(Arg8)-Vasopressin (free acid)

Arginine vasopressin induces vascular, inotropic and arrhythmogenic effects in the heart. Existing evidence, obtained indirectly, suggests that these effects occur through paracrine endothelial mechanisms. To demonstrate this, vasopressin was confined to the intravascular space by covalent coupling to high molecular weight (2x10(6) Da, vasopresin-dextran) dextran. Isolated guinea pig hearts were infused with equivalent concentrations of vasopressin and vasopressin-dextran. The negative inotropic and coronary vasopressor effects of vasopressin-dextran were similar to those evoked by vasopressin; in both cases effects were reversible. Free dextran had no effect on vascular resistance nor in ventricular developed pressure. The inotropic and vascular effects of both vasopressin and vasopressin-dextran were blocked by the vasopressin receptor antagonist [Adamantaneacetyl(1), o-Et-D-Tyr(2), Val(4), Aminobutyryl(6), Arg(8,9)]vasopressin (Adam-vasopressin), indicating that the effects of the two agonists were vasopressin receptor-mediated. To elucidate possible endothelial intermediaries of these effects, isolated guinea pig hearts were infused simultaneously with vasopressin or vasopressin-dextran and several inhibitors either of synthesis or blockers of receptors of possible endothelial mediators. Only reactive blue 2, a P(2y) purinoceptor antagonist, and suramin, a P(2y) and a P(2x) purinoceptor antagonist, caused a total reversal of vascular and inotropic effects of vasopressin and vasopressin-dextran. Pyridoxalphosphate-6-Azophenyl-2'-4'disulphonic acid, a P(2x) purinoceptor antagonist, was without effect. Our results provide direct evidence that the short-term cardiac effects of vasopressin are due to selective activation of intravascular purinoceptors and suggest that an intermediary of these effects is ATP.

[Arg8]Vasopressin (AVP) has an antilipolytic action on adipocytes, but little is known about the mechanisms involved. Here, we examined the involvement of the V1a receptor in the antilipolytic effect of AVP using V1a receptor-deficient (V1aR-/-) mice. The levels of blood glycerol were increased in V1aR-/- mice. The levels of ketone bodies, such as acetoacetic acid and 3-hydroxybutyric acid, the products of the lipid metabolism, were increased in V1aR-/- mice under a fasting condition. Triacylglyceride and free fatty acid levels in blood were decreased in V1aR-/- mice. Furthermore, measurements with tandem mass spectrometry determined that carnitine and acylcarnitines in serum, the products of beta-oxidation, were increased in V1aR-/- mice. Most acylcarnitines were increased in V1aR-/- mice, especially in the case of 2-carbon (C2), C10:1, C10, C14:1, C16, C18:1, and hydroxy-18:1-carbon (OH-C18:1)-acylcarnitines under feeding rather than under fasting conditions. The analysis of tissue C2-acylcarnitine level showed that beta-oxidation was promoted in muscle under the feeding condition and in liver under the fasting condition. An in vitro assay using brown adipocytes showed that the cells of V1aR-/- mice were more sensitive to isoproterenol for lipolysis. These results suggest that the lipid metabolism is enhanced in V1aR-/- mice. The cAMP level was enhanced in V1aR-/- mice in response to isoproterenol. The phosphorylation of Akt by insulin stimulation was reduced in V1aR-/- mice. These results suggest that insulin signaling is suppressed in V1aR-/- mice. In addition, the total bile acid, taurine, and cholesterol levels in blood were increased, and an enlargement of the cholecyst was observed in V1aR-/- mice. These results indicated that the production of bile acid was enhanced by the increased level of cholesterol and taurine. Therefore, these results indicated that AVP could modulate the lipid metabolism by the antilipolytic action and the synthesis of bile acid via the V1a receptor.

Solubilized noncovalent complexes of [Arg8]-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor [pro-AVP/BNPII-(20-1)], a region that includes the AVP sequence at residues 1-9. The AVP-related AS peptide previously was shown to bind selectively to AVP. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarily at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, [3H]AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. When this fraction was eluted through AS pro-AVP/BNPII-(20-1) silica, a complex containing [3H]AVP was bound and separated from the major, unretarded membrane protein fraction as well as from free AVP. Chemical crosslinking of [3H]AVP complex, SDS/PAGE of the products, and analysis of gel slices by scintillation counting led to detection of two major radiolabeled bands of 31 and 38 kDa. Covalent labeling was blocked when unlabeled AVP was added as a competitor before crosslinking. A third radiolabeled protein band of 15 kDa was found when the receptor complex was solubilized from rat liver membranes in the absence of the protease inhibitor phenylmethylsulfonyl fluoride. Covalently crosslinked [3H]AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photo-affinity labeling, we conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein-receptor complexes in other systems as well.