Wilbur H. Sawyer

125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT: a selective oxytocin receptor ligand

An oxytocic antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid,2-O-methyltyrosine,4-threonine, 8-ornithine,9-tyrosylamide]vasotocin (d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT [corrected], was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. 125I-labelling was performed with 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl-glycoluril. Iodination resulted in an increased affinity for rat uterine oxytocin receptors. A considerably lower affinity for rat vascular V1- and renal V2-receptors was found, resulting in a highly specific oxytocin receptor ligand. 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT [corrected] was demonstrated to bind selectively to one population of binding sites in rat uterus and ventral hippocampal membrane preparations. Dissociation constants ranged between 0.03 and 0.06 nM. After 3 days of exposure autoradiography revealed binding in regions known to contain oxytocin receptors as well as labelling in some new regions, while no binding was found in the lateral septum, a structure containing mainly [8-arginine]vasopressin receptors. The high specific radioactivity of 125I-labelling allowed important reductions in membrane protein amount, gain in precision of binding analysis as well as considerably lower exposure times for autoradiography.

Arginine vasopressin and a vasopressin antagonist peptide: Opposite effects on extinction of active avoidance in rats

Systemic injection or arginine vasopressin (AVP) (1 micrograms/rat) significantly prolonged extinction of a pole-jump, active avoidance response in rats; lateral ventricular injection of 1000-fold less AVP (1 ng/rat) produced similar results. A new AVP analogue, [1-deaminopenicillamine-2-(O-methyl)tyrosine]arginine vasopressin (dPTyr-(Me)AVP), is known to antagonize behavioral and vascular effects of exogenous AVP at molar ratios of 5 : 1. At a dose of 100 micrograms/rat (subcutaneously) dPTyr-(Me)AVP produces, by itself, a behavioral effect opposite to that of exogenous AVP, namely a facilitation of extinction. Injections of dPTyr-(Me)AVP into the lateral ventricle were ineffective except at a dose of 10 micrograms/rat. These results confirm previous reports of the effect of vasopressin on delaying extinction of avoidance behavior, but suggest a site of action distant from the lateral ventricle.

A radioiodinated linear vasopressin antagonist: A ligand with high affinity and specificity for V1a receptors

A linear vasopressin antagonist, Phaa‐D‐Tyr(Me)‐Phe‐Gin‐Asn‐Arg‐Pro‐Arg‐Tyr‐NH2 (Linear AVP Antag) (Phaa = Phenylacetyl), was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. This antagonist appeared to be a highly potent anti‐vasopressor peptide with a pA2 value in vivo of 8.94. It was demonstrated to bind to rat liver membrane preparations with a very high affinity (K d = 0.06 nM). The affinity for the rat uterus oxytocin receptor was lower (K i = 2.1 nM), and affinities for the rat kidney‐ and adenohypophysis‐vasopressin receptors were much lower (K i 47 nM and 92 nM, respectively), resulting in a highly specific vasopressin V18 receptor ligand. Autoradiographical studies using rat brain slices showed that this ligand is a good tool for studies on vasopressin receptor localization and characterization.

DESIGN, SYNTHESIS AND SOME USES OF RECEPTOR-SPECIFIC AGONISTS AND ANTAGONISTS OF VASOPRESSIN AND OXYTOCIN

Selective agonists and antagonists are powerful tools for studies on AVP and OT receptors and on the physiological and pathophysiological roles of AVP and OT. Here we show how some of these peptides and their radiolabelled derivatives were designed. We also present examples of the currently available cyclic and linear OT and AVP agonists and antagonists from our laboratories.

Selective Agonists and Antagonists of Vasopressin

Since the original synthesis of (arginine-) vasopressin (AVP) in 1954 (du Vig-neaud et al, 1954b), hundreds of analogues of VP and of oxytocin (OX) have been synthesized; their properties have been reported in numerous publications (see e.g., Berde and Boissonnas, 1968; Sawyer and Manning, 1973; Sawyer et al., 1981a; Manning et al., 1981a). Space considerations do not permit the inclusion of all the published analogues of VP and OX. Since the focus of this volume is on vasopressin, this chapter deals primarily with analogues—agonists and antagonists—of VP.

Design of potent antagonists of the vasopressor response to arginine-vasopressin.

As part of a program in which we are attempting to design and synthesize antagonists of the vasopressor response to arginine-vasopressin (AVP), [1-deaminopenicillamine]arginine-vasopressin (dPAVP), [2-O-methyl)tyrosine]-arginine-vasopressin [Tyr(Me)AVP], and [1-deaminopenicillamine,2-(O-methyl)tyrosine]arginine-vasopressin [dPTyr(Me)AVP] were synthesized by the solid-phase method and assayed for vasopressor, antidiuretic, and oxytocic activities. Tyr(Me)AVP has a vasopressor potency of 9.7 +/- 0.5 units/mg and an antidiuretic potency of 386 +/- 36 units/mg. These values are 2.5 and 120%, respectively, of the corresponding potencies of AVP. The analogue is an antagonist of the in vitro response to oxytocin (pA2 = 7.44 +/- 0.12). dPAVP has an antivasopressor pA2 of 7.45 +/- 0.11. Its antidiuretic potency is 42.2 +/- 2 units/mg, 2.5% that of its parent, 1-[deamino]arginine-vasopressin (dAVP). It is an antagonist of the in vitro response to oxytocin (pA2 value = 6.93 +/- 0.10). dPTyr(Me)AVP has an antivasopressor pA2 of 7.96 +/- 0.05 and an antidiuretic potency of 3.5 +/- 0.5 units/mg. It is also an antagonist of the in vitro oxytocic response to oxytocin (pA2 value = 7.61 +/- 0.14). It is thus one of the most potent vasopressor antagonists reported to date.

Inhibition of oxytocin release by morphine and its analogs.

Summary Two synthetic morphine analogs, butorphanol and oxilorphan have been found to inhibit suckling induced oxytocin (OT) release in lactating mice. Morphine when tested in a similar experimental set-up produced even greater inhibition. Naloxone could effectively block inhibitions induced both by butorphanol and by morphine. When injected alone, naloxone did not produce any inhibition. These results indicate that (a) butorphanol, oxilorphan and morphine inhibit suckling induced OT release in mice, (b) the analgesic property of these compounds is responsible for the inhibition, and (c) inhibition produced by morphine and butorphanol is specific in nature since it could be reversed by naloxone.

Electrolyte balance in hypophysectomized goldfish, Carassius auratus L

Abstract Hypophysectomized goldfish ( Carassius auratus L.) kept for 3 weeks in fresh water show a sharp decrease (35% or more) in plasma Na and Cl concentrations. Due to expansion of the apparent Na space, however, total exchangeable Na drops only about 20%. Flux analyses, using 22 Na, show that hypophysectomized fish are in strongly negative Na balance. Urinary Na concentration rises but urine flow decreases to about 55% of normal. The rate of urinary Na loss is not significantly greater than that of intact or sham-operated fish. Negative Na balance is thus due to increased extrarenal Na loss. Repeated injections of sheep prolactin partially prevent the decline in plasma electrolyte concentrations. ACTH injections are ineffective in this respect. Survival of hypophysectomized goldfish in fresh water for long periods appears to be due largely to their ability to withstand dilution of the internal medium.

Sodium exchanges in goldfish (Carassius auratus L.) adapted to a hypertonic saline solution

Abstract 1. 1. Goldfish were adapted to diluted sea water (190 m-equiv./1 of Na) which was hyperosmotic to plasma from fresh-water-adapted fish. Fish survive well in this medium but plasma Na levels rise to equal those of the environment and exchangeable Na increases by 74 per cent. 2. 2. Fish in this hyperosmotic medium increase Na outflux about twenty times. They also increase their Na influx and drinking rates and decrease free-water excretion. 3. 3. Despite such adjustments, goldfish do not osmoregulate effectively. The goldfish appears to be stenohaline chiefly because it lacks the ability to excrete enough Na actively across the gills to balance the increased Na intake from the hyperosmotic environment.

Endocrine adaptation to osmotic requirements of the environment: Endocrine factors in osmoregulation by lungfishes and amphibians

Abstract Arginine vasotocin (AVT) occurs in the neurohypophyses of lungfishes and amphibians. Injected AVT is diuretic in lungfishes and antidiuretic in anuran amphibians. A new radioimmunoassay for AVT has been used in an effort to define whether AVT has endocrine functions related to osmoregulation and to attempt to demonstrate the factors that influence its release. Preliminary experiments on bullfrogs suggest that decreased blood volume or pressure is a more important stimulus for the release of AVT than plasma hyperosmolality. Factors other than AVT appear important in controlling water excretion by bullfrogs.