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The activation of platelet V1-receptors by vasopressin (0.01-1 microM) induces the rapid formation of inositol phosphates, 1,2-diacylglycerol and phosphatidic acid, indicating inositol phospholipid hydrolysis by phospholipase C. Vasopressin immediately induces the formation of inositol bisphosphate and inositol trisphosphate. Accumulation of inositol 1-monophosphate and inositol 4-monophosphate occurs later after a time lag of 15 s. Low concentrations (10-100 nM) of vasopressin only activate phospholipase C, whereas high concentrations (1 microM) induce activation of phospholipase C and subsequently the production of arachidonate metabolites. Cyclo-oxygenase metabolites are associated with further activation of phospholipase C, release reaction and irreversible platelet aggregation. Vasopressin requires for its action extracellular Mg2+, but not Ca2+. The described platelet changes are not induced by 1-desamino-[8-D-arginine]vasopressin, a V2-receptor agonist, and are blocked by a specific V1-receptor antagonist. The results indicate that platelets possess a V1-receptor that is coupled to polyphosphoinositide hydrolysis by phospholipase C, leading to the formation of 1,2-diacylglycerol and inositol trisphosphate. Those compounds may act as second messengers for platelet responses induced by vasopressin, whereas endoperoxides and thromboxane A2 stimulated by vasopressin may serve as amplifiers for platelet activation.

Vasopressin may act in the brain as a neurotransmitter or neuromodulator to influence blood pressure, memory, body temperature and brain development. In order to localize probable central nervous system sites for these actions, we have used 125I-labelled 1-d(CH2)5, 7-sarcosine-8-arginine vasopressin, a specific V1-receptor antagonist, and in vitro autoradiography to map brain vasopressin binding sites. High levels of binding were found in the choroid plexus, blood vessels, lateral septum, bed nucleus of stria terminalis, accumbens nucleus, central nucleus of amygdala, stigmoid hypothalamic nucleus, suprachiasmatic nucleus, arcuate nucleus, nucleus of the solitary tract, area postrema and parts of the hippocampus, thalamus, superior colliculus, and inferior olivary nuclei. Many of these regions are known to be vasopressin-sensitive and to contain vasopressin fibres. Significantly there was no binding to the paraventricular nor the supraoptic nuclei. Displacement of the radioligand from the lateral septum with unlabelled vasopressin analogues gave a rank order of potencies: d(CH2)5-D-Tyr2(Et)Val4-desGly9-arginine-vasopressin approximately equal to d(CH2)5-Tyr2-(Me)arginine-vasopressin approximately equal to arginine-vasopressin approximately equal to d(CH2)5-Sar7-arginine-vasopressin greater than [1-deamino, 8-D-arginine]-vasopressin approximately equal to oxytocin much greater than vasopressin4-9, consistent with binding to V1 receptor subtype. These studies confirm and extend previous findings of V1 receptors in the rat brain. In particular, several new regions of vasopressin receptor binding have been identified, possibly due to the advantages of a radioiodinated ligand with high receptor affinity without binding to neurophysins. Future study of these regions may prove fruitful in elucidating the central actions of vasopressin.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.

Corticotropin-releasing factor (CFR) was bioassayed and arginine vasopressin (AVP) radioimmunoassayed from punched-out hypothalamic nuclei. The highest concentration of CFR was found in the median eminence (ME), followed by the paraventricular nucleus (PVN), supraoptic nucleus (SON), suprachiasmatic nucleus (SCN), arcuate nucleus (ARC), dorsomedial nucleus (DMN) and ventromedial nucleus (VMN). The AVP concentration was in the order of ME, SON, PVN, SCN, ARC, VMN and DMN. Sephadex G-25 gel filtration of the ME extracts showed one peak for AVP and two peaks for CRF. One CRF peak appeared on the void volume (big CRF) and the other (small CRF) was coeluted with AVP. Gel filtration of the PVN and SON extracts showed one peak for AVP but three or four peaks for CRF. The addition of anti-AVP serum (AVP-AS) to pituitary cell cultures reduced the CRF activities of AVP and ME extracts by approximately 80 and 40%, respectively. When the small CRF fraction of ME extracts was treated with AVP-AS on affinity chromatography, the unbound fraction (AVP-free) still showed significant CRF activity. Re-examination of CRF concentration using AVP-AS showed that it was still highest in ME, but was significantly higher in PVN than in SON, SCN and ARC. These results suggest that the PVN is an important nucleus for producing corticotropin-releasing hormone.

Publisher Summary This chapter focuses on the similarities and differences between vasopressin or oxytocin receptors in the brain and peripheral receptors for these peptides. The chapter also discusses the transduction mechanisms activated by vasopressin and oxytocin receptors in the brain and peripheral nervous system. Vasopressin and oxytocin meet most of the criteria that are applied to assign a role as a neurotransmitter or a neuromodulator to a given compound. It is recognized that vasopressin and oxytocin are synthesized at multiple sites in the brain in neurons projecting to several areas. A calcium-dependent release of oxytocin and (or) vasopressin can be evoked by potassium or veratridine in various extrahypothalamic areas of the brain. No direct effect of vasopressin on the adenylate cyclase activity of nervous tissue can be demonstrated. Vasopressin stimulates inositol lipid breakdown in hippocampal slices and isolated sympathetic ganglia.

The Brattleboro rat carries as a recessive trait the inability to synthesize hypothalamic vasopressin and its related neurophysin but is able to synthesize oxytocin and its neurophysin. Brattleboro rats homozygous for this trait have no immunologically detectable circulating vasopressin and manifest a complete syndrome of diabetes insipidus, which is corrected with vasopressin replacement therapy. Such a defect could be due to absence of the gene encoding vasopressin, the presence of an abnormal gene, or a variety of transcriptional or posttranscriptional abnormalities. We report here that the hypothalamus of the Brattleboro rat contains detectable, although markedly reduced, levels of an mRNA indistinguishable in size with and similar in sequence to authentic vasopressin mRNA. Corresponding levels of oxytocin mRNA were the same in Brattleboro and normal rat hypothalami. These findings indicate that the Brattleboro rat expresses a vasopressin gene, but at a reduced level.

We report the solid phase synthesis of a series of 16 linear analogues of the cyclic antagonist of the antidiuretic (V2) and the vasopressor (V1) responses to arginine vasopressin (AVP), d(CH2)5[D-Tyr(Et)2, Val4]AVP(A). Peptide 1, the linear precursor of (A), (CH2)5(SH)-CH2-CO-D-Tyr(Et)-Phe-Val-Asn-Cys-Pro-Arg-Gly-NH2 was modified at position six with alpha-L-aminobutyric acid (Abu) to give peptide 2. Further modifications of the Abu6 analogue (No. 2) at position one by substituting cyclohexylacetic acid (Caa), cyclohexylpropionic acid (Cpa), 1-adamantaneacetic acid (Aaa), phenylacetic acid (Phaa), tert.-butylacetic acid (t-Baa), isovaleric acid (Iva), propionic acid (Pa), L-penicillamine (P), tert.-butoxycarbonyl (Boc) or omitting any substituent at this position, and/or in combination with Arg-NH2(9), Ala-NH2(9), D-Arg8-Arg-NH2(9), and desGly9 modifications yielded the remaining 14 peptides. All 16 peptides were examined for agonistic and antagonistic potencies in AVP V2 and V1 assays in rats. Apart from the Cpa analogue and the analogue lacking any substituent in the 1-position, all exhibit substantial V2 and V1 antagonism. A number are as potent as (A) as V2 antagonists. With an anti-V2 pA2 = 8.11 +/- 0.07, Aaa-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (No. 6) is as potent as any cyclic AVP V2 antagonist reported to date. The PaI analogue of No. 6 exhibits promising anti-V2/anti-V1 selectivity. These findings prove conclusively that a ring structure is not a requirement for recognition of or for binding to AVP V2 or V1 receptors. This discovery thus offers a promising new approach to the design of peptide and non-peptide antagonists of AVP and perhaps also to other cyclic peptides such as somatostatin, atrial-natriuretic factor, insulin, and the recently discovered endothelin. Some of these linear antagonists may be of value as pharmacological tools and as therapeutic agents.

Vasopressin can alter hippocampal corticosterone receptor number, as a congenital absence of a peptide in the Brattleboro rat results in decreased concentrations of the receptor; this deficit can be reversed with des-glycinimide arginine vasopressin (dGVP), a centrally-acting VP analogue. We examined whether vasopressin might regulate less dramatic fluctuations of hippocampal corticosterone receptor number in the normal rat. Administration of dGVP failed to alter the rate of or extent of down- or up-regulation of hippocampal corticosterone receptors by circulating corticosterone, suggesting that alteration of neural VP content is not a mediating step in such regulation.

Publisher Summary The chapter reviews available pharmacological and biochemical data on vasopressin receptors in mammals and discusses the validity of the different criteria that can be used to distinguish vasopressin isoreceptors. A comparison of vasopressin receptors in mammals is presented with respect to (1) the kinetics of hormonal binding, (2) the nature of their primary effectors, (3) the nature of the putative modulators of receptor-effector coupling, (4) the known physiocochemical characteristics of solubilized receptors, and (5) respective recognition patterns. There are two types of vasopressin receptors, V1 and V2, distinguished on the basis of their functional coupling either to adenylate cyclase (V2 receptors) or to the cellular mechanisms involved in the regulation of calcium entry in the cell and (or) mobilization of cellular calcium (V1 receptors). Specific vasopressin binding sites are characterized in isolated cells or membrane fractions derived from three vasopressin-responsive tissues: kidney, liver, and blood vessels. Unlike vasopressin receptors triggering the tubular effects (increase in water and solute transport) of vasopressin on the kidney, the receptors present on hepatocytes and vascular smooth muscle cells are not functionally coupled to adenylate cyclase. The primary involvement of a rise in cytosolic cell calcium in the glycogenolytic response of hepatocytes and in the contractile response of vascular smooth muscle cells to vasopressin is established. Several other biological effects of vasopressin such as a platelet aggregation and corticotropin release by the adenohypophysis are also elicited by calcium-dependent, CAMP-independent mechanisms.

The location, cytology and projections of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig were investigated using specific antisera against vasopressin, oxytocin or neurophysin in the unlabeled antibody enzyme immunoperoxidase method. Light microscopic examination of the neurons of the supraoptic and paraventricular nuclei shows that hormone is transported not only in axons, but also in processes having the characteristics of dendrites. Neurons were found to contain only vasopressin or oxytocin; all neurons containing neurophysin appear to contain either vasopressin or oxytocin. In the neural lobe, vasopressin and oxytocin terminals are intermingled. In the median eminence, vasopressin and oxytocin fibers are intermingled in the internal zone. In a caudal portion of the median eminence, a number of vasopressin and neurophysin (but few oxytocin) axons enter the external zone from the internal zone, and surround portal capillaries. In the supraoptic nucleus, vasopressin neurons outnumber oxytocin neurons with a ratio of at least 5:1. The paraventricular nucleus is separated into two distinct groups of neurons, a lateral group consisting of only vasopressin neurons, and a medial group consisting of only oxytocin neurons. In addition to axons passing to the neurohypophysis, a number of axons appear to interconnect the supraoptic and paraventricular nuclei.

Publisher Summary This chapter describes the application of immunohistochemical techniques, which has revealed a vasopressin pathway to the zona externa that arises from the paraventricular nucleus and is separate from the one to the posterior pituitary. The chapter also discusses a similar corticotropin-releasing factor (CRF) pathway, which is apparently different from that containing vasopressin. It has been established, by immunohistochemical techniques, that there is a vasopressin neurosecretory system that originates in the paraventricular nucleus and terminates on the hypophysial portal system. It appears to be a separate pathway from the magnocellular projection to the posterior pituitary gland. Release of vasopressin into the portal blood in concentrations high enough to participate in ACTH release may arise from the secretory terminals. Glucocorticoid deficiency has been found to be associated with the increase in RNA turnover in paraventricular nucleus vasopressin neurons, in vasopressin biosynthesis, and in the content of vasopressin in the cell bodies. The marked increase in the numbers of vasopressin terminals in the zona externa of the median eminence between days 5 and 21 following adrenalectomy is not due to the sprouting of pre-existing terminals. However, sprouting occurs in response to a unilateral lesion of the paraventricular nucleus in adrenalectornized animals. Within three weeks, vasopressin fibres from the intact side reinnervate the denervated side of the zona externa. These results suggest a complex interaction between this vasopressin neurosecretory system and adrenal glucocorticoid levels.

We have previously shown that arginine vasopressin (AVP) directly inhibits testicular steroidogenesis in vitro. In the present study, binding of neurohypophysial peptides to interstitial cells of the rat testis was studied using [3H]AVP as the ligand. Interstitial cells were obtained from adult rat testis after collagenase dispersion and were incubated with [3H]AVP in the presence or absence of unlabeled AVP. Binding equilibrium was reached by 60 min at 4 C, while incubation at higher temperatures (23 and 37 C) resulted in an apparent decrease in binding. Scatchard plot analysis of equilibrium binding data revealed the existence of one class of high affinity, low capacity binding sites (Kd = 1.0 +/- 0.3 nM; maximal binding = 8.5 fmol/10(6) cells). In addition, the rate constants of association and dissociation were calculated to be 0.024 nM-1 min-1 and 0.009 min-1, respectively. Addition of naturally occurring neurohypophysial hormones as well as their synthetic analogs inhibited [3H]AVP binding to testis cells, resulting in parallel displacement curves. The order of potencies for the native peptides was: AVP = lysine vasopressin = arginine vasotocin (IC50, 5 X 10(-10) M) greater than oxytocin = mesotocin (IC50, 4 X 10(-7) M) greater than isotocin = glumitocin (IC50 greater than 10(-6) M). Furthermore, two potent vasopressor antagonists, d(CH2)5Tyr(Me)AVP ([1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]AVP) and dPTyr(Me)AVP ([1-deaminopenicillamine-2-(O-methyl)tyrosine]AVP) competed for [3H]AVP binding with a higher affinity (IC50, approximately 10(-11) M) than native AVP. In contrast, a selective antidiuretic agonist, dDAVP (1-deamino-8-D-AVP), only competed weakly for receptor binding, while a specific oxytocic agonist, (Thr4,Gly7)oxytocin, did not affect AVP binding. These results suggested that the testis may contain the V1 receptor subtype. Studies on the intratesticular distribution of AVP receptors indicated minimal binding to cells derived from the seminiferous tubule, while most of the AVP-binding sites sediment with enriched fractions of Leydig cells after Metrizamide density gradient centrifugation. AVP-binding sites were also found in rat liver, kidney, and anterior pituitary (10.7, 2.6, and 1.7 fmol/mg protein), whereas adrenal, cerebellum, prostate, and hypothalamus were devoid of AVP-binding sites. Thus, we have demonstrated the presence of high affinity, stereospecific receptors for AVP in the interstitial cell compartment of the rat testis. These V1 receptors may mediate the direct inhibitory action of neurohypophysial hormones on testicular Leydig cell steroidogenesis.

Vasopressin secretion is stimulated by hyperosmolality, hypovolemia, or hypotension and is inhibited by hypoosmolality, hypervolemia, or hypertension. These osmotic and hemodynamic influences are mediated by neuronal afferents that originate in separate osmoreceptors or baroreceptors but ultimately converge to act on the same neurosecretory neurons. Functionally, the two control systems are integrated in such a way that osmoregulation is altered but not disrupted by hemodynamic influences. In patients with uncomplicated essential hypertension, basal as well as osmotically stimulated vasopressin is completely normal. The vasopressin response to an acute reduction in blood pressure is also normal if the values are expressed relative to the change in pressure. However, if the plasma vasopressin response is plotted as a function of absolute blood pressure, the line describing the relationship lies well to the right of normal. Thus, although it is completely intact, the baroregulatory mechanism appears to be reset to a higher level in essential hypertension. These results suggest that increased secretion of vasopressin does not contribute to the genesis or maintenance of uncomplicated, untreated essential hypertension but may antagonize the therapeutic effect of some antihypertensive drugs. If so, antagonists of V1 receptors may be useful as second-line adjunctive therapy for this condition.

In the dorsal and ventral portions of the bed nucleus of the stria terminalis of the rat numerous cell bodies immunoreactive for vasopressin and neurophysin II were found after colchicin pretreatment. These cells are predominantly multipolar but sometimes also bipolar, and have a width and length of approximately 9 and 16 microns, respectively. In the homozygous Brattleboro rat, which is deficient in vasopressin, no immunoreactive vasopressin was found in these cells. Following incubation with anti-oxytocin and anti-bovine neurophysin I, only magnocellular immunoreactive cell bodies were found in the septal region. The consequences of these results concerning the vasopressin fiber pathways in the brain are discussed.

It is generally accepted that hypothalamic factors are released from the median eminence into the hypophysial portal vessels and exert a powerful control on the secretion of anterior pituitary hormones1. The brain sites involved in the synthesis of hypothalamic factors—particularly those that control ACTH secretion2,3—are, however, not yet established. It is also not known whether these sites are targets for pituitary hormone action, although recent anatomical4 and physiological studies5,6 suggest that pituitary hormones may reach the brain. For these reasons, we co-cultured7,8 various hypothalamic explants from rat brain for 4 weeks with anterior pituitary. We report that during the third and fourth week in vitro, there is an approximately 10-fold increase in bioactive ACTH9,10 in the medium of co-cultures prepared from the paraventricular nucleus and anterior pituitary, and that this increase is highly correlated with an increase (up to 10-fold) in concentrations of immuno-reactive11,12 arginine vasopressin (AVP). Hormone levels did not increase when basal hypothalamus was co-cultured with anterior pituitary, paraventricular nucleus with posterior pituitary, or supraoptic nucleus with anterior pituitary. Thus, we propose a specific interaction between anterior pituitary and paraventricular nucleus that results in a mutual enhancement ofeach other's peptide production.

Arginine-vasopressin (AVP) microinjected into an area extending from the diagonal band of Broca to the anterior hypothalamus of the rat evokes severe motor disturbances, including barrel rotations and myoclinic/myotonic movements. These disturbances do not occur after administration of an artificial physiological solution or of oxytocin. Injection of this peptide into other areas of the brain does not cause these effects. This action of vasopressin is dose-related, can be prevented by the prior administration of an AVP receptor antagonist and involves a ‘sensitization’ process. It is possible that AVP, acting in this mediobasal region of the forebrain, might be involved as a causative agent in some convulsive disorders.

The existence of vasopressin-sensitive neurons in the nucleus of the solitary tract of the rat and the presence in this brain area of vasopressin binding sites were investigated using extracellular single- unit recordings from brain-stem slices and light microscopic autoradiography. About 45% of the recorded neurons responded to vasopressin at 5–2000 nM by a reversible, concentration-dependent increase in firing rate. The action of vasopressin was direct, was suppressed by a vasopressor antagonist, and was mimicked by a vasopressor agonist. Oxytocin was 10–100 times less efficient than vasopressin and a specific antidiuretic agonist was without effect. Using light microscopic autoradiography and 3H-arginine vasopressin as a ligand, high-affinity vasopressin binding sites were found distributed over the whole rostrocaudal extent of the nucleus of the solitary tract. Binding was displaced by unlabeled vasopressor agonist but not by unlabeled antidiuretic agonist. Thus, the nucleus of the solitary tract contains V1-type vasopressin receptors which are, at least in part, located on neuronal membranes and whose activation generates bioelectrical signals. Solitary tract vasopressin-sensitive neurons may be the target of a vasopressinergic innervation originating in the hypothalamic paraventricular nucleus and could be involved in the central regulation of cardiovascular functions.

The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only 1 20 of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.

Arginine vasopressin (AVP) is a potent vasopressor and antidiuretic neurohormone. However, when administered intravenously to humans, AVP causes forearm vasodilation. This effect has been attributed to sympathetic withdrawal, secondary to AVP-induced sensitization of baroreceptors. The possibility that AVP also causes forearm vasodilation directly has not been examined. Accordingly, the direct effect of AVP was determined by studying the forearm blood flow (FBF) response to intraarterial (IA) AVP infusion (0.01-1.0 ng/kg per min). Infusion of IA AVP increased FBF (96%) in the infused arm, but not the control arm, in a dose-dependent manner. The role of specific AVP V1 receptors in mediating this FBF response was determined before and after pretreatment with a V1 antagonist (AVP-A). AVP-A alone had no effect on FBF, but coadministration of AVP and AVP-A potentiated the vasodilatory response (223%). IA infusion of the V2 agonist, 1-desamino[8-D-arginine] vasopressin, caused a dose-dependent increase in FBF. These findings suggest that AVP causes direct, dose-dependent vasodilation in the human forearm that may be mediated by V2 vasopressinergic receptors. In contrast, AVP infusion caused digital vasoconstriction that was blocked by AVP-A, whereas dDAVP did not affect digital blood flow. Thus, AVP induces regionally selective vascular effects, with concurrent forearm vasodilation and digital vasoconstriction.

The objective of this study was to determine the effects of i.v. nicotine on plasma arginine vasopressin (AVP), plasma osmolality, and behavior in the conscious monkey. Adult, female, chronically prepared monkeys (Macaca mulatta) were studied with i.v. infusion of 5% dextrose and water in control experiments without change in parameters. Nicotine infusion (100 mug/kg/min) in 14 experiments produced a significant increase in plasma AVP from control levels of 0.6 +/- 0.5 muU/ml to end-of-infusion levels of 35 +/- 17 muU/ml (p less than 0.001). During the 15-20 min of nicotine infusion, a behavioral sequence of restlessness, yawning, retching, salivation and chewing accompanied AVP release. Plasma osmolality remained unchanged. Pretreatment of the monkeys with promethazine (Phenergan) and diphenhydramine (Benadryl) at 1-5 mg/kg reduced both the plasma AVP increase and the behavioral effects. These results provide conclusive evidence that nicotine can release large amounts of AVP in the monkey.

Vasopressin and oxytocin exert pronounced effects on behaviour by a direct action on the brain. A single injection of vasopressin results in a long-term inhibition of extinction of a conditioned avoidance response suggesting that vasopressin triggers a long-term effect on the maintenance of a learned response, probably by facilitation of memory processes. In addition vasopressin improves passive avoidance behaviour, delays extinction of appetitive discrimination tasks, affects approach behaviour to an imprinting stimulus in ducklings, improves copulation rewarded behaviour of male rats in a T-maze, prevents or reverses amnesia induced by electroconvulsive shoch. CO2 inhalation, pentylenetetrazol or puromycin. The majority of these effects of vasopressin in the various and sometimes relatively complex tasks may be explained by stimulatory influences of this neuropeptide on memory processes. Generally oxytocin exerts effects which are opposite to those of vasopressin and it has been suggested that oxytocin may be an amnesic neuropeptide. Various limbic system structures seem to act as the anatomical substrate for the bevavioural effects of vasopressin. In particular the amygdala, the dentate gyrus of the hippocampal complex, the ventral hippocampus and the dorsal septum seem to be involved. Evidence has been obtained from experiments with homozygous diabetes insipidus rats and from experiments in which antisera were applied that endogenous vasopressin and oxytocin play a physiological role in brain processes related to memory. It appears that highly active fragments can be generated from vasopressin and experiments in which a fragment of vasopressin ([pGlu4, Cyt6]AVP-(4–8)) as well as an AVP-antagonist were used, reveal that the vasopressin receptors mediating the behavioural effects are situated in the brain and differ in specificity from the peripheral (blood pressure) vasopressin receptors. Generally the clinical data obtained so far with vasopressin treatment are in agreement with the results from animal experiments and they support the notion on the involvement of vasopressin in memory function. The sometimes reported conflicting results on vasopressin effects in certain patients (Korsakoff or Alzheimer) may have to do with the wide-spread pathology in these diseases.

The data highlighted here suggest that the CRH effects of vasopressin and oxytocin are mediated by one and the same hypophysial receptor which has unique pharmacological specificity. The nomenclature for this receptor type is not established; both V3(53) and V1 beta 39 have been proposed. The former proposal is logical if the pharmacology of ligand recognition is emphasized, whereas the latter designation takes into account that transmembrane signalling from V1 receptors occurs via coupling proteins Go and Gi but not Gs. Such issues are best resolved after cDNA cloning of the genes for the receptors: in the meanwhile the working definition V3 seems more convenient. Several studies show that pituitary V3 receptors are regulated by the concentration of vasopressin in hypophysial portal blood and the amount of glucocorticoid hormones in the circulation (see Ref. 9 for review). Work in this area should clarify further the intracellular mechanism of the CRH action of vasopressin, as well as the factors that determine the responsiveness of corticotrophs to various secretagogues. Most recently, it has been shown that vasopressin is a potent thyrotropin-releasing hormone. This finding extends further the growing concept that there is considerable "cross-talk" between the classical neuroendocrine axes. These were previously thought to be separated by the hypothalamic organization of "final common pathways" of neuroendocrine motoneurons in the hypothalamus, each producing a unique neurohormone to regulate a single type of adenohypophysial cell. It seems that the days of the validity of this hypothesis are numbered, and an important task will be to determine the possible physiological significance of the "cross-talk" within the hypothalamo-pituitary unit in the regulation adrenocortical function.

We investigated in conscious rats, whether vasopressin, whose release was stimulated by isoprenaline (i.m.), caused the simultaneous increase in plasma beta-endorphin-like immunoreactivity (beta-EI). The increase in plasma beta-EI following isoprenaline administration was diminished by about 50% in rats pretreated with a vasopressin antagonist and also in rats with a hereditary absolute lack of vasopressin. We conclude that the isoprenaline-induced release of beta-EI is mediated in part by vasopressin.

Extracellular recordings were obtained in anaesthetized rats from single neurons located in various structures around the rostral end of the third ventricle, known to harbour integrative neurons sensing deficiencies in and originating corrective responses for water-electrolyte balance. Once arginine vasopressin (AVP) responsive neurons were located, a selective antidiuretic agonist (binding to V2 receptors) and either V1 (presser response related) or V2 (antidiuretic) antagonists were iontophoretically applied. Neurons in this region did not respond to the V2 agonist and only the VI antagonist was able to block the response to AVP. It is assessed that the investigated region has neurons equipped only with receptors of the VI type. Interestingly, a number of these neurons also responded to angiotensin II (All), oxytocin and to blood pressure changes. The integrative neuronal population of parasagittal rostrodiencephalic neurons seem therefore to sense indices of haemodynamic changes including their neuro-hormonal signals within the brain such as All and AVP which bind to V1 (pressor response related) receptors.

Perfusion of rat brain followed by immersion fixation with 2.5% glutaraldehyde-1% paraformaldehyde, purification of the first antisera and application of the unlabelled antibody enzyme method were used to specifically identify vasopressin and oxytocin containing cells and fibres. The conventional sites of production of these hormones were confirmed as follows: supraoptic and paraventricular nuclei, suprachiasmatic nucleus (only vasopressin), and other cells and cell groups of the hypothalamus. Fibres from the suprachiasmatic nucleus spread out in various direction, and probably project to the nucleus praeopticus periventricularis, organum vasculosum laminae terminalis and in the direction of the supraoptic nucleus. Oxytocin and vasopressin containing pathways could be traced from the paraventricular nucleus to the lateral ventricle, the stria terminalis and the stria medullaris. Some of the oxytocin and vasopressin containing tracts appear to continue onto ther septum. The possible importance of these morphological findings for the behavioural effects of vasopressin and oxytocin is discussed.

The anatomical distribution and pharmacological characteristics of the different types of neurohypophysial hormone binding sites were compared in the forebrains and pituitary glands of Long-Evans rats and its mutant Brattleboro strain, genetically deficient in vasopressin. Quantitative autoradiography on sections incubated in the presence of 5 nM of either [ 3 H]oxytocin or [ 3 H]vasopressin revealed the presence of the same types of sites in the brains of both strains but noticeable variations in their densities were found in several areas. In the forebrain, oxytocin/vasopressin sites, which bind both peptides with similar high nanomolar affinities, had the same locations and densities in the ventral subiculum, in several nuclei of the amygdala, the bed nucleus of the stria terminalis and the olfactory tubercle. The density of such sites was, in contrast, lower in the ventromedial hypothalamic nucleus of the Brattleboro rat. Selective vasopressin sites which bind [ 3 H]vasopressin with a nanomolar-range affinity and [ 3 H]oxytocin with a much lower affinity showed more variations. They were not found in the Brattleboro rat thalamus but were highly concentrated in several thalamic nuclei in the Long-Evans rat. Conversely, their densities were higher in the dopaminergic A13 cell group of the zona incerta and the suprachiasmatic nucleus of the Brattleboro rat. Their densities were similar in the lateral septal nucleus and in the fundus striati of both strains. In the hypothalamo-neurohypophysial system, [ 3 H]oxytocin and [ 3 H]vasopressin binding occurred in the Long-Evans rat with characteristics different from those found in other brain areas. In the Brattleboro rat, no [ 3 H]vasopressin binding and only low [ 3 H]oxytocin binding, restricted to the magnocellular nuclei, were found. While these results clearly demonstrate differences in the densities and distribution of neurohypophysial hormone binding sites between the Brattleboro mutant and its strain of origin, whether or not these variations are directly related to the deficience of vasopressin in the Brattleboro rat brain remains to be demonstrated.

The corticotropin-releasing activity of synthetic corticoliberin increases 75–90% when it is incubated in the presence of arginine vasopressin with rat anterior pituitary cell suspensions; this suggests a synergistic control of ACTH secretion by vasopressin and corticoliberin.

In rat L5 dorsal root ganglia 50% of neurons contained arginine vasopressin-like immunoreactivity and 38% oxytocin-like immunoreactivity, the oxytocin entirely coexisting with the arginine vasopressin. Staining of alternate mirror-image sections with RT97 (an antibody to neurofilament protein, and a marker for large light neurons) and with arginine vasopressin antiserum showed that the two were entirely complementary, thus establishing arginine vasopressin as a marker for all small dark neurons. Mirror-image staining also showed that neurons containing substance P-like immunoreactivity and those containing fluoride-resistant acid phosphatase activity were each contained within the arginine vasopressin-positive population. Arginine vasopressin-like immunoreactivity was axonally transported in the dorsal root and (in greater quantity) in sciatic nerve. Arginine vasopressin-like immunoreactivity was present also in laminae I and II of the dorsal horn of the spinal cord and this reactivity was absent in animals which had been treated neonatally with capsaicin, suggesting that it was contained in primary afferent terminals. These results are discussed in terms of their implications for the classification of primary afferent neurons and of a possible physiological role for arginine vasopressin in these neurons.

Median eminence corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) and pituitary and peripheral plasma adrenocorticotropin (ACTH) and AVP were measured in male Wistar rats 1 and 2 weeks after bilateral adrenalectomy (ADX), sham operation (SHAM) or dexamethasone-treatment (DEX). Median eminence AVP content was unchanged 1 week after ADX but was significantly elevated 2 weeks after ADX, whereas CRF activity was reduced at 1 week after ADX and returned to control range at 2 weeks. Anterior pituitary ACTH content was elevated but posterior pituitary AVP content was reduced at 1 and 2 weeks after ADX. Plasma ACTH was greatly elevated in ADX rats and reduced in DEX rats, whereas plasma AVP did not differ significantly between these two groups or the control group. When ADX and SHAM rats were laparotomized under ether, plasma ACTH increased greatly, but this elevation was prevented by DEX treatment. The plasma AVP level was elevated in all three groups 2.5 min after onset of stress but returned to the basal range at 20 min. Median eminence CRF and AVP and pituitary ACTH and AVP were not significantly changed after onset of stress. These results indicate that the vasopressin and CRF-ACTH responses were not consistent in the median eminence, pituitary and peripheral plasma and suggest that vasopression is not involved in the feedback and acute stress mechanism of CRF-ACTH secretion. However, we have to measure CRF activity and AVP concentration in the hypophysial portal blood to confirm this conclusion.

Vasopressin was virtually absent from 5 microdissected hypothalamic areas and from the posterior pituitary glands of homozygous Brattleboro rats. The oxytocin concentration was normal in all these areas except for the arcuate nucleus, where it was absent. These results support the concept that Brattleboro rats have a specific defect in biosynthesis of vasopressin. The significance of the absence of oxytocin from the arcuate nucleus in these rats remains to be determined.

Anesthetized rats of different strains show a hypotensive response to administration of an antagonist of the V1 receptors for vasopressin [d(CH2)5DAVP]. Such an effect is not seen in conscious, water-replete animals or in Long-Evans rats challenged with a subcutaneous injection of polyethylene glycol (PEG) to cause isosmotic hypovolemia. However, Long-Evans rats experiencing a similar volume reduction due to water deprivation show hyperosmolality and exhibit a small hypotensive response to d(CH2)5DAVP. Inhibition of the renin-angiotensin system following administration of d(CH2)5DAVP causes a greater hypotension in PEG-treated than in water-deprived Long-Evans rats. In both experimental conditions, the fall in blood pressure is greater than when captopril administration precedes that of d(CH2)5DAVP, indicating that prolonged administration of d(CH2)5DAVP may be interfering with mechanisms other than those mediated by peripheral V1 receptors. However, administration of d(CH2)5DAVP and captopril to water-deprived Long-Evans rats rarely causes the profound hypotension seen in water-deprived Brattleboro rats given captopril alone. In some adrenalectomized Wistar rats, following withdrawal of salt supplementation, the hypotensive response to d(CH2)5DAVP is the greatest seen in any experimental model. These results indicate that AVP is overtly involved in the support of blood pressure in various hypotensive states and, more importantly, may be responsible for the maintenance of a "normal" blood pressure in some conditions. However, the involvement of AVP in cardiovascular regulation in the majority of normotensive conditions is intriguingly subtle.

Vasopressin and oxytocin were demonstrated by means of the unlabeled antibody enzyme method in the rat central nervous system. Vasopressin and oxytocin fibers of the magnocellular nuclei were found to run towards the amygdala, hippocampus, and spinal cord. From the parvocellular suprachiasmatic nucleus only vasopressin containing fibers were found to run towards the organum vasculosum lamina terminalis, lateral septum, and lateral habenular nucleus. Immunoelectron microscopy, using the preembedding staining technique, frequently demonstrated vasopressin-containing synapses on neuronal structures in the lateral septum, lateral habenular nucleus, and amygdala. These results suggest a putative neurotransmitter role for these neuropeptides.

Publisher Summary This chapter discusses the rationale for a therapeutic interest in vasopressin antagonists and describes the current status of animal models for the discovery of antagonists. The chapter describes the structural features present in antagonists. The identification of receptor-selective antagonists has furthered the characterization of the physiological and pathophysiological roles of vasopressin. Although vasopressin has been implicated in many biological processes, its principal physiological action is the regulation of body fluid tonicity and volume. The anti-diuretic or V2 receptor is located on the basolateral membranes of renal collecting duct epithelia and is coupled to adenylate cyclase via stimulatory guanine nucleotide regulatory proteins. A second receptor subtype, the vasopressor or V1 receptor, is located in the vasculature and liver and was recently identified in the adenohypophysis, the brain hippocampus, and seminal vesicles of mammals. V1 receptors mediate the activation of phosphatidylinositol bisphosphate hydrolysis that, via the formation of inositol 1,4,5-trisphosphate, causes an increase in free cytosolic calcium. If the clinically observed agonism is the result of intrinsic agonist activity, the problem would be simplified if was possible to use the extensive structure–activity relationships (SAR) for V2 receptor agonists to predict the residual intrinsic activity in antagonists. However, a number of reports have appeared that indicate that agonists and antagonists at the V2 receptor have substantively different pharmacophores. The therapeutic utility of V1 and V1/OT receptor antagonists has been evaluated in clinical studies. The therapeutic utility of V2 receptor antagonists has not been tested because the only V2 antagonist to be evaluated in man to date exhibited agonist activity.

Neurohypophysial peptides were applied by superfusion to rat hippocampal slices. The peptides, arginine vasopressin, lysine vasopressin, arginine vasotocin and oxytocin, increased the activity of 88% of spontaneously active cells in the CA1 region and induced firing in many neurones that were not spontaneously active. The peptide sensitive cells appeared to be pyramidal cells rather than interneurones. The four peptides were found to be of roughly equivalent potency, producing a reversible, dose-dependent response in the range 10 −9 to 10 −6 M. Most of the cells were tested with more than one peptide and were always found to respond either to all or to none of them. The analogue [7-glycine]oxytocin and the deamino, dicarba derivatives of oxytocin and vasopressin were about as active as the parent compounds, but the oxytocin fragment prolyl-leucyl-glycinamide had no effect, and desglycinamide vasopressin was extremely weak. Responses to the peptides could be blocked by “specific” antagonists. The results suggest that all of the peptides are acting upon a single class of receptor.

Arginine vasopressin is reported to have an excitatory effect on hippocampal neurons in the slice preparation. However, vasopressin also has a classic vasopressor action on mammalian blood vessels. We used the rat hippocampal slice to examine the effects of this peptide on central neural and vascular targets. The hippocampus is densely vascularized and pyramidal cells are enmeshed in a network of microvessels. Vasopressin increased the excitability of impaled neurons without substantially altering membrane potential or resistance. The peptide also caused pronounced vasoconstriction in penetrating microvessels when applied at micromolar concentrations. The concerted action of vasopressin on neurons and blood vessels and the physical proximity of these cell types suggest mechanisms whereby these responses may be associated.

Hepatic plasma membranes of female obese mice C57 BL-6 orl ob/ob (ob/ob mice) completely lack vasopressin (VP) receptors of the V1 type whereas kidney VP receptors are normally expressed and functionally coupled to adenylate cyclase. To discover if these alterations are linked to a genetic defect of the V1 receptor, we have studied the binding of VP on liver and kidney membranes of two other models, female diabetic mice C57 BL-6 orl db/db (db/db mice) and female Zucker rats Fatty/orl fa/fa (fa/fa rats), which exhibit different temporal pattern of obesity, hyperinsulinemia and insulin resistance. In addition, since VP is known to exert its vascular response through stimulation of V1 receptors, we have studied the reactivity of VP of isolated tail artery in the three different models, ob/ob and db/db mice and fa/fa rats, and in their respective controls. In all cases, VP kidney receptors and VP vascular reactivity are normal. db/db mice exhibit a marked decrease in hepatic VP receptors whereas a 50% decrease was observed in 32 week fa/fa rats. Angiotensin II and prazosin binding sites are still present as well as the adenylate cyclase response to glucagon. These results suggest that the specific alteration in liver VP receptors is not related to a defect in V1 receptor genetic expression but is specific for liver and appears to parallel the level of hyperinsulinemia and/or insulin resistance.

Previous studies using the isolated rat hypothalamus in vitro have shown that both acetylcholine and 5-hydroxytryptamine (5-HT) stimulate the secretion of bioactive corticotrophin-releasing factor (CRF). However, the CRF complex consists of a number of bioactive substances, and in this study we examined the effect of acetylcholine and 5-HT on the release of immunoreactive (ir)-CRF-41 and ir-arginine vasopressin (AVP) in vitro. Acetylcholine caused a dose-dependent (10 pmol–10 nmol/l) release of both neuropeptides, and the effect was partially antagonized by both atropine and hexamethonium. Nicotine (0·1–10 μmol/l) also stimulated the release of both peptides, whereas bethanacol had no effect on AVP release, but had a variable action on CRF-41 release. 5-HT caused a dose-dependent (10 pmol–1 nmol/l) stimulation of CRF-41 release without any effect on AVP release, and this effect was antagonized by cyproheptadine, suggesting the participation of specific 5-HT receptors. Journal of Endocrinology (1989) 122, 713–718

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are perisinusoidal cells believed to participate in the regulation of hepatic blood flow because of their contractile properties and presence of receptors for several vasoactive factors. It is unknown whether HSCs have receptors for vasopressin, one of the most potent endogenous vasoconstrictors. This study investigated the existence of receptors for and the effects of arginine vasopressin (AVP) on cultured human HSCs. METHODS: intracellular calcium concentration ([Ca2+]i) and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a CCD imaging system (Photometrics, Tucson, AZ). AVP-specific binding was measured with [3H]AVP. Mitogen-activated protein kinase (MAPk) activity and DNA synthesis were measured by in vitro phosphorylation of myelin basic protein and [3H]thymidine incorporation, respectively. Parallel experiments were performed in vascular smooth muscle cells. RESULTS: AVP elicited a dose-dependent increase in [Ca2+]i and contraction of HSCs. Moreover, AVP increased MAPk activity, DNA synthesis, and cell number. These effects were similar to those observed in vascular smooth muscle cells and were blocked by a V1 receptor antagonist. The existence of V1 receptors was further confirmed by binding studies. CONCLUSIONS: Human HSCs have V1-vasopressin receptors that induce effects similar to those observed in vascular smooth muscle cells. AVP may play a role in the regulation of HSC function. (Gastroenterology 1997 Aug;113(2):615-24)

The push-pull perfusion technique, in conjunction with specific radioimmunoassays, was used to monitor the release of both arginine vasopressin (AVP) and oxytocin (OXT) within distinct limbic brain areas of conscious female rats. In pregnant rats near term, the release of AVP was greater than that of virgin rats in both the ventral (p less than 0.001) and mediolateral (p less than 0.001) septal areas; similarly, release of OXT increased in the ventral septal area (p less than 0.01) at this time. In contrast, no changes in the levels of either peptide occurred in the dorsal hippocampus. In parturient rats, AVP release tended to decrease in the septal areas but increased fivefold in the dorsal hippocampus (p less than 0.001) compared to pregnant animals. In contrast, OXT levels assayed in the same perfusates did not differ from those observed in pregnant animals. Plasma levels of AVP in pregnant rats (p less than 0.05) and of OXT in parturient animals (p less than 0.01) were found to be increased over levels in virgin rats. The regionally different and peptide-specific changes in release pattern of AVP and OXT in virgin, pregnant and parturient rats may be of physiological significance in antipyresis and behaviors accompanying parturition.

Plasma vasopressin sensitizes the baroreceptor reflex, whereas vasopressin given into the cerebral ventricle overrides the baroreceptor reflex by means of sympathetic stimulation. To test the hypothesis that arginine vasopressin stimulates two different receptor subtypes (V1 and V2) in the central nervous system, we measured the baroreceptor reflex (change in pulse interval vs change in blood pressure) after administering methoxamine (10-300 micrograms/kg i.v.) in conscious rats. Animals were pretreated either with a V1 vasopressin receptor antagonist administered intravenously or intracerebroventricularly, or with a V2 receptor antagonist administered intravenously. The central V1 antagonist caused sensitization of the baroreceptor reflex, whereas the intravenous V2 antagonist attenuated it. The intravenous V1 vasopressin antagonist had no effect on baroreceptor reflex sensitivity. When the experiments were repeated in rats with hereditary diabetes insipidus, neither antagonist influenced the baroreceptor reflex. Volume expansion lowered circulating vasopressin levels and also attenuated the baroreceptor reflex--effects similar to those observed with the intravenous V2 antagonist. We conclude that vasopressin sensitizes the baroreceptor reflex through V2 receptors accessible from the blood and inhibits the reflex through V1 receptors in the brain that cannot be reached from the blood. These observations suggest a direct interaction between hormonal and neuronal vasopressin in cardiovascular control.

Enucleation-induced adrenal regeneration is a classic model to investigate adrenocortical proliferation in vivo, which is dependent not only on pituitary ACTH release, but also on various other neural and endocrine signals. Arginin-vasopressin (AVP), mainly acting via V1 receptors, regulates hypothalamic-hypophyseal-adrenal axis function, acting on both its central and peripheral branches. Here, we studied whether endogenous AVP system modulates rat adrenal regeneration. Reverse transcription-polymerase chain reaction (PCR) detected only the mRNAs of V1a and V1b receptors in normal and regenerating adrenals. The expression was very low, and semi-quantitative conventional and real-time PCR showed that it was down-regulated in regenerating adrenals in relation to the time elapsed from enucleation. AVP (three subcutaneous injections 28, 16 and 4 h before sacrifice) raised metaphase index at day 5, but not at day 8 of regeneration. Unexpectedly, both V1-receptor and V2-receptor antagonists increased metaphase index at days 5 and 8 of regeneration. Neither AVP nor AVP-receptor antagonists affected plasma levels of corticosterone in rats bearing regenerating adrenals. It is concluded that AVP, acting via V1 receptors located in adrenals, exerts a stimulating effects on adrenal regeneration. Due to the down-regulation of V1-receptor expression in regenerating adrenals, this effect is very weak and is easily overcome by a tonic inhibitory action of endogenous AVP systems probably involving extra-adrenal indirect mechanisms.

Angiotensin II (ANG II) and vasopressin (AVP) act together with the mechanical effect of aortic constriction in the onset of acute aortic coarctation hypertension. Blockade of ANG II and AVP V1 receptors demonstrated that ANG II acts on the prompt (5 min) rise in pressure whereas AVP is responsible for the maintenance (30-45 min) of the arterial pressure elevation during aortic coarctation. Hormone assays carried out on blood collected from conscious rats submitted to aortic constriction supported a role for ANG II in the early stage and a combined role for both ANG II and AVP in the maintenance of proximal hypertension. As expected, a role for catecholamines was ruled out in this model of hypertension, presumably due to the inhibitory effect of the sinoaortic baroreceptors. The lack of afferent feedback from the kidneys for AVP release from the central nervous system in rats with previous renal denervation allowed ANG II to play the major role in the onset of the hypertensive response. Median eminence-lesioned rats exhibited a prompt increase in proximal pressure followed by a progressive decline to lower hypertensive levels, revealing a significant role for the integrity of the neuroaxis in the maintenance of the aortic coarctation hypertension through the release of AVP. In conclusion, the important issue raised by this model of hypertension is the likelihood of a link between some vascular territory - probably renal - below the coarctation triggering the release of AVP, with this vasoconstrictor hormone participating with Ang II and the mechanical effect of aortic constriction in the acute aortic coarctation hypertension