Characterization of arginine vasopressin actions in human uterine artery: lack of role of the vascular endothelium.

1. The effect of arginine vasopressin (AVP) on human uterine artery rings, both intact and denuded of endothelium, was investigated. 2. Initially, AVP (63 pM-32 nM) induced concentration-dependent contraction of human uterine artery (pD2 = 8.92 +/- 0.01). Removal of the endothelium did not affect the concentration-response curve for AVP (pD2 = 8.83 +/- 0.03). 3. In contrast, human uterine arteries, both intact and denuded of endothelium, did not respond to the addition of 1-desamino-8-D-arginine vasopressin (dDAVP, 1 nM-1 microM). 4. In both types of preparations, [d(CH2)5Tyr(Me)AVP (1-10 nM) and [d(CH2)5,D-Ile2,Ile4]AVP (300 nM-3 microM) produced parallel rightward shifts of the curves for AVP. The Schild plots constrained to a slope of unity gave the following -log KB values: [d(CH2)5Tyr(Me)]AVP vs. [d(CH2)5,D-Ile2,Ile4]AVP 9.66 vs. 6.69 and 9.61 vs. 6.80 for human uterine artery, intact and denuded of endothelium, respectively. 5. The pKA values for AVP itself also did not differ between preparations: 6.56 and 6.43 for human uterine artery with and without endothelium, respectively. In both types of preparations, the receptor reserve (KA/EC50) was considerably greater than unity (intact vs. denuded: 228 vs. 244). 6. It is concluded that, in human uterine artery, AVP induces contractions that are not modulated by the endothelium. It is likely that AVP acts as a full agonist on human uterine artery, regardless of the endothelial condition. On the basis of differential antagonists affinity and affinity of AVP itself, it is probable that vasopressin receptors involved in AVP-induced contraction in human uterine arteries belong to the V1a or V1a-like subtype.     

Mitogenic effect of arginine vasopressin on adult rat cardiac fibroblast: involvement of PKC-erk1/2 pathway

Arginine vasopressin (AVP) has been implicated in the pathophysiology of cardiac hypertrophy. We previously demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extend our investigation to adult rat CFs to explore whether AVP could induce adult rat CFs proliferation and, if so, to identify the underlying mechanisms. We found that AVP stimulated cell proliferation, an effect abolished by V1 receptor antagonist, d(CH2)5[Tyr2(Me), Arg8]-vasopressin, but not V2 receptor antagonist, desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin. AVP also activated extracellular signal-regulated kinase 1/2 (erk1/2), a response mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but abolished by depleting cellular PKC through chronic PMA incubation. Calphostin C, an inhibitor of PKC, attenuated and PMA mimicked the effect of AVP on cell proliferation, whereas Ca2+ chelating agent 1,2-bis(2-aminophenoxy)ethane N, N, N', N'-tetraacetic acid (BAPTA) had no effect. Further, AVP downregulated protein expression of p27Kip1, increased cyclins D1, A, and E expressions and induced cell cycle progression through G0/G1 into S stage. Antisense oligonucleotides against cyclins D1, A, and E decreased cell number in the presence of AVP. Whereas antisense treatment against p27Kip1 and overexpression of p27Kip1 exerted a stimulatory and inhibitory effect, respectively. Inhibiting erk1/2 activation by PD98059 abolished the effect of AVP on cell proliferation, cell cycle regulatory proteins, and cell cycle progression. These results suggest that AVP is a mitogen for adult rat CFs via the mediation of V1 receptor and PKC-erk1/2 pathway.     

Neonatal treatment with vasopressin antagonist dP[Tyr(Me)2]AVP, but not with vasopressin antagonist d(CH2)5[Tyr(Me)2]AVP, inhibits body and brain development and induces polyuria in the rat

Two vasopressin antagonists, d(CH2)5[Tyr(Me)2]AVP and dP[Tyr(Me)2]AVP, were given to Wistar rats from postnatal day 1 to 21 in order to investigate the influence on development and later diuresis. The latter antagonist significantly reduced body growth from day 3 postnatally onwards. At postnatal day 35 body, total brain, cerebellar and kidney weights were significantly reduced compared with controls. Diuresis, measured at one month of age, was four- to five-fold higher than the control group. Combined treatment with vasopressin failed to abolish the weight disturbances or polyuria. However, animals treated with the vasopressin antagonist d(CH2)5[Tyr(Me)2]AVP did not show developmental or diuretic deficits. Allometric analysis of brain/body relationship of the young animals indicated a disturbance of brain development by dP[Tyr(Me)2]AVP. Although the body and brain growth retardation induced by dP[Tyr(Me)2]AVP supports the hypothesis of a role for vasopressin in brain ontogeny, it can also be the result of a nonAVP-related toxic effect, since it could not be prevented by concomitant treatment with vasopressin.     

Distinct receptor subtypes mediate arginine vasopressin-dependent ACTH release and intracellular calcium mobilization in rat pituitary cells

In the present study, adrenocorticotropic hormone (ACTH) release and intracellular calcium ([Ca(2+)](i)) increase induced by arginine vasopressin (AVP) were characterized in collagenase-dispersed and 3-day cultured rat anterior pituitary cells. AVP and the selective vasopressin V(1b) receptor agonist, [1-deamino-4-cyclohexylalanine]AVP (d[Cha(4)]AVP) induced ACTH release with nanomolar potencies in both cell preparations, and produced a maximal stimulation that was about 1.5 fold greater in the 3-day cultured cells, indicating that the vasopressin V(1b) receptor-ACTH release pathway is enhanced over time in culture. In dispersed cells, AVP, oxytocin and d[Cha(4)]AVP induced [Ca(2+)](i) increases with nanomolar potencies. The selective vasopressin V(1a) receptors antagonist, SR49059 (100 nM), together with the selective oxytocin receptors antagonist (d(CH(2))(5)(1)Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH(2)(9)-vasotocin (100 nM), inhibited the maximal AVP response by ~70%, without affecting the response to d[Cha(4)]AVP, suggesting that the V(1b) receptor was only partially responsible for the AVP-induced [Ca(2+)](i) increase. In contrast, in 3-day cultures, AVP induced an increase in [Ca(2+)](i), while oxytocin and d[Cha(4)]AVP did not. The response to AVP was completely antagonized by SR49059, whereas the vasopressin V(1b) receptor antagonists, SSR149415 and (d(CH(2))(5)(1)Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH(2)(9))-vasotocin had no effect, indicating that the [Ca(2+)](i) increase was mediated exclusively by vasopressin V(1a) receptors. In conclusion, the enhancement of vasopressin V(1b) receptor-mediated ACTH release and the lack of a detectable vasopressin V(1b) receptor coupling to [Ca(2+)](i) increase in cultured cells suggests the activation of a different/additional signaling pathway in the molecular mechanism of ACTH release.     

Involvement of neurokinins in the non-cholinergic response to activation of 5-HT3 and 5-HT4 receptors in guinea-pig ileum.

1. The effects of vasopressin and deamino-8-D-arginine vasopressin (DDAVP, desmopressin) were studied in artery rings (0.8-1 mm in external diameter) obtained from portions of human omentum during the course of abdominal operations (27 patients). 2. In arterial rings under resting tension, vasopressin produced concentration-dependent, endothelium-independent contractions with an EC50 of 0.59 +/- 0.12 nM. The V1 antagonist d(CH2)5Tyr(Me)AVP (1 microM) and the mixed V1-V2 antagonist desGly-d(CH2)5D-Tyr(Et)ValAVP (0.01 microM) displaced the control curve to vasopressin to the right in a parallel manner without differences in the maximal responses. In the presence of indomethacin (1 microM) the contractile response to vasopressin was significantly increased (P < 0.01). 3. In precontracted arterial rings, previously treated with the V1 antagonist, d(CH2)5Tyr(Me)AVP (1 microM), vasopressin produced endothelium-dependent relaxation. This relaxation was reduced significantly (P < 0.05) by indomethacin (1 microM) and unaffected by the V1-V2 receptor antagonist desGly-d(CH2)5D-Tyr(Et)ValAVP (1 microM) or by NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM). 4. The selective V2 receptor agonist, DDAVP, caused endothelium-independent, concentration-dependent relaxations in precontracted arterial rings that were inhibited by the mixed V1-V2 receptor antagonist, but not by the V1 receptor antagonist or by pretreatment with indomethacin or L-NAME. 5. Results from this study suggest that vasopressin is primarily a constrictor of human mesenteric arteries by V1 receptor stimulation; vasopressin causes dilatation only during V1 receptor blockade. The relaxation appears to be mediated by the release of vasodilator prostaglandins from the endothelial cell layer and is independent of V2 receptor stimulation or release of nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasopressin and stress-induced antinociception in the mouse.

1. Arginine vasopressin produced antinociception in the hot-plate test after intracerebroventricular injection (0.5 micrograms) and in the acetic acid abdominal constriction test after intraperitoneal injection (0.1 mg kg-1). 2. The antinociception produced by arginine vasopressin was sensitive to deamino(CH2)5Tyr(Me) arginine vasopressin (0.5 micrograms i.c.v.; 0.1 mg kg-1 i.p.) but not to naloxone (5 micrograms i.c.v.; 2 mg kg-1 i.p.) 3. Arginine vasopressin when administered by the intracerebroventricular route, but not by the intraperitoneal route, produced characteristic behaviour which was sensitive to deamino(CH2)5Tyr(Me) arginine vasopressin (0.5 micrograms, i.c.v.). 4. A 3 min swim at 20 degrees C produced antinociception on the hot-plate which was sensitive to naloxone (0.4 mg kg-1, i.p.) but not to deamino(CH2)5Tyr(Me) arginine vasopressin (0.5 micrograms, i.c.v.). 5. The reduction in the number of acetic acid-induced abdominal constrictions produced by a 30 s swim at 30 degrees C was not sensitive to either naloxone (2 mg kg-1, i.p.) or deamino(CH2)5Tyr(Me) arginine vasopressin (0.1 mg kg-1, i.p.). 6. Arginine vasopressin, at high doses, is antinociceptive in the mouse but does not appear to mediate stress-induced antinociception in this species.     

Solid-phase synthesis of 16 potent (selective and nonselective) in vivo antagonists of oxytocin

We describe the synthesis and some pharmacological properties of 16 new in vivo antagonists of oxytocin. These are based on modifications of three peptides: A, B, and C. A is our previously reported potent and selective antagonist of the vasopressor (V1 receptor) responses to arginine-vasopressin (AVP)/weak oxytocin antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-O-methyltyrosine]arginine-vasopressin (d(CH2)5[Tyr(Me)2]AVP. B reported here, the Ile3 analogue of A, is d(CH2)5[Tyr(Me)2]AVT (5 below) and C is our previously reported potent nonselective oxytocin antagonist/AVP V1 antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O- methyltyrosine,8-ornithine]vasotocin (d(CH2)5[Tyr(Me)2]OVT). The following substitutions and deletions, alone or in combination, were employed in A, B, and C: 1-deaminopenicillamine (dP); D-Tyr(Alk)2 (where Alk = Me or Et), D-Phe2; Val4, Thr4; delta 3-Pro7; Lys8, Cit8; desGly9, desGly-NH2(9), Ala-NH2(9); Leu-NH2(9); Arg-NH2(9). The 16 new analogues are (1) d(CH2)5[D-Tyr(Me)2]AVP, (2) d(CH2)5[D-Tyr(Me)2, Val4,delta 3-Pro7]AVP, (3) d(CH2)5[D-Tyr-(Et)2, Val4,Lys8]VP, (4) d(CH2)5[D-Tyr(Et)2,Val4,Cit8]VP, (5) d(CH2)5[Tyr(Me)2]AVT, (6) d(CH2)5[Tyr(Me)2,Lys8]VT, (7) dP[Tyr(Me)2]AVT, (8) dP[Tyr(Me)2,Val4]AVT, (9) d(CH2)5[D-Tyr(Me)2, Val4]AVT, (10) d(CH2)5[D-Phe2,Val4]AVT, (11) d(CH2)5[Tyr(Me)2,Thr4]OVT, (12) d(CH2)5[Tyr(Me)2,Thr4,Ala-NH2(9)]OVT, (13) d(CH2)5[Tyr(Me)2,Thr4,Leu-NH2(9)]OVT, (14) d(CH2)5[Tyr(Me)2,Thr4,Arg-NH2(9)]OVT, (15) desGly-NH2(9),d(CH2)5[Tyr(Me)2,Thr4]OVT, (16) desGly9,d(CH2)5[Tyr(Me)2,Thr4]OVT. 1-4 are analogues of A, 5-10 are analogues of B, and 11-16 are analogues of C. Their protected precursors were synthesized either entirely by the solid-phase method or by a combination of solid-phase and solution methods (1 + 8 or 8 + 1 couplings). All analogues were tested in rats for agonistic and antagonistic activities in oxytocic (in vitro, without and with Mg2+, and in vivo) assays as well as by antidiuretic and vasopressor assays. All analogues exhibit potent oxytocic antagonism in vitro and in vivo. With an in vitro pA2 (in the absence of Mg2+) = 9.12 +/- 0.09, dP[Tyr(Me)2]AVT is (7) one of the most potent in vitro oxytocin antagonists reported to date. Fifteen of these analogues (all but 6) appear as potent or more potent in vivo oxytocin antagonists than C (pA2 = 7.37 +/- 0.17). Analogues 1-9 and 14 are potent AVP V1 antagonists. Their anti-V1 pA2 values range from 7.92 to 8.45. They are thus nonselective oxytocin antagonists.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurohypophyseal hormone receptors in the septum are implicated in social recognition in the rat

The effects on social recognition memory of (Arg(8))-vasopressin (AVP-(1-9), [pGlu(4), Cyt(6)]AVP-(4-8) and oxytocin locally administered into the rat's septum were investigated. In the behavioural paradigm used, a juvenile intruder was presented to an adult resident male rat twice for 5 min, with an inter-exposure interval of 120 min. Peptide-free residents investigated the juvenile during the second encounter as long as during the first encounter. Intraseptal injection just after the first encounter with graded doses of (Arg(8))-vasopressin, [pGlu(4),Cyt(6)]AVP-(4-8) or oxytocin caused a decrease of social investigation, as compared to placebo treatment, indicating facilitation of social recognition. The least effective dose was 100pg, 200pg and 300pg respectively. The action of vasopressin was blocked by both d(CH(2))(5)[Tyr(Me)(2)]AVP and d(CH(2))(5)[D-Ile(2)Ile(4)]AVP, V(1) and V(2) vasopressinergic receptor antagonists, but not by desGly(NH(2))(9)-d(CH(2))(5)[Tyr(Me)(2)Thr(4)]-OVT, an oxytocinergic receptor antagonist. None of the antagonists blocked the oxytocin-facilitating action on social recognition. They also did not affect social recognition when injected alone. The effects of vasopressin seem to be mediated by vasopressinergic receptors dissimilar to those found in the periphery, while the receptors involved in the action of oxytocin remain to be elucidated.     

Central and systemic effects of a vasopressin V1 antagonist on MAP recovery after haemorrhage in rats

The present study examined the effects of central and peripheral administration of a vascular (V1) vasopressin (AVP) receptor antagonist on blood pressure, heart rate, and AVP levels in conscious rats. Rats subjected to rapid arterial haemorrhage were administered the AVP V1 antagonist [d(CH2)5Tyr(Me)AVP] either 5 min pre- or 20 min posthaemorrhage. Mean arterial blood pressure (MAP) was monitored for 45 min, after which the animals were killed and selected brain regions and plasma taken for AVP measurement. Intravenous (i.v.) administration of d(CH2)5Tyr(Me)AVP at 10 micrograms kg-1, but not 100 ng kg-1, significantly reduced MAP between 20 and 45 min posthaemorrhage compared with saline-treated controls. In contrast, administration of d(CH2)5Tyr(Me)AVP at 100 ng kg-1 intracerebroventricularly caused an attenuated MAP recovery to haemorrhage comparable with the effect of the antagonist at 10 micrograms kg-1 i.v. Haemorrhage caused a marked increase in circulating AVP levels, which was further enhanced in rats treated with the V1 antagonist at 10 micrograms kg-1 i.v., but no change in AVP levels of selected brain regions. The results indicate a role for AVP in MAP recovery following haemorrhage which may be centrally mediated.     

Synthesis and some pharmacological properties of 18 potent O-alkyltyrosine-substituted antagonists of the vasopressor responses to arginine-vasopressin

Using the Merrifield solid-phase method, we have synthesized 18 new 2-O-alkyltyrosine-substituted analogues (where alkyl = methyl and ethyl) of the arginine-vasopressin (AVP) vasopressor antagonists [1-deaminopenicillamine]-arginine-vasopressin (dPAVP), [1-(beta-mercapto-beta,beta-diethylpropionic acid)]arginine-vasopressin (dEt2AVP), and [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)]arginine-vasopressin (d(CH2)5AVP) and of their 8-D-arginine (d(R2)DAVP) analogues, their 4-valine (dR2VAVP) analogues, and their 4-valine,8-D-arginine (d(R2)VDAVP) analogues [where R = CH3 or C2H5 and 2R = (CH2)5]. These analogues were tested for agonistic and antagonistic activities in in vivo rat vasopressor and rat antidiuretic and in vitro rat uterus assay systems. Although many exhibit very low antidiuretic activities, none of the new analogues antagonize antidiuretic responses to AVP. They exhibit no evident pressor activities and are in fact all highly effective antagonists of the vasopressor responses to AVP. They are also potent antagonists of the in vitro oxytocic responses to oxytocin, both in the absence and in the presence of Mg2+. These analogues together with their corresponding antivasopressor pA2 values are as follows: 1. dPTyr(Et)AVP, 8.40 +/- 0.08; 2. dEt2Tyr(Me)AVP, 8.53 +/- 0.06; 3. dEt2Tyr(Et)AVP, 8.46 +/- 0.08; 4. d(CH2)5Tyr(Et)AVP, 8.47 +/- 0.04; 5. dPTyr(Me)DAVP, 8.31 +/- 0.08; 6. dPTyr(Et)DAVP, 8.27 +/- 0.06; 7. dEt2Tyr(Me)DAVP, 8.57 +/- 0.03; 8. dEt2Tyr(Et)DAVP, 8.33 +/- 0.06; 9. d(CH2)5Tyr(Me)DAVP, 8.41 +/- 0.05; 10. d(CH2)5Tyr(Et)DAVP, 8.45 +/- 0.05; 11. dPTyr(Me)VAVP, 8.36 +/- 0.07; 12. dPTyr(Et)VAVP, 8.07 +/- 0.13; 13. dEt2Tyr(Me)VAVP, 8.29 +/- 0.08; 14. dEt2Tyr(Et)VAVP, 8.42 +/- 0.06; 15. dPTyr(Me)VDAVP, 7.84 +/- 0.06; 16. dPTyr(Et)VDAVP, 8.46 +/- 0.03; 17. dET2Tyr(Me)VDAVP, 8.35 +/- 0.10; 18. dEt2Tyr (Et)VDAVP, 8.19 +/- 0.07. Seven of these analogues are clearly more potent vasopressor antagonists than their respective unalkylated tyrosine-containing parents. In the remaining 11, antagonistic potency was not changed significantly. In no instance did 2-O-alkyltyrosine substitution decrease antagonistic potency. With pA2 values equal to or greater than 8.40, nine of these antagonists (numbers 1-4, 7, 9, 10, 14, and 16) are among the most potent vasopressor antagonists reported to date. They could thus serve as additional valuable pharmacological tools in studies on the roles of AVP in the control of blood pressure in normal and in pathophysiological conditions. These findings may also provide useful clues to the design of more potent and selective antagonists of AVP.     

Hemodynamic effects of antagonists of the vasoconstrictor action of vasopressin in conscious dogs

Two antagonists of the pressor action of arginine-vasopressin (AVP) were studied in conscious, normally hydrated dogs: 1-deaminopenicillamine-4-valine-8-D-arginine-vasopressin, or dPVDAVP, and 1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid)2-(O-methyl)tyrosine arginine-vasopressin, or d(CH2)5Tyr(Me)AVP. We first examined the hemodynamic effects of these antagonists when given alone. The infusion of dPVDAVP, 200 ng/kg/min, increased cardiac output (measured with an aortic electromagnetic flowmeter) by 23% and heart rate by 27%, leaving arterial pressure unchanged. Most of the change in cardiac output reflected a large increase in skeletal muscle blood flow, as determined by radioactive microspheres. The injection of d(CH2)5Tyr(Me)AVP, 10 micrograms/kg, had little effect on cardiac output, arterial pressure, and heart rate. We then examined the ability of the two antagonists to block the hemodynamic responses to injections of AVP. In the absence of the antagonists, AVP induced dose-related increases in mean arterial pressure and total peripheral resistance, as well as decreases in heart rate and cardiac output. The antagonist dPVDAVP shifted the dose-response curves to the right without changing their slope. On the contrary, the hemodynamic response to AVP was strikingly modified following blockade with d(CH2)5Tyr(Me)AVP. Cardiac output and heart rate increased, whereas total peripheral resistance decreased, for doses of AVP between 25 and 400 ng/kg. It is concluded that some antagonists of the pressor action of vasopressin may influence hemodynamics of conscious dogs by effects other than competitive antagonism at the level of vascular receptors.     

Synthesis and some pharmacological properties of potent and selective antagonists of the vasopressor (V1-receptor) response to arginine-vasopressin.

We report the solid-phase synthesis of eight position-9-modified analogues of the potent V1-receptor antagonist of arginine-vasopressin, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-methyltyrosine]arginine-vasopressin (d(CH2)5Tyr(Me)AVP) (1-8) and five position-9-modified analogues of the closely related beta,beta-dimethyl less potent V1 antagonist, [1-deaminopenicillamine,2-O-methyltyrosine]arginine-vasopressin (dPTyr(Me)AVP) (9-13). In d(CH2)5Tyr(Me)AVP the C-terminal Gly-NH2 was replaced by (1) ethylenediamine (Eda), (2) methylamine (NHMe), (3) Ala-NH2, (4) Val-NH2, (5) Arg-NH2, (6) Thr-NH2, (7) Gly-Eda, (8) Gly-N-butylamide (Gly-NH-Bu); in dPTyr(Me)AVP the C-terminal Gly-NH2 was replaced by (9) Ala-NH2, (10) Val-NH2, (11) Thr-NH2, (12) Arg-NH2, and (13) Tyr-NH2. All 13 analogues were tested for agonistic and antagonistic activities in in vivo rat vasopressor (V1-receptor) and rat antidiuretic (V2-receptor) assays. They exhibit no evident vasopressor agonism. All modifications in both antagonists were well-tolerated with excellent retention of V1 antagonism and striking enhancements in anti-V1/anti-V2 selectivity. With anti-V1 pA2 values of 8.75, 8.73, 8.86, and 8.78, four of the analogues of d-(CH2)5Tyr(Me)AVP (1-3 and 6) are equipotent with d(CH2)5Tyr(Me)AVP (anti-V1 pA2 = 8.62) but retain virtually none of the V2 agonism of d(CH2)5Tyr(Me)AVP. They are in fact weak V2 antagonists and strong V1 antagonists with greatly enhanced selectivity for V1 receptors relative to that of d(CH2)5Tyr(Me)AVP. With anti-V1 pA2 values respectively of 8.16, 8.05, 8.04, 8.52, and 8.25, all five analogues (9-13) of dPTyr(Me)AVP are at least as potent V1 antagonists as dPTyr(Me)AVP (pA2 = 7.96) and three of these (9, 12, 13) actually show enhanced V1 antagonism over that of dPTyr(Me)AVP. In fact, the Arg-NH2(9) analogue (12) is almost equipotent with d(CH2)5Tyr(Me)AVP. These new V1 antagonists are potentially useful as pharmacological tools for studies on the cardiovascular roles of AVP. Furthermore the analogues of dPTyr(Me)AVP may be useful in studies on the role(s) of AVP in the V1b-receptor-mediated release of ACTH from corticotrophs.     

Receptor-mediated stimulation of taurocholate efflux from the rat hepatocyte and the ex vivo perfused rat liver

The peptide hormone, arginine-vasopressin[( Arg8]vasopressin, AVP), stimulates efflux of the bile salts taurocholate and glycocholate from the rat hepatocyte in suspension via its association with the V1 receptor on the hepatic cell membrane. At a concentration ratio of 5:1 (antagonist to hormone), the V1 vasopressin antagonist, (dCH2)5Tyr(Me)AVP, inhibits the vasopressin induced efflux of taurocholate by approximately 82%, and of glycocholate, by approximately 85%. In contrast, the V2 antagonist (d(CH2)5[D-Ile2,Ala4]AVP, does not interfere with the stimulation of taurocholate and glycocholate efflux by vasopressin. In the isolated perfused rat liver, vasopressin (5 X 10(-10) M) causes an immediate increase of 55 +/- 12% over baseline in [14C]taurocholate secretion and a corresponding increase in bile flow. A more gradual and prolonged increase in [14C]taurocholate secretion, reflecting an increased biliary concentration of [14C]taurocholate, is observed beginning 6 min after vasopressin, reaching a plateau of 23 +/- 12% over baseline by 14 min and returning to baseline by 30 min. The mean rate of 14C secretion during the 30 min following administration of vasopressin (non-steady state) is increased by 14.3 +/- 6.4% over pre-infusion steady-state baseline (P less than 0.05). Prior administration of the V1 receptor antagonist d(CH2)5Tyr(Me)AVP attenuates these effects of vasopressin. The combination of these in vitro and in vivo findings suggest that vasopressin may play a role in regulating bile salt efflux. Furthermore, these studies in the isolated hepatocyte and the intact liver may provide a unique approach for defining biochemical changes associated with bile salt transport from the hepatic cell.     

Comparison of vasopressin binding sites in human uterine and vascular smooth muscle cells.

Several studies indicate that oxytocin and vasopressin receptors in the human uterus are heterogeneous. We have investigated whether oxytocin and vasopressin bind to separate receptors or one class of receptors in human uterine smooth muscle cells. [3H]d(CH2)5Tyr(Me)AVP, the vasopressin V1A receptor selective radioligand, was used for comparison of vasopressin binding sites in human uterine and vascular smooth muscle cell membranes. Both membrane preparations exhibited one class of high-affinity binding sites with Kd values of 6.44 and 0.47 nM, Bmax values of 166 and 34.8 fmol/mg protein for uterine and vascular smooth muscle cells, respectively. In vascular preparations, the selective vasopressin V1A receptor antagonist, SR 49059 ((2S) 1-[(2R 3S)-(5-chloro-3-(2-chlorophenyl)- -(3.4-dimethoxybenzenesulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2- carbonyl]-pyrrolidine-2-carboxamide), showed high affinity with Ki value of 0.98 nM, confirming that these receptors belong to the vasopressin V1A receptor subtype. On the contrary, in uterine preparations, binding of [3H]d(CH2)5Tyr(Me)AVP was more effectively displaced by oxytocin and the oxytocin receptor selective antagonist, L-371257, (1-[1-[4-[ N-Acetyl-4-piperidinyl)oxy]2-methoxybenzoyl]piperidin-4-yl]- 4H-3,1-benzoxazin-2(1H)-one), than vasopressin and SR 49059, suggesting that binding may be due to cross-reaction with the oxytocin receptors. These results suggest that human uterine smooth muscle cells express only a high density of oxytocin receptors.     

Labelling of vasopressin and oxytocin receptors from the human uterus

Four labelled ligands, [3H]arginine vasopressin ([3H]AVP), [3H]oxytocin ([3H]OT), [3H]d(CH2)5[Tyr(Me)2]AVP ([3H]VPA), and [125I]d(CH2)5[Tyr(Me)2-Thr4-Orn8-Tyr(NH2)9]OT([125I]OTA] and nine unlabelled analogues exhibiting enhanced selectivity for rat oxytocin (OT) and vasopressin (VP) receptors were used to characterize OT and VP receptors on myometrial membranes from non-pregnant and pregnant human uteri. On membranes from non-pregnant uteri, [3H]AVP, [3H]VPA, and [125I]OTA labelled with high affinity (Kd values: 3.2, 2 and 0.8 nM, respectively) a major and apparently homogeneous population of sites, the ligand selectivity of which resembled that of rat V1a VP receptors. On membranes from pregnant and non-pregnant uteri, [3H]OT labelled a single population of high-affinity sites that could be distinguished from VP receptors on the basis of ligand selectivity. Several analogues (in particular [125I]OTA) that are highly selective for rat OT receptors exhibited a much less pronounced selectivity for human OT receptors. Experiments with [3H]VPA allowed detection of VP receptors on myometrical membranes from pregnant uteri and confirmed that only OT but not VP receptors increase during pregnancy in humans.     

Antagonism of vasopressin-induced coronary artery constriction by the vasopressin antagonist d(CH2)5Tyr(Me)-AVP

d(CH2)5Tyr(Me)-AVP is a potent inhibitor of the systemic vasoconstrictor action of vasopressin (AVP). In order to examine the effectiveness of this agent in blocking AVP-induced coronary vasoconstriction, 11 pentobarbital-anesthetized mongrel dogs were instrumented for the measurement of left circumflex (LCX) coronary blood flow, systemic arterial blood pressure, heart rate, lead II electrocardiogram, left ventricular end-diastolic pressure, left ventricular developed pressure, and left ventricular +/- dP/dt. Direct injection of AVP (0.01-1 microgram) into the LCX produced a dose-dependent decrease in coronary artery blood flow (maximum reduction: 60.5 +/- 8.1% after 1 microgram), - dP/dt (maximum reduction: 41.8 +/- 5.3% after 1 microgram), and +dP/dt (maximum reduction: 14.6 +/- 5.3%), whereas a dose-dependent increase in left ventricular end-diastolic pressure was observed (maximum increase: 62.6 +/- 20.2%). No significant changes occurred in heart rate, mean blood pressure, or left ventricular developed pressure. Intravenous administration of d(CH2)5Tyr(Me)-AVP reduced (1 microgram/kg) or abolished (5 micrograms/kg) the effects of AVP on coronary blood flow +/-dP/dt and left ventricular end-diastolic pressure. In addition, doses of 1,2, and 5 micrograms/kg of d(CH2)5Tyr(Me)-AVP alone produced increases of LCX blood flow of 5.1 +/- 1.5, 2.0 +/- 0.7, and 6.8 +/- 1.7 ml/min, respectively (p less than 0.05). We conclude that d(CH2)5Tyr(Me)-AVP is effective in preventing the coronary artery constriction and hemodynamic sequelae of intracoronary administered AVP.     

Evidence that apomorphine induces penile erection and yawning by releasing oxytocin in the central nervous system

Oxytocin (10 and 30 ng) injected into a lateral ventricle (i.c.v.) or the dopamine agonist apomorphine (40 and 80 micrograms/kg) injected subcutaneously induced repeated episodes of penile erection and yawning in male rats. The concomitant administration of the two substances did not produce any further increase in the number of penile erection and yawning episodes. Penile erection and yawning induced by either oxytocin or apomorphine were antagonized in a dose-dependent manner by i.c.v. pretreatment with the oxytocin antagonists [d(CH2)5Tyr(Me)-Orn8]vasotocin, [Pen1,Phe(Me)2,Thr4,Orn8]oxytocin and [d(CH2)5Tyr(Me)-Arg8]vasopressin, with a rank order of potency that follows their antioxytocic activity. (i.e. [d(CH2)5Tyr(Me)-Orn8]vasotocin congruent to [Pen1,Phe(Me)2,Thr4,Orn8]-oxytocin greater than [d(CH2)5Tyr(Me)-Arg8]vasopressin). The results suggest that apomorphine induces penile erection and yawning by releasing oxytocin in the central nervous system.     

Thermoregulatory actions of centrally-administered vasopressin in the rat

Dependent upon the route and/or site of administration, arginine vasopressin (AVP) evoked a number of thermoregulatory actions in the conscious rat. Infused into a lateral cerebral ventricle, arginine vasopressin produced short-lasting hypothermia of rapid onset. Injected into the preoptic area, arginine vasopressin caused long-lasting hyperthermia of rapid onset that was antagonized by the prior administration of a V1 receptor antagonist, [d(CH2)5 Tyr(Me)AVP]. Injections of arginine vasopressin into the nucleus accumbens, ventral septal area, substantia innominata and the dorsomedial hypothalamus were without effect on body temperature. Although the antipyretic action of arginine vasopressin within the ventral septal area has been well documented, these findings provide further evidence that this peptide exerts additional thermoregulatory actions that are both neuroanatomically and functionally specific.     

Amygdalar injections of vasopressin and its antagonist do not disrupt the circadian rhythm of food and water intake in the rat

There is an indication that areas of the brain other than the suprachiasmatic nuclei (SCN), the known neural circadian pacemaker, are involved in the control of circadian rhythms. The present study investigated the role of amygdala in the circadian rhythms of food and water intake. Vasopressin and its antagonist d(CH2)5Tyr(Me)AVP were injected into the amygdala bilaterally through chronically implanted stainless steel cannulae. The results of the study have shown that neither vasopressin nor its antagonist d(CH2)5Tyr(Me)AVP alters the circadian rhythm of food and water intake thereby showing that vasopressinergic neurons/projections to amygdala are not involved in the control of circadian rhythms of food and water intake and amygdala is not likely to be an additional oscillator.     

INTERACTION OF VASOPRESSIN, ANGIOTENSIN AND α-ADRENERGIC SYSTEM IN SODIUM DEPLETION IN THE RAT

1. The role of vasopressin in cardiovascular adaptation to sodium depletion was examined in rats after 6 days on a low sodium diet. Studies were performed after selective or combined blockade with d(CH2)5 Tyr(Me)AVP (AVPA), enalaprilat (CEI) and phentolamine (PHENTOL). AVPA alone had no effect on systemic haemodynamics or regional blood flow distribution. After CEI or PHENTOL pretreatment, AVPA led to significant falls in peripheral resistance and increases in cardiac output and renal blood flow. In sodium depletion, endogenous vasopressin acts as a vasoconstrictor hormone, particularly in the kidney, when either the renin-angiotensin or alpha-adrenergic system is inhibited.