoiodinated antagonist was used as a reference for identification. 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP binding appeared to take place with a dissociation constant of 0.28 +/- 0.09 nM (Kd +/- SD) to V1 vasopressin receptors on rat liver membranes.     

A Low Affinity Vasopressin V2-Receptor in Inherited Nephrogenic Diabetes Insipidus

Abstract

Congenital nephrogenic diabetes insipidus (NDI) is an X-linked inherited disorder characterized by renal resistance to the antidiuretic hormonal action of vasopressin. This study describes the molecular basis of nephrogenic diabetes insipidus in a dog family. Kidney membranes prepared from NDI-affected male huskies were examined for vasopressin binding and response. Compared to membranes from unaffected canines, those from the kidney inner medulla of NDI-dogs possessed normal V₂-receptor numbers, but with 10–fold lower affinity for [Arg⁸] vasopressin (AVP). Adenylate cyclase stimulation by AVP in contrast to that by forskolin or GTP-analogues was similarly reduced in a dose responsive manner. The NDI-affected dogs showed antidiuretic responses to very high doses of V₂–specific agonists, consistent with their possessing V₂–receptors of lower affinity. Prolonged treatment with V₂–agonists, 1–deamino [D-Arg⁸] VP (dDAVP) and 1–deamino [Va]⁴, Sar⁷] AVP (dVSAVP), rendered the NDI-affected dogs near normal in terms of water intake and urine osmolality.     

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.     

[3H]Vasopressin binding to rat hippocampal synaptic plasma membrane: Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [³H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (K_d = 2.8 nM) and dDAVP has a low affinity (K_d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe²Orn⁸VT are at least as active as AVP in inhibiting [³H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.     

Inhibition of hepatic alpha 1-adrenergic effects and binding by phorbol myristate acetate

Treatment of isolated hepatocytes with the tumor-promoting agent, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and dose-dependent, non-competitive inhibition of alpha 1-adrenergic responses, including the activation of phosphorylase, increase in Ca2+ efflux, increase in free cytosolic Ca2+, and release of myo-inositol-1,4,5-P3. The actions of [8-arginine] vasopressin (AVP) on liver cells were also inhibited by PMA, but the inhibition could be overcome by high AVP concentrations. No significant inhibition of beta-adrenergic and glucagon-mediated activation of phosphorylase was induced by PMA and no inhibitory or synergistic effects of PMA were observed on the dose-dependent activation of phosphorylase by the Ca2+ ionophore A23187. In radioligand binding studies, PMA did not directly interfere with [3H]prazosin specific binding, the displacement of [3H]prazosin by (-)-norepinephrine nor with [3H]AVP specific binding to purified liver plasma membranes. Plasma membranes prepared from livers perfused with PMA exhibited a 30-44% reduction in [3H]prazosin binding capacity. Under identical conditions [3H]AVP binding was unchanged. The alpha 1-receptors remaining in membranes from PMA-treated livers had equivalent affinities for [3H]prazosin and (-)-norepinephrine, and were unaffected in terms of coupling to guanine nucleotide-regulating proteins as indicated by the ability of guanosine 5'-(beta, gamma-imido)triphosphate to promote the conversion of the remaining alpha 1-receptors into a low affinity state. These data indicate that tumor promoters are potent antagonists of alpha 1-adrenergic and vasopressin (low dose) responses in liver. It is proposed that PMA acting via protein kinase C (which presumably mediates the action of PMA) exerts its inhibitory action on alpha 1-adrenergic responses at the alpha 1-adrenergic receptor itself and also at a site close to or before myo-inositol-1,4,5-P3 release.     

Functional relations of crab molt-inhibiting hormone and neurohypophysial peptides

Biological and immunological relationships between molt-inhibiting hormone (MIH) activity in eyestalk ganglia extracts of the crab, Cancer antennarius Stimpson, and peptides of the vasopressin-oxytocin family were assessed. Lysine vasopressin (LVP), arginine vasopressin (AVP), vasotocin (VT), and oxytocin (OT) mimicked MIH action by inhibiting ecdysteroid production of Y-organ segments in vitro with the relative potencies LVP greater than AVP greater than VT much much greater than OT. The inhibitory effect was reversible and specific (6 other peptides did not alter Y-organ activity). MIH and LVP increased Y-organ cyclic adenosine 3',5' monophosphate (cAMP) levels dose-dependently and with identical time course in which the rise in cAMP preceded inhibition of ecdysteroid production. The synthetic vasopressin antidiuretic agonist 1-deamino-8-D-AVP (dDAVP) inhibited Y-organ steroidogenesis dose-dependently; the vasopressin analog ([1(B-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine[AVP) (d(CH2)5Tyr(Me)AVP), a vasopressor antagonist, had no effect on basal or MIH-suppressed steroidogenesis. AVP antiserum abolished the inhibitory action of MIH, LVP, and AVP. Competitive binding curves for MIH, LVP, AVP, VT, and OT with the AVP antiserum suggested that MIH is most closely related to LVP. MIH may be structurally related to the vasopressins and act on Y-organ cells via type V2 (cAMP-linked) receptors.     

Vasopressin antisense peptide interactions with the V1 receptor

The molecular recognition hypothesis, that peptide ligands and their receptor binding sites are encoded by complementary nucleotide sequences, was tested for arginine vasopressin (AVP) and its V1 receptor. Binding of [125I] [d(CH2)5,Sar7]AVP (a selective V1 vasopressin antagonist radioligand) or [3H]AVP to rat liver plasma membranes was inhibited by peptides known to bind to V1 receptors but not by the AVP complementary peptide (Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala) (PVA). Rabbit anti-PVA antibodies were nonimmunoreactive with any protein in rat liver membranes or in a partially purified preparation from rat liver containing reconstitutable vasopressin binding activity. Furthermore, there was no suppression of the AVP pressor effect by PVA in vivo using a rat blood pressure bioassay. These findings do not support the hypothesis that the V1 receptor binding site is encoded by the antisense DNA strand to AVP.     

Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits

To identify and characterize V1 vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3-mercapto-3,3-cyclopentamethylene-propionic acid (Mca) and N-methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V1 antagonists: [Mca1, D-Tyr2, MeAla7, Lys8]VP (1), [Mca1, MeAla7, Arg8, Lys9]VP (2), [Mca1, MeAla7, Arg8, D-Lys9]VP (3). Introduction of the photoreactive 4-azidophenylamidino group into the side-chain of Lys8 in analogue 1 or into Lys9 in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono-iodination at Tyr2 with 125I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V1 receptor (Kd = 0.9-1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2--3 pmol V1 receptor/mg protein, the analogues labelled specifically two proteins with the relative molecular masses (Mr) of 30,000 and 38,000. These results and the results of a recent study using 3H-labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Biol. Chem. 260, 15051-15054] provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V1 receptor. Furthermore the radioiodinated photoreactive V1 antagonists should be helpful to identify V1 receptor proteins in membranes of other cell types.     

Subcellular Localization and Characterization of Vasopressin Binding Sites in the Ventral Septal Area, Lateral Septum, and Hippocampus of the Rat Brain

[Arg8]-Vasopressin (AVP) has been shown to exert characteristic central physiological actions in the ventral septal area of the rat brain. This study reports the characterization of receptors for AVP in synaptic plasma membranes prepared from the ventral septal area, the lateral septum, and the hippocampus. Binding of [3H]AVP was temperature and time dependent, linearly related to protein concentration, saturable, and specific. Scatchard plot analysis suggested the presence of a population of binding sites in the three brain areas with dissociation constants and maximal binding capacities, respectively, of 1.06 +/- 0.39 nM and 24.0 +/- 7.01 fmol/mg of protein (mean +/- SEM; n = 3 for the ventral septal area, 0.92 +/- 0.13 nM and 47.0 +/- 4.96 fmol/mg of protein (n = 3) for the lateral septum, and 0.91 +/- 0.14 nM and 25 +/- 5.02 fmol/mg of protein (n = 3) for the hippocampus. In all three brain regions, the rank order of potencies of several vasopressin analogs, unrelated peptides, and other compounds for competitive displacement of ligand indicated a receptor with properties resembling those of the V1-like receptor for AVP. These data document the presence of a high-affinity, V1-like vasopressin receptor in the rat ventral septal area for which the pharmacological properties are similar to those previously reported in physiological studies.     

Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney

Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.7 +/- 0.3 and 2.3 +/- 0.2 nM, respectively. In competition binding experiments, the solubilized fraction exhibited the same pharmacological profile as the membranes. Chemical crosslinking of [125I]vasopressin to the solubilized fraction followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated a 62-kDa band which was specifically labeled with [125I]vasopressin. Vasopressin binding sites from the solubilized fractions were resolved by gel filtration and ultracentrifugation on a sucrose gradient. In addition, agonist high affinity binding to V2 receptors and its sensitivity to guanine nucleotides were preserved even after solubilization in the absence of prebound agonist prior to solubilization. Addition of guanine nucleotides such as GTP gamma S decreased the specific binding of [3H]arginine vasopressin to these solubilized fractions in a dose-dependent manner, suggesting the solubilization of a V2 receptor-G protein complex. [32P]ADP ribosylation of the solubilized fraction by cholera and pertussis toxins revealed specifically labeled proteins with molecular weights of 42,000-43,000 and 39,000-41,000, respectively, on sodium dodecyl sulfate polyacrylamide gels. Furthermore [35S]GTP gamma S binding to these solubilized fractions was enhanced by vasopressin, confirming that a significant proportion of the vasopressin receptors must be closely coupled to G proteins even when these receptors are solubilized in the absence of agonist. These results are in contrast with those reported for beta, alpha 2 adrenergic and D2 dopaminergic receptor systems, but in agreement with D1 dopaminergic and A1 adenosine receptors. The molecular mechanism responsible for this difference remains to be determined.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

Solubilization of rat liver vasopressin receptors as a complex with a guanine-nucleotide-binding protein and phosphoinositide-specific phospholipase C.

Vasopressin V1 receptors were solubilized from rat liver plasma membranes with the detergent lysophosphatidylcholine. [[3H]Arginine]vasopressin (AVP) binding to the solubilized preparations was specific and saturable, with a dissociation constant of 0.6 nM. Cross-linking of [125I]vasopressin to the solubilized fraction, studied by SDS/polyacrylamide-gel-electrophoretic analysis, demonstrated the presence of a 65 kDa band which was specifically labelled with [125I]vasopressin. Specific binding of [3H]AVP to these solubilized receptors was decreased by guanine nucleotides, but not by adenosine 5'-[beta gamma-imido]triphosphate. Addition of vasopressin increased specific binding of 35S-labelled guanosine 5'-[gamma-thio]triphosphate (GTP[35S]) to the solubilized fractions, indicating co-solubilization of GTP-binding protein(s) [G-protein(s)] and vasopressin receptors. The solubilized fraction was insensitive to both cholera- and pertussistoxin treatment. Immunoblotting of the solubilized fraction with antibodies specific for a phosphoinositide-specific phospholipase C (PI-PLC I) demonstrated the presence of a 60 kDa protein. Anti-PI-PLC I antiserum immunoprecipitated solubilized vasopressin-binding sites from rat liver (V1), but not solubilized vasopressin-binding sites from hog kidney (V2). Similar results were obtained with an anti-PI-PLC I IgG affinity column. The solubilized (V1) receptors were enriched by ion-exchange and high-performance gel-filtration liquid chromatography. Vasopressin-binding activity was co-eluted with PI-PLC I and GTP[S]-binding activity on a DEAE-Sepharose column. The major vasopressin- and GTP[35S]-binding activities were co-eluted with PI-PLC I activity at approx. 240 kDa suggesting that vasopressin receptors from rat liver membranes can be solubilized as a complex of receptor-coupler-effector by using the detergent lysophosphatidycholine.     

Effects of the substitution of photoreactive groups in positions 4 and 8 of vasopressin

In an effort to synthesize potent vasopressin analogs containing photoreactive groups, we prepared, by solid phase synthesis, three analogs with proline or hydroxyproline substitutions in positions 4 and/or 7, lysine in positions 4 or 8, and beta-mercaptopropionic acid in position 1. From these three parent analogs, 1-desamino[4-proline,8-lysine]VP, 1-desamino[4-hydroxyproline,8-lysine]VP, and 1-desamino[4-lysine,7-hydroxyproline]AVP, we then prepared the corresponding azido compounds using the epsilon-amino group of lysine as the attachment point. These six analogs were then assayed for antidiuretic and pressor activities in rats. One of the resulting analogs, 1-desamino[4-lysine(N epsilon-4-azidobenzoyl),7-hydroxyproline)]AVP has the highest antidiuretic activity of any photoreactive compound reported to date.     

Probing the adenosine receptor with adenosine and xanthine biotin conjugates

Biotin-containing analogs of a potent agonist (N6-phenyladenosine) and a potent antagonist (1,3-dipropyl-8-phenylxanthine) of adenosine receptor activity have been synthesized. A spacer chain to the biotin moiety is attached in both cases to the para-position of the phenyl ring. Two biotin conjugates of N6-phenyladenosine differing only in the length of the spacer chain bind to the adenosine receptor and to avidin simultaneously. The shorter-chain derivative was more potent in inhibiting binding of N6-[3H]cyclohexyladenosine to rat cerebral cortical membranes (Ki of 11 nM in the absence of avidin, 36 nM for the avidin complex). Three biotin conjugates of 1,3-dipropyl-8-phenylxanthine bound competitively to the adenosine receptor, but only in the absence of avidin. The results are interpreted in terms of the possible orientation of the ligands at the receptor binding site.     

Theoretical conformational analysis of six arginine vasopressin analogs with the L-naphthylalanine in position 3.

Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with L-2-Nal in position 3 causes moderate antiduretic activity, whereas [Mpa1, (L-1-Nal)3, (D-Arg)8] VP and [Mpa1, (L-2-Nal)3, (D-Arg)8] VP are potent and selective V2 agonists. Moreover [(L-2-Nal)3, (D-Arg)8] VP is among the most potent and selective antagonists of V1a receptors. In this study we carried out conformational calculations on [(L-1-Nal)3] AVP, [(L-2-Nal)3] AVP, [(L-1-Nal)3, (D-Arg)8] VP, [(L-2-Nal)3, (D-Arg)8] VP, [Mpa1, (L-1-Nal)3, (D-Arg)8] VP, [Mpa1, (L-2-Nal)3, (D-Arg)8] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low-energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest-energy conformations in the presence and absence of water had beta-turns at residues Phe3-Gln4 and Gln4-Asn5 and Gln4-Asn5, respectively. The conformation at the Gln4-Asn5 turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).     

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function

A novel mutant of the LLC-PK₁ renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V₂ receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK₁ cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5′-[β,γ-imino] triphosphate, 8-bromo adenosine-3′,5′-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK₁ and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V₂ receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V₂ receptor gene using DNA-transfer.     

Effect of a new V1 antagonist (OPC-21268) on vascular action of vasopressin in cultured rat vascular smooth muscle cells.

The effect of 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl-3,4-dihydro-2(1 H)- quinolinone) (OPC-21268) on vascular action of arginine vasopressin (AVP) was examined in cultured rat vascular smooth muscle cells (VSMC) by the measurement of cytosolic free calcium concentration [( Ca2+]i) and the AVP V1 receptor study. The preincubation of cells with OPC-21268 for 10 min inhibited the AVP-induced mobilization of [Ca2+]i in a dose-dependent manner but did not affect the angiotensin II-induced mobilization of [Ca2+]i. The receptor study revealed that OPC-21268 blocks the binding of AVP to the receptor in VSMC in a similar way to the V1 structural antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-2-O-methyltyrosine]AVP: d(CH2)5Tyr(Me)AVP. Lineweaver-Burk plot showed that OPC-21268 is the competitive AVP V1 receptor antagonist. These results therefore indicate that OPC-21268 specifically blocks the vascular action of AVP mediated through the competitive inhibition of AVP binding to the receptors in VSMC.     

Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland. Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)5(1)[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding.(ABSTRACT TRUNCATED AT 250 WORDS)