Arginine Vasopressin Binding Sites Research Articles

Oxytocin-Induced Analgesia and Scratching Are Mediated by the Vasopressin-1A Receptor in the Mouse

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) contribute to the regulation of diverse cognitive and physiological functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal cord, or systemically. Here, we characterized the phenotype of oxytocin receptor (OTR) and vasopressin-1A receptor (V1AR) null mutant mice in a battery of pain assays. Surprisingly, OTR knock-out mice displayed a pain phenotype identical to their wild-type littermates. Moreover, systemic administration of OXT dose-dependently produced analgesia in both wild-type and OTR knock-out mice in three different assays, the radiant-heat paw withdrawal test, the von Frey test of mechanical sensitivity, and the formalin test of inflammatory nociception. In contrast, OXT-induced analgesia was completely absent in V1AR knock-out mice. In wild-type mice, OXT-induced analgesia could be fully prevented by pretreatment with a V1AR but not an OTR antagonist. Receptor binding studies demonstrated that the distribution of OXT and AVP binding sites in mouse lumbar spinal cord resembles the pattern observed in rat. AVP binding sites diffusely label the lumbar spinal cord, whereas OXT binding sites cluster in the substantia gelatinosa of the dorsal horn. In contrast, quantitative real-time reverse transcription (RT)-PCR revealed that V1AR but not OTR mRNA is abundantly expressed in mouse dorsal root ganglia, where it localizes to small- and medium-diameter cells as shown by single-cell RT-PCR. Hence, V1ARs expressed in dorsal root ganglia might represent a previously unrecognized target for the analgesic action of OXT and AVP.

Mapping the Binding Site of Six Nonpeptide Antagonists to the Human V2-Renal Vasopressin Receptor

Whereas arginine vasopressin binds to its receptor subtypes V(1)R and V(2)R with equal affinity of approximately 2 nM, nonpeptide antagonists interact differently with vasopressin receptor subtypes. The V(2)R antagonist binding site was mapped by site-directed mutagenesis at six selected amino acid positions, K100D, A110W, M120V, L175Y, R202S, and F307I, predicted to be involved in antagonist binding differences between V(2) R and V(1)R. These mutations did not alter the affinity for arginine vasopressin. However, the affinity for six nonpeptide receptor antagonists SR121463B [1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4[(2 morpholinoethoxy)cy-clohexane]indoline-2-one, phosphate monohydrate cis-isomer], SR49059 [(2S)1-[(2R3S)-(5-chloro-3-(2 chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide], SSR149415 [(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine carboxamide, isomer(-)], OPC21268 [1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone], OPC41061 [(+/-)-4'-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide], and OPC31260, [(+/-)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-1,2, 3,4,5-tetrahydro-1H-benzazepine monohydrochloride], was altered to varying degrees, resulting in differences up to 6000-fold. Replacement of the small alanine for the bulky tryptophan in position 110 resulted in a reduced affinity for all six antagonists. In contrast, replacement of the large methionine for the smaller valine in position 120 caused a dramatic increase in affinity, up to a K(i) of 7 fM for OPC31260. Molecular modeling revealed that the binding sites for arginine vasopressin and the nonpeptide antagonists are partially overlapping. Whereas arginine vasopressin binds on the extracellular surface of V(2) R, the nonpeptide antagonists penetrate deeper into the transmembrane region of the receptor, in particular OPC21268. The mutagenesis data point to significant differences in the shape of the V(1)R and V(2)R antagonist binding pockets. The most important factor determining the specificity of nonpeptide antagonists seems to be the shape of the binding pocket on the receptor.

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium.

The cerebrospinal fluid (CSF)-generating choroid plexus (CP) has many V1 binding sites for arginine vasopressin (AVP). AVP decreases CSF formation rate and choroidal blood flow, but little is known about how AVP alters ion transport across the blood-CSF barrier. Adult rat lateral ventricle CP was loaded with 36Cl-, exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of 36Cl-. Effect of AVP at 10(-12) to 10(-7) M on the Cl- efflux rate coefficient (in s-1) was quantified. Maximal inhibition (by 20%) of Cl- extrusion at 10(-9) M AVP was prevented by the V1 receptor antagonist [beta-mercapto-beta, beta-cyclopentamethyleneproprionyl1,O-Me-Tyr2,Arg8]vasopressin. AVP also increased by more than twofold the number of dark and possibly dehydrated but otherwise morphologically normal choroid epithelial cells in adult CP. The V1 receptor antagonist prevented this AVP-induced increment in dark cell frequency. In infant rats (1 wk) with incomplete CSF secretory ability, 10(-9) M AVP altered neither Cl- efflux nor dark cell frequency. The ability of AVP to elicit functional and structural changes in adult, but not infant, CP epithelium is discussed in regard to ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

Oxytocin and vasopressin receptors in human and uterine myomas during menstrual cycle and early pregnancy.

The purpose of this study was to determine the specificity and concentration of oxytocin (OT) and arginine vasopressin (AVP) binding sites in non-pregnant (NP) human and rhesus monkey endometrium, myometrium and fibromyomas, and to determine the cellular localization of OT receptor (OTR). Besides [3H]AVP, [125I]LVA, a specific VP1 receptor subtype antagonist, was used to determine vasopressin receptor (VPR) concentrations. Samples were obtained from 42 pre-menopausal and three pregnant women (5, 13 and 35 weeks gestation), and several NP and pregnant monkeys. Specificity of binding was assessed in competition experiments with unlabelled agonists and antagonists of known pharmacological potency. Cellular localization of OTR was determined by immunohistochemistry. In NP human uterine tissues, [3H]AVP was bound with higher affinity and greater binding capacity than [3H]OT, whereas in pregnant women and in NP and pregnant rhesus monkeys, uterine OT binding capacity was greater. OT and AVP binding sites discriminated very poorly between OT and AVP; [125I]LVA binding sites were more selective than [3H]AVP. Their ligand specificity and binding kinetics indicated the presence of two distinct populations of binding sites for OT and AVP in primate uterus. Endometrium of NP women and monkeys had low OTR and VPR concentrations. Myometrial and endometrial OTR and VPR were down-regulated in midcycle and in early human pregnancy, they were up-regulated in the secretory phase and second half of pregnancy. Immunoreactive OTR in NP uterus was localized in patches of myometrial muscle cells and small numbers of endometrial epithelial cells.

Autoradiographic localization of arginine vasopressin binding sites in the brain of adult and developing Brazilian opossums.

We are utilizing the Brazilian short-tailed opossum, Monodelphis domestica, to study the development of the vasopressinergic system. Earlier studies demonstrated that arginine vasopressin-like immunoreactivity was present very early in the Brazilian opossum brain, suggesting a role for vasopressin in the developing central nervous system of mammals. In this study, we have utilized [3H]arginine vasopressin autoradiography to describe the distribution of arginine vasopressin binding sites in adult and developing Brazilian opossum brains. In general, arginine vasopressin binding patterns in adult opossum brains resembled those of other species. However, we found very few labelled areas in neonatal Brazilian opossum brains. At birth, only the ventral tegmental area and the nucleus of the solitary tract were labelled. Binding was not evident in the forebrain until 25 days of postnatal age. The anterior pituitary was heavily labelled from birth onward, but binding in the brain itself remained at low levels until 35 days postnatal. Heavy binding was observed in only a few areas of the brain in adults, including the dorsal part of the lateral septal nucleus, the suprachiasmatic nucleus, the dorsal and median raphe, the nucleus of the solitary tract, and the caudal part of the spinal trigeminal nucleus. Surprisingly, arginine vasopressin binding sites in the Brazilian opossum appeared much later than arginine vasopressin immunoreactivity and, in many cases, after neurogenesis was complete. These findings suggest that the arginine vasopressin binding sites are not playing a developmental role in opossums, although the peptide is present at an early age.

Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site.

Most G-protein-coupled receptors have conserved cysteine residues in their C-terminal cytoplasmic domain that appear to be generally palmitoylated. An example is the human arginine vasopressin V2 receptor with cysteine residues at positions 341 and 342. Site-directed mutagenesis of the putative palmitoylation site was used to study the significance of palmitoylation for the V2 receptor. A multifunctional expression plasmid was constructed by cloning the V2 receptor cDNA into the vector pCDNAI.Neo. The resulting plasmid allowed site-directed mutagenesis experiments without subcloning, and stable and transient expression of the V2 receptor in Ltk- and COS.M6 cells respectively. The conserved cysteine residues Cys-341 and Cys-342 were placed by serine residues, yielding the single mutants C-341S and C-342S and the double mutant C-341S/C-342S. Functional expression in stably transfected Ltk- cells showed that the affinity of the three mutant receptors for arginine vasopressin was not altered. In contrast with the activation of adenylate cyclase through beta 2 adrenergic receptors, arginine vasopressin stimulated adenylate cyclase to the same extent and with similar EC50 values in both wild-type and mutant receptors. Transient expression of the C-341S/C-342S mutant receptor in COS.M6 cells confirmed an unaltered affinity of the mutant receptor for arginine vasopressin. However, the number of arginine vasopressin-binding sites on the cell surface was reduced by 30%, suggesting that the transport of the mutant receptor to the cell surface was impaired. In addition, the decrease in detectable arginine vasopressin-binding sites on the cell surface following pre-exposure to hormone was reduced, indicating that the sequestration/internalization of the mutant receptor on the cell surface was affected. The present data indicate that palmitoylation of the V2 receptor is important for intracellular trafficking and/or sequestration/internalization but not for agonist binding or activation of the Gs/adenylate cyclase system.

Identification and characterization of [ 125I] arginine vasopressin binding sites on human peripheral blood mononuclear cells

Arginine vasopressin (AVP) is a nonapeptide that has been shown to be released from the posterior pituitary during stress. Although noted primarily for its hemodynamic and homeostatic properties. AVP also appears to have an effect on the immune system. It may modulate cellular immunity via its enhancement of the autologous mixed lymphocyte response (AMLR), an effect which we have demonstrated to occur over a wide dose range with a maximum at 10 −7 M. In this study, we examined the binding of [ 125I]AVP, and AVP analogues to human peripheral blood mononuclear cells (PBMC). AVP inhibited [ 125I]AVP (0.2 nM) binding on PBMC in a dose-dependent manner with maximal inhibition being reached at 10 −8 M. Specific [ 125I]AVP binding, as defined as that which could be displaced by 1 × 10 −6 M AVP, was saturable, time-dependent, and linear to cell concentration. Specific binding reached saturation at approximately 1000 pM in 45 minutes. From Scatchard analysis of saturation experiments it appeared to be a homogeneous population of binding sites with K D of approximately 0.5 nM and B max of approximately 7.6 fmole / 8×10 6 cells, corresponding to approximately 527 binding sites/cell. There was a good correlation between AVP binding and cell number. AVP failed to dissociate completely from its binding sites in 60 minutes, perhaps because of the formation of a high-affinity ligand-binding site complex. From competitive binding studies with various AVP antagonists and analogues, it was found that the AVP binding site appeared to be V 1-like. AVP binding occured predominantly on B-cells and macrophages. Having provided evidence for the existence of specific, high affinity, and saturable V 1-like AVP binding sites, we suggest a potential modulatory role for AVP in the communication between the neuroendocrine and immune systems.

Localization and characterization of binding sites for vasopressin and oxytocin in the brain of the guinea pig

Using autoradiography on film, specific binding sites for arginine-vasopressin (AVP) and for oxytocin (OT) were localized in various areas of the brain of adult male guinea pigs. Vasopressin binding sites were detected with [ 3H]AVP r with [ 325I]VPA, a recently synthetized linear vasopressin antagonist radiolabelled with 125I. [ 125I]VPA and [ 3H]AAP yielded similar results, thus suggesting that AVP binding ites present in the guinea pig brain are V 1 type receptors. Supporting evidence on this was obtained in competing studies using s structural analogues allowing to discriminate V 1 receptors from V 2 and from OT receptors. Oxytocin binding sites were labeleed with [ 3H]OT or wwith the iodinated OT a antagonist [ 125I]OTA; both ligands yielded similar results. The localization in the guinea pig brain of AVP binding sites differed from that of OT binding sites. AVP binding sites were mainly detected in the olfactory bulb and throughout the cerebral cortex. Oxytocin binding sites were most noticeable in the hypothalamic ventromedial nucleus, in the amygdaloid complex and in restricted areas of the cerebral cortex. A comparison of the present data with those previously described in the rat, the mouse, the human and the hamster brain suggests hat similar binding sites are present in these species, but that their anatomical distribution differs markedly. These data are discussed in relation to immunocytochemical and electrophysiological data whit suggest that binding sites detected by autoradiography may represent, at least in part, functional neuronal receptors.

Oxytocin and vasopressin binding sites in human and bovine ovaries

Human ovarian tissue and bovine ovarian stroma and follicles bound tritiated oxytocin and tritiated arginine vasopressin with similar affinity, whereas bovine corpora lutea bound tritiated oxytocin only. Competition for the binding of tritiated oxytocin by various agonists and antagonists was suggestive of receptor function. The number of oxytocin binding sites varied cyclically in all tissues. In bovine ovarian stroma and corpora lutea the concentrations were lowest on day 14 and highest on days 17, 19, and 21 after ovulation, with a striking peak in the luteal concentration on day 19. In human ovarian tissues the concentrations also were highest in samples obtained in late luteal phase. In large follicles the concentration of oxytocin binding sites was highest on the day of estrus and lowest on day 7. In bovine ovary the number of arginine vasopressin binding sites was -50% lower than oxytocin binding sites and the cyclic variations were not significant. Human ovarian tissue had similar numbers of oxytocin and arginine vasopressin binding sites. Because bovine ovaries produce oxytocin and argine vasopressin the results suggest a paracrine or autocrine role for these neuropeptides in luteolysis and ovulation. Although their synthesis in human ovaries is still controversial the presence of binding sites suggests a physiologic role in the regulation of human ovarian function as well.

3H]Arginine-vasopressin binding sites in the CNS of the golden hamster

Tritiated arginine 8-vasopressin ([ 3H]AVP) was used to identify specific binding sites for this neuropeptide in the CNS of the golden hamster. [ 3H]AVP binding sites were concentrated in the dorsal, ventral and lateral septum, bed nucleus of the stria terminalis (BNST), central amygdala, hippocampus, dorsal thalamic nuclei, anterior cingulate gyrus and endopiriform nucleus. Septal-BNST [ 3H]AVP binding sites were further characterized using membrane preparations. Saturation analysis in septal-BNST membranes resulted in a B max of 12.4 ± 0.82 fmol/mg protein and a K d of 2.5 ± 0.36 nM. Pharmacological studies indicate that the binding site in the septum-BNST membranes shows selectivity similar to a V 1-type vasopressin receptor. Binding characteristics were similar to those obtained from crude septal membranes prepared from adult Long-Evans rat.

Neurohypophyseal Hormone Receptors in the Rat Thymus, Spleen, and Lymphocytes

Receptor sites for the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) have been identified and characterized in some tissues involved in immune function in the rat. Novel radioiodinated ligands for the detection of neurohypophyseal hormone receptors, with a high specific radioactivity and affinity, enabled the selective detection of OT receptors in the thymus and vasopressin (VP) receptors in the spleen. OT receptors were detected in thymic membrane preparations and on thymocytes, which had a ligand selectivity similar to that of uterine OT receptors. AVP receptors of the V1 pressor type were present in a splenic membrane preparation. Specific AVP-binding sites, probably of the V1 type, were also present on splenic lymphocytes. Binding sites for AVP and OT could not be detected on mononuclear cells in peripheral blood of the rat. This study demonstrates that the use of the newly developed radioiodinated AVP and OT receptor ligands, with high specific radioactivity and affinity, enables the selective characterization of receptor sites for the neurohypophyseal hormones, even in the thymus, where previously no binding sites could be detected.

Binding of Four Oxytocin Analogues to Myometrial Oxytocin and Arginine-Vasopressin Binding Sites in Pregnant Women

The binding of 3H-oxytocin (3H-OT) and 3H-arginine-vasopressin (3H-AVP) and the displacement from binding sites by four oxytocin analogues were studied in myometrial membrane preparations from full-term pregnant women. Specific 3H-OT binding was saturable with a maximal binding capacity of 76.1 fmol/mg DNA, and a dissociation constant of 0.5 pM. Corresponding values regarding 3H-AVP was 148.6 fmol/mg DNA and 0.7 pM. The oxytocin analogues tested demonstrated a high specific binding to the OT and AVP receptor sites; in fact, the affinity of the analogues to the 3H-AVP binding sites was higher than to the 3H-OT binding sites. The order of potency between the analogues was CAU greater than CAM greater than CAP greater than CAO and CAP greater than CAU greater than CAO greater than CAM for the OT and AVP binding sites, respectively. The displacement of oxytocin and arginine-vasopressin, respectively, from the myometrial receptor sites indicate partly separate binding sites for oxytocin and AVP and might implicate that AVP can be of importance in regulating myometrial activity in pregnancy. The results on oxytocin analogues imply that other pharmacological tests must be performed for quantification of the relaxing effects on the uterus and to determine the optimal analogue for clinical trials in preterm labor and dysmenorrhoea.

Reduction of arginine vasopressin binding sites in mouse lateral septum by treatment with 6-hydroxydopamine

The neuropeptide arginine vasopressin modulates neuroadaptive processes, including memory consolidation and functional tolerance to ethanol, by actions at CNS V 1 receptors. Noradrenergic systems play a role in these actions of the peptide. To assess whether vasopressin may act presynaptically on catecholamine neurons, vasopressin receptors were measured by quantitative autoradiography in the lateral septum, an area that is innervated by catecholaminergic neurons and has a high density of V 1 receptors, of control and 6-hydroxydopamine-treated mice. Vasopressin receptors were distributed non-uniformly throughout the lateral septum, with greater binding in the more caudal regions. Treatment with 6-hydroxydopamine lowered septal catecholamine levels and vasopressin binding, with a greater effect on binding in the intermediate and caudal portions of the lateral septum. Pretreatment with desmethylimipramine reversed the depletion of norepinephrine, and attenuated the effect of 6-hydroxydopamine on vaspressin binding in the intermediate region, but was less effective in the caudal region of the lateral septum. The results suggest that a portion of septal vasopressin receptors are localized on the terminals of noradrenergic and, possibly, dopaminergic neurons, consistent with the hypothesis that certain neuroadaptive responses to vasopressin could be mediated by modulation of neurotransmitter release. In contrast to the results with 6-hydroxydomapine, treatment of mice with 5,7-dihydroxytryptamine, to destroy serotonergic terminals, did not alter vasopressin binding in the lateral septum.

Vasopressin receptors in human pregnant myometrium and decidua: Interactions with oxytocin and vasopressin agonists and antagonists

Saturation analysis and competition experiments were performed to identify and characterize [3H]arginine vasopressin binding to human myometrium and decidua in late pregnancy. [3H]Arginine vasopressin bound with affinity similar to that of [3H]oxytocin to both tissues (dissociation constant 1 to 2 nmol/L). The concentration of [3H]arginine vasopressin binding sites was high, particularly in decidua, but in all instances was about 50% to 60% of [3H]oxytocin binding. Analogs with selective oxytocic potency (4-threonine oxytocin, isotocin) had high affinity to both [3H]arginine vasopressin and [3H]oxytocin binding sites, as did analogs with both oxytocic and vasopressor activity (vasotocin). Analogs with selective antidiuretic activity (1-deamino-8-D-arginine vasopressin) showed drastically reduced affinity to [3H]oxytocin binding sites and relatively low but significantly higher affinity to [3H]arginine vasopressin binding sites. A new oxytocin antagonist (RW22164 or [1-deamino-2D-tyrosine-(O-ethyl)-4-threonine-8-ornithine]oxytocin) competitively bound to both binding sites. Its affinity to [3H]oxytocin binding sites was greater than to [3H]arginine vasopressin binding sites whereas the relative affinities of a predominantly vasopressor antagonist [Manning compound) were reversed, suggesting the presence of distinct receptors for oxytocin and arginine vasopressin in pregnant human myometrium and decidua.

Identification and characterization of arginine vasopressin receptors in the clonal murine Leydig-derived TM3 cell line.

Specific arginine vasopressin (AVP) binding sites were identified and characterized using Leydig cell membranes prepared from a clonal murine Leydig-derived cell line, TM3. 3H-AVP binding data analyses demonstrated that the radioligand binds to a high affinity, low capacity, homogeneous class of sites with a dissociation constant of 0.5 nM. Characterization of these AVP binding sites included competition studies. Displacement of 3H-AVP binding with high affinity by unlabelled AVP, LVP and the V1 antagonist, d(CH2)5Tyr(Me)AVP, indicated that the Leydig cell AVP receptor is of the V1 type. Furthermore, AVP did not increase adenylate cyclase activity in TM3 membranes, a finding consistent with the V1 type of AVP receptor. No competition with 3H-AVP was found with the V2 agonist, dVDAVP, or the selective oxytocin agonist, [Thr4,Gly7]oxytocin. No specific binding for oxytocin was found in Leydig cell membranes. No specific binding for either 3H-AVP or 3H-oxytocin was observed in membranes prepared from the Sertoli cell line or peritubular cell line. These findings indicate that murine Leydig cells have specific AVP binding sites of the V1 type. These AVP sites are not coupled to the adenylate cyclase system.

Solubilization and purification of the Ni-stimulated arginine-vasopressin binding site of rat brain membranes.

Arginine vasopressin binding sites on rat brain membranes were solubilized and purified by affinity chromatography. Membrane protein solubilized with CHAPS bound arginine vasopressin (AVP) only in the presence of divalent cations. Specific binding to the solubilized tissue was maximally stimulated by Ni2+, and markedly stimulated by Co2+ (30% of maximal binding with Ni2+), Zn2+ (18%), and Fe2+ (11%), parallel to the effects of these ions on the binding of AVP to neural membranes. Binding to solubilized tissue was not stimulated by Mg2+, Cu2+, Mn2+, or Ca2+. In the presence of Ni2+, binding of AVP to solubilized tissue was reversible, and the dissociation constant (10.5 nM), pH optimum, and time course were virtually identical to those of the membrane-bound AVP binding site. Purification of solubilized AVP-binding proteins by affinity chromatography on AVP-sepharose followed by gel electrophoresis yielded a major band of 55 kdalton molecular weight when purified in the presence of 5 mM Mg2+, or a major band of 62 kdaltons when purified in the presence of 1-5 mM Ni2+ or 10 microM Zn2+. By means of a new binding assay involving conjugation of the 62 kdalton fraction to brain membranes, the extent of purification of AVP binding activity was 150-fold in the presence of Ni2+. We suggest that the 62 kdalton protein is a component of the Ni-stimulated AVP binding site.

Reduction of Specific Arginine-Vasopressin Binding Sites of Renal Medulla Membranes in Lithium Treated Rats

To elucidate the early effects of lithium treatment on the kidney, arginine-vasopressin (AVP) binding sites in the renal medulla were measured by radiolabeled receptor assay (RRA) in rats injected intraperitoneally with lithium chloride (4 mmol/kg body weight) for 7 days. After the 8th day, urine volume of rats injected lithium (Li group) increased and was significantly larger than that of the control rats that were injected physiological saline (control group). On the same day, the AVP binding capacities of renal medulla membranes of the Li group (Bmax = 28.9 +/- 7.2 fmol/100 micrograms protein) were significantly lower than that of the control group (Bmax = 50.0 +/- 6.3 fmol/100 micrograms protein), but there was no difference in the dissociation constant between the two groups. Our findings suggested that lithium-induced diabetes insipidus was due to a reduction in the number of AVP binding sites in the kidney.

Autoradiographic localization of peripheral benzodiazepine, dihydroalprenolol and arginine vasopressin binding sites in the pituitaries of control, stalk transected and Brattleboro rats.

The autoradiographic distribution of [3H]arginine vasopressin, [3H]spiperone, [3H]GABA, [3H]dihydroalprenolol and the peripheral-type benzodiazepine ligand [3H]Ro5-4864 were examined in the rat pituitary before and after pituitary stalk transection. Stalk transection produced dramatic changes in the cellular architecture of the pars nervosa. Glial fibrillary acidic protein, an astrocyte marker reported in pituicytes, increased after stalk transection, whereas neurofilament, a marker for neuronal innervation, was lost. These structural changes demonstrated a successful stalk transection, permitting interpretation of changes in the densities of several [3H]-ligands over the three lobes. [3H]Ro5-4864 binding was markedly increased, suggesting that this site was located on the pituicytes. Conversely [3H]spiperone and [3H]arginine vasopressin binding density over the pars nervosa decreased. In the mutant diabetes insipidus rat (Brattleboro), which lacks pituitary vasopressin, [3H]arginine vasopressin binding was undetectable in the pars nervosa. [3H]dihydroalprenolol and [3H]GABA binding sites were unchanged by the lesion. These results are discussed in terms of the occurrence of functional acceptors on pituicytes and their possible role in neurohydrophyseal secretions.

Two classes of arginine vasopressin binding sites on rat brain membranes.

Specific binding sites for arginine vasopressin (AVP) were demonstrated on rat brain membranes using [3H]AVP of high specific activity. At pH 7.4 in the presence of 5 mM MgCl2, one class of sites was measured with a KD of 0.56 nM and a Bmax of 4.3 fmol/mg protein. At pH 8.0 in the presence of MgCl2, two distinct sites were observed, having KD values of 0.42 and 13 nM and Bmax values of 5.6 and 68 fmol/mg protein, respectively, and similar results were obtained at pH 7.4 after repeatedly freezing and thawing the membranes. Binding increased with pH, apparently representing increased occupancy of the high capacity, lower affinity site. Binding to the lower affinity site was also enhanced by freezing and thawing membranes, or by adding 5 mM NiCl2 or 10 microM ZnCl2 to the incubation medium, whereas binding to the high affinity site was dependent on the addition of Mg. AVP was over 35 times more active in displacing 0.4 nM AVP than oxytocin or arginine-vasotocin, and 10,000 times more active than somatostatin. A number of other peptides had no effect on [3H]AVP binding at concentrations up to 10(-5) M. Autoradiography and regional dissection studies revealed a marked concentration of high affinity AVP-binding sites in the supraoptic and paraventricular nuclei of the hypothalamus, and Mg significantly enhanced the binding in these regions.

Topography of binding sites for neurohypophyseal hormones in rat brain

Binding sites for arginine-vasopressin (AVP) and oxytocin (OT) were detected in neuroanatomically defined regions of rat brain by using in vitro incubation of 32 μm brain sections with the tritium-labelled ligands and autoradiography with LKB Ultrofilm. Sites were quantified by computerized densitometry of the film images. The highest amount of [ 3H]AVP-labelled binding sites was observed in the ventral subiculum of the hippocampus, central amygdala nucleus, lateral septum, olfactory nucleus and nucleus tractus solitarii, while the dentate gyrus contained a large amount of low affinity binding. The binding pattern of [ 3H]OT was more discrete. Relatively high concentrations of OT binding sites were observed in the central amygdala, ventral subiculum and olfactory nucleus. The lateral septum and the nucleus tractus solitarii were virtually devoid of displaceable OT binding while no significant association of OT with the dentate gyrus was found. The topography of binding sites for neurohypophyseal hormones corresponds to the localization of responsive sites in behavioural, biochemical and electrophysiological studies.