pituitrin and pressinoic-acid

Methodology is described for characterization of the kinetics and equilibria of thiol/disulfide interchange reactions of the disulfide bonds in the neurohypophyseal peptide hormones arginine vasopressin and oxytocin and the related peptides pressinoic acid and tocinoic acid. Thiol/disulfide interchange reaction mixtures are analyzed by reversed-phase high-performance liquid chromatography. The effect of mobile-phase composition and pH on the HPLC capacity factors for the native disulfide and reduced dithiol forms of each peptide was examined. In each case, the capacity factor decreases as the acetonitrile content of the mobile phase increases. For each disulfide/dithiol peptide pair, the capacity factor is larger for the dithiol form of the peptide, indicating that the hydrophobic side chains of the linear peptide are more accessible for interaction with the hydrophobic stationary phase. To illustrate application of the methodology, rate and equilibrium constants are reported for the thiol/disulfide interchange reactions of cysteine with arginine vasopressin at pH 7.0. Cysteine reacts with arginine vasopressin to form two mixed disulfides, which in turn react with another molecule of cysteine to give the dithiol form of arginine vasopressin and cystine. Rate and equilibrium constants were determined for each step by analysis of reaction mixtures by HPLC. The results are compared to rate and equilibrium constants for reaction of cysteine with oxidized glutathione.

Topics: Amino Acid Sequence; Arginine Vasopressin; Chromatography, High Pressure Liquid; Disulfides; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Oxidation-Reduction; Oxytocin; Pituitary Gland, Posterior; Sulfhydryl Compounds; Vasopressins

Arginine vasopressin (AVP), a nine-amino acid neurohypophyseal hormone, is capable of replacing the helper cell requirement for IFN-gamma production by Lyt-2+ mouse splenic lymphocytes. We present data here showing that the AVP helper signal occurs via interaction with a novel R on splenic lymphocytes and involves primarily the N-terminal six-amino acid cyclic ring (pressinoic acid) with the C-terminal three-amino acid end of AVP playing a minor role. Pressinoic acid was capable of providing help at concentrations similar to those of AVP, whereas oxytocin and isoleucine pressinoic acid were 10- and 100-fold less effective, respectively. Isoleucine pressinoic acid has the same structure as pressinoic acid except for the substitution of isoleucine for phenylalanine in position 3 of the sequence. Consistent with the function data, R binding competitions with splenic lymphocyte membrane preparations showed that AVP and pressinoic acid competed similarly with [3H]AVP, whereas oxytocin and isoleucine pressinoic acid were much less effective competitors. Further characterization of the AVP lymphocyte R was performed using AVP analogues having well defined agonist and antagonist activities on either V1 (vasopressor) R or V2 (antidiuretic) R. The AVP helper signal was blocked by the V1 antagonist [d(CH2)1(5) Tyr(methyl)]AVP but not by another V1 antagonist, [d(CH2)1(5)D-Tyr(ethyl)2Val4]AVP. Both V1-R antagonists were able to block [3H]AVP binding to the V1-R on liver cells, whereas only the V1 antagonist that blocked AVP help was able to compete effectively for the spleen AVP-R. Neither a V2 agonist nor a V2 antagonist had any effect on AVP help in IFN-gamma production. These data strongly indicate the presence of a novel AVP-R on spleen lymphocytes, which is related to the classic V1-R on liver cell membranes.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Arginine Vasopressin; Binding, Competitive; Female; Interferon-gamma; Mice; Mice, Inbred C57BL; Receptors, Angiotensin; Receptors, Vasopressin; Structure-Activity Relationship; T-Lymphocytes, Helper-Inducer; Vasopressins

Plasma concentrations of immunoreactive vasotocin (AVT) and mesotocin (MT) were measured periodically before and subsequent to spontaneous oviposition in conscious chickens. The concentrations of AVT and MT approximately an hour prior to oviposition were 5.2 +/- 1.1 microU/ml and 14.7 +/- 5.1 pg/ml, respectively. Plasma AVT levels increased abruptly at oviposition (25.1 +/- 3.3 microU/ml) and decreased to 5.0 +/- 0.6 microU/ml within 30 min postoviposition. Significant changes in MT were not observed. The data indicate that AVT is selectively released during oviposition. The uterus was removed immediately after oviposition and the oxytocic potencies of several peptides were tested on muscle strips in vitro. The order of oxytocic potencies was AVT greater than or equal to arginine vasopressin (AVP) much greater than MT = pressinoic acid. Partially purified membranes were prepared from separate portions of the uteri used in the oxytocic assay. [3H]arginine8 vasopressin, [3H]AVP, bound to membranes saturably (Bmax = 17 fmol/mg protein) and with high affinity (Kd = 0.7 nM). The rank order of potency of the peptides in displacing [3H]AVP from the binding sites was the same as in the oxytocic assay which suggests that the [3H]AVP binding sites in uterine membranes represent physiological receptors that interact with AVT during oviposition.

Topics: Animals; Arginine Vasopressin; Binding, Competitive; Biological Assay; Cell Membrane; Chickens; Female; Kinetics; Myometrium; Oviposition; Oxytocin; Uterine Contraction; Vasopressins; Vasotocin

The use of carboethoxysulfenyl chloride for disulfide bond formation and concomitant cyclization of five peptides was investigated. Even though cyclic peptides were obtained very rapidly and in good yields when cyclization was performed in aqueous media at different pHs (4 to 7), the final crude peptides were found to contain closely related impurities which, in the case of somatostatin and pressinoic acid, were not generated by air oxidation. This observation may limit the use of carboethoxysulfenyl chloride to those cases where other methods of disulfide bond formation prove inadequate.

Topics: Disulfides; Hydrogen-Ion Concentration; Oligopeptides; Somatostatin; Sulfenic Acids; Urotensins; Vasopressins

To characterize the V2 receptor (for antidiuretic hormone), we have studied the effect of a number of neurohypophysial hormone analogues on cyclic AMP (cAMP) accumulation and short-circuit current in cultured epithelia formed by A6 cells. A6 is the designation of a continuous cell line derived from the kidney of Xenopus laevis. The order of potency for stimulating cAMP accumulation and short-circuit current in A6 epithelia is like that for stimulating water permeability in toad urinary bladder. As anticipated, arginine vasotocin (AVT), the antidiuretic hormone of Amphibia, is more potent than arginine vasopressin (AVP), the antidiuretic hormone of most mammals. The two hormones differ only in the third amino acid (Phe-3 in AVP is a substitution for Ile-3 in AVT). However, there are a number of striking differences in the responsiveness of these amphibian V2 receptors and mammalian V2 receptors to changes in the 7th, 8th, and 9th amino acids where AVT and AVP are identical. 1) Substitution of Lys-8 for Arg-8 in AVP results in marked loss of potency in Amphibia, whereas there is only modest loss of potency in mammals. 2) Desglycinamide AVP is nearly as potent as AVP in Amphibia, whereas it is inactive in mammals. 2) Tocinoic acid, lacking amino acids 7, 8, and 9, has activity in Amphibia, but pressinoic acid, lacking the same three amino acids, is inactive.

Topics: Amino Acids; Animals; Arginine Vasopressin; Cell Line; Cyclic AMP; Dose-Response Relationship, Drug; Epithelium; Kidney; Lypressin; Oxytocin; Peptides; Permeability; Pituitary Gland, Posterior; Receptors, Angiotensin; Receptors, Cell Surface; Receptors, Vasopressin; Vasopressins; Vasotocin; Xenopus laevis

Arginine vasopressin consists of a 20-membered, disulfide-linked macrocyclic ring system called pressinoic acid to which is attached a COOH-terminal tripeptide. The molecular conformation of pressinoic acid has been determined from single crystal x-ray diffraction data. The 20-membered macrocyclic ring, stabilized by two intramolecular hydrogen bonds, has a type I beta-bend centered on Gln4 and Asn5 and a highly distorted type II' bend centered on Phe3 and Gln4. In vasopressin the Asn5 side chain extends away from the macrocyclic ring system and hydrogen bonds to the terminal tripeptide, but in pressinoic acid the Asn5 side chain lies over the molecule and forms a strong hydrogen bond to the nitrogen of Tyr2. The absence of pressor activity in pressinoic acid may be a result of both the loss of the COOH-terminal tripeptide and the incorrect orientation of the Asn5 side chain. Whether this class of hormones has pressor or oxytocic activity is determined by the orientation of the Tyr2 side chain, that is, whether it is extended away from or over the ring system, respectively. In pressinoic acid, the Tyr2 side chain is in the expected "pressor conformation," that is, extended away from the ring system, and is stabilized through a hydrophobic interaction with the Phe3 side chain. Thus, the conformation of the pressinoic acid molecule partly explains the activity of vasopressin-like hormones.

Topics: Arginine Vasopressin; Models, Molecular; Molecular Conformation; Vasopressins; X-Ray Diffraction