cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells.

The relationship between activation of the cAMP-dependent protein kinase (cAMP-PK) and ligand binding and internalization by the vasopressin renal (V2-type) receptor of LLC-PK1 renal epithelial cells was examined. Upon cAMP-PK activation through 1 h treatment with the cAMP analogue 8-bromo-cAMP (BrcA), a marked reduction in V2-receptor steady state number and internalization in LLC-PK1 cells was effected. In cells treated for 17 h with BrcA and hence down-regulated for cAMP-PK, the V2-receptor number was normal but internalization was markedly reduced. Cells of the LLC-PK1 mutant FIB4, which possesses about 10% parental cAMP-PK catalytic subunit activity, exhibited lower V2-receptor steady state number and internalization in comparison to untreated LLC-PK1 cells. A negative correlation was thus evident between cAMP-PK activation and V2-receptor number, and internalization. Phosphorylation by cAMP-PK may effect ligand-independent removal of receptor from the plasma membrane.     

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

We have previously demonstrated that apical Na⁺ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971–6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na⁺ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882–C888); however, it is unknown if the Na⁺ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for modulation of Na⁺ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for regulation of Na⁺ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na⁺ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na⁺ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na⁺ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 ± 5.42%) when compared to control (unstimulated) (f = 14.2 ± 0.77%) and vasopressin-treated (20 min) (f = 12.7 ± 2.4%) monolayers. The twofold increase in mobile fraction of Na⁺ channels corresponds to the average increase in Na⁺ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na⁺ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na⁺ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na⁺ channels.Supported by NIH grants DK37206 (DJB), NS26733 and NS28072 (KJA), DK46705 (PRS) and AHA New York Affiliate grant 91007G (LCS).     

The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.     

Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor.

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.     

N-glycosylation plays a role in biosynthesis and internalization of the adenylate cyclase stimulating vasopressin V2-receptor of LLC-PK1 renal epithelial cells: an effect of concanavalin A on binding and expression.

The role of N-glycosylation in the function and biosynthesis of the vasopressin V2-receptor in LLC-PK1 renal epithelial cells was examined using various lectins and inhibitors operating at different steps of the glycosidic pathway. Tunicamycin, which blocks all N-glycosylation, and castanospermine, which inhibits glycosidase I and hence blocks formation of high-mannose-type N-glycosylated intermediates, resembled one another in affecting V2-receptor biosynthesis and internalization in a concentration-dependent manner. In contrast, swainsonine, an inhibitor of mannosidase II and hence of complex-type oligosaccharide formation, had no effect. Interestingly, the alpha-D-mannose/alpha-D-glucose-specific lectin concanavalin A, (Con A), in contrast to the beta-D-galactose-specific lectin ricin, had a marked effect on the V2-receptor in LLC-PK1 cells, increasing both receptor numbers up to twofold in vivo and specific [3H]AVP binding up to 50% in vitro in a concentration-dependent manner. The concentrations inducing half-maximal response were about 0.2 and 20 micrograms/ml for the in vivo and in vitro responses, respectively, implying distinct effects on V2-expression and ligand binding. That the in vitro effect on binding was due to a direct effect on the V2-receptor could be shown by the lack of a Con A effect on [3H]AVP binding in membranes prepared from LLC-PK1 cells down-regulated for the V2-receptor or from cells of the LLC-PK1 V2-receptor deficient mutant M18. All results were consistent with a functional role for N-glycosylation of the V2-receptor in LLC-PK1 cells.     

Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.     

Administration of oxytocin affects vasopressin V2 receptor and aquaporin-2 gene expression in the rat.

Oxytocin (OT) binds to the vasopressin V2 receptor (V2R) because of its structural similarity to arginine vasopressin (AVP). Though the affinity of OT for V2R is low, it is known that OT causes antidiuresis. To clarify the effect of OT as an agonist of V2R, we investigated the influence of acute elevation of plasma OT levels on the rat mRNA expression of V2R and aquaporin-2 (AQP2), the water channel regulated by V2R. The plasma OT level increased from 11.1+/-1.6 pg/ml to 331.0+/-67.9 pg/ml by 1 h after subcutaneousinjection of 20 microg OT. V2R mRNA expression decreased to 68.3+/-4.1% of the control at 3 h, and AQP2 mRNA expression increased to 239.3+/-26.8% of the control at 6 h. The plasma AVP level did not change significantly during the experiment. The influence of a subcutaneous injection of 20 microg OT on V2R and AQP2 mRNA expression is comparable to that of 10 microg AVP that we documented in the previous study. In conclusion, OT can downregulate V2R mRNA expression and upregulate AQP2 mRNA expression in the collecting duct as an agonist of the V2R like AVP.     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Interaction between the keratin cytoskeleton and eEF1Bgamma affects protein synthesis in epithelial cells

Eukaryotic elongation factor-1 (eEF1) is essential for peptide-chain elongation during translation. We report that its gamma subunit (eEF1Bgamma) specifically binds, and bundles, keratin intermediate filaments. Disrupting this interaction depresses translation by approximately 20% and selectively increases 80S ribosomes in epithelial cells, an outcome recapitulated by RNA interference-mediated silencing of eEF1Bgamma. These findings extend the emerging relationship between keratin proteins and the translational machinery.     

Linking membrane microdomains to the cytoskeleton: regulation of the lateral mobility of reggie-1/flotillin-2 by interaction with actin

The reggies/flotillins are oligomeric scaffolding proteins for membrane microdomains. We show here that reggie-1/flotillin-2 microdomains are organized along cortical F-actin in several cell types. Interaction with F-actin is mediated by the SPFH domain as shown by in vivo co-localization and in vitro binding experiments. Reggie-1/flotillin-2 microdomains form independent of actin, but disruption or stabilization of the actin cytoskeleton modulate the lateral mobility of reggie-1/flotillin-2 as shown by FRAP. Furthermore, reggie/flotillin microdomains can efficiently be immobilized by actin polymerisation, while exchange of reggie-1/flotillin-2 molecules between microdomains is enhanced by actin disruption as shown by tracking of individual microdomains using TIRF microscopy.     

External Ca2+ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel whose dysfunction is associated with the onset of autosomal dominant polycystic kidney disease (ADPKD). PC2 contributes to Ca²⁺ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to the external Ca²⁺ regulation of PC2 channel function. In this study, we explored the effect of external Ca²⁺ on endogenous PC2 in wild type LLC-PK1 renal epithelial cells. We obtained whole cell currents at different external Ca²⁺ concentrations, and observed that the basal whole cell conductance in normal Ca²⁺(1.2 mM), decreased by 30.2% in zero (nominal) Ca²⁺ and conversely, increased by 38% in high external Ca²⁺(6.2 mM). The high Ca²⁺-increased whole cell currents were completely inhibited by either PC2 gene silencing, or intracellular dialysis with active, but not denatured by boiling, PC2 antibody. Exposure of cells to high Ca²⁺ was also associated with relocation of PC2 to the plasma membrane. To explore whether a Ca²⁺ sensing receptor (CaSR) was implicated in the external Ca²⁺ modulation of PC2 currents, we tested the effect of the CaSR agonists, spermine and the calcimimetic R-568, which largely mimicked the effect of high Ca²⁺ under Ca²⁺-free conditions. The CaSR agonist gentamicin also increased the PC2 currents in the presence of normal Ca²⁺. The presence of CaSR was confirmed by immunocytochemistry, which partially colocalized with the intracellular PC2 protein, in an external Ca²⁺-dependent manner. The data support a novel Ca²⁺ sensing mechanism for PC2 expression and functional regulation in renal epithelial cells.     

Basal-lateral transport and transcellular flux of methyl alpha-D-glucoside across LLC-PK1 renal epithelial cells

The characteristics of methyl alpha-D-glucoside transport by the LLC-PK1 cell line are extended by a study of the interaction of this glucose analog with the basal-lateral membrane of these cells: 1 mM methyl alpha-D-glucoside enters LLC-PK1 cells across the basal-lateral membrane 10-times more slowly than when entering across the apical membrane; neither 10 mM glucose nor 10 mM methyl alpha-D-glucoside affect the rate of methyl glucoside uptake at the basal lateral membrane; 0.1 mM phlorizin in the apical hemichamber significantly decreases the rate at which methyl glucoside enter the cell when methyl glucoside is present in the basal-lateral hemichamber; the methyl glucoside transcellular flux ratio, Ja/Jb (apical to basal vs. basal to apical) is 15, whereas Ja/Jb for D-mannitol equals 1; and basal-lateral to apical fluxes (Jb) of mannitol consistently exceed those of methyl glucoside. These results demonstrate that methyl glucoside, unlike glucose, is accumulated within these cells to a high degree because of the limited ability of methyl glucoside to exit the cells by a carrier-mediated pathway. They also raise the important caveat for any studies with glucose (and other low-molecular-weight substrates) by showing that a monosaccharide presented to one surface of these cells will traverse the cell sheet (in part) by the intercellular route and will enter the cell at the unintended cell surface. The ability of the tight junctions of this intercellular route to discriminate between open-chain molecules, such as mannitol, vs. closed ring structures, like methyl glucoside, is also described.     

Cationic amino acid transport by two renal epithelial cell lines: LLC-PK1 and MDCK cells

LLC-PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC-PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans-stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.     

Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK1 cells

We have previously demonstrated that a cultured porcine kidney cell, LLC-PK(1), maintains the characteristics of a polar renal epithelial cell in culture, and responds to salmon calcitonin and [arginine]vasopressin by increasing cyclic AMP content. To demonstrate the usefulness of this cell line as a model for the study of the biochemical events distal to cyclic AMP production, the activation of cyclic AMP-dependent protein kinase was examined. Intact cells in monolayer demonstrated progressive increases in cyclic AMP content and activation of protein kinase in response to [arginine]vasopressin (2-200nm) and salmon calcitonin (0.03-30nm) with both hormones fully activating the enzyme at a cell cyclic AMP content of 35pmol/mg of protein. Of the total cyclic AMP-dependent protein kinase activity, 80% was found in the 27000g supernatant fraction of sonicated cell material, and this soluble protein kinase could be fully activated by hormone. Conversely, the 27000g pellet contained a significant proportion of cyclic AMP-independent protein kinase and only 20% of total cell cyclic AMP-dependent protein kinase; the latter showed little response to hormone. On the basis of DEAE-cellulose chromatography, type II protein kinase was the predominant isoenzyme in both soluble and particulate fractions of the LLC-PK(1) cells and the soluble fractions of rat and guinea-pig renal medulla. Thus, the LLC-PK(1) cell line can serve as a model for hormonal modulation of protein kinase and as a potential source for defining the endogenous substrates for these enzymes.     

A novel C-terminal motif is necessary for the export of the vasopressin V1b/V3 receptor to the plasma membrane

Little is known about endoplasmic reticulum (ER) export signals, particularly those of members of the G-protein-coupled receptor family. We investigated the structural motifs involved in membrane export of the human pituitary vasopressin V1b/V3 receptor. A series of V3 receptors carrying deletions and point mutations were expressed in AtT20 corticotroph cells. We analyzed the export of these receptors by monitoring radioligand binding and by analysis of a V3 receptor tagged with both green fluorescent protein and Myc epitopes by a novel flow cytometry-based method. This novel method allowed us to quantify total and membrane-bound receptor expression. Receptors lacking the C terminus were not expressed at the cell surface, suggesting the presence of an export motif in this domain. The distal C terminus contains two di-acidic (DXE) ER export motifs; however, mutating both these motifs had no effect on the V3 receptor export. The proximal C terminus contains a di-leucine (345)LL(346) motif surrounded by the hydrophobic residues Phe(341), Asn(342), and Leu(350). The mutation of one or more of these five residues abolished up to 100% of the receptor export. In addition, these mutants colocalized with calnexin, demonstrating that they were retained in the ER. Finally, this motif was sufficient to confer export properties on a CD8alpha glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)(2)LL(X)(3)L, in the C terminus of the V3 receptor.     

Molecular dynamics of a vasopressin V2 receptor in a phospholipid bilayer membrane

Molecular dynamics simulations were carried out for a V2 receptor (V2R) model embedded in a dimyristoylphosphatidylcholine (DMPC) bilayer. Both free and ligand-bound states of V2R were modeled. Our initial V2R model was obtained using a rule-based automated method for GPCR modeling and refined using constrained simulated annealing in vacuo. The docking site of the native vasopressin ligand was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. The primary purpose of this work was to investigate the usefulness of MD simulation of an integral membrane protein like a GPCR receptor, upon inclusion of a carefully parameterized surrounding lipid membrane and water. Physical properties of the system were evaluated and compared with the fully hydrated pure DMPC bilayer membrane. The solvation interactions, individual lipid-protein interaction and fluctuations of the protein, the lipid, and water were analyzed in detail. As expected, the membrane-spanning helices of the protein fluctuate less than the peripheral loops do. The protein appears to disturb the local lipid structure. Simulations were carried out using AMBER 4.1 package upon constant number-pressure-temperature (NPT) conditions on massively parallel computers Cray T3E and IBM SP2.     

Expression of vasopressin V2 receptor in Xenopus laevis oocytes by porcine kidney cell line (LLC-PK1) messenger RNA.

Vasopressin V2 receptor was expressed in Xenopus laevis oocytes which were injected with poly(A) +RNA from porcine kidney cell line LLC-PK1. Pharmacological antagonism of the expressed V2 receptor was observed between arginine vasopressin and two potent and selective vasopressin antagonists: [d(CH2)5, D2-Phe2 Ile4, Ala9-NH2]arginine vasopressin and [d(CH2)5,D-Ile2, Ile4]arginine vasopressin. Activation constant for arginine vasopressin concentration was 1.32 x 10(-10)M. The nucleotide length of the mRNA encoding for vasopressin V2 receptor was deduced to be approximately 2 kilobases.