Synthesis and biological activity of a biotinylated vasopressin analog

To study vasopressin receptor-mediated endocytosis using electronmicroscopy methods and to develop avidin affinity columns for receptor purification, we synthesized and tested the biological properties of a biotinylated vasopressin (VP) analog [1-(2-mercapto) propionic acid] 8-[lysine-N6-biotin] VP (B-MLVP). B-MLVP was prepared by coupling biotin to the epsilon amine of the lysine residue in [1-(2-mercapto) propionic acid] 8-(lysine) VP (MLVP). The structure of HPLC purified B-MLVP was confirmed by fast atom bombardment mass spectrometry. B-MLVP effectively competed for arginine vasopressin (AVP) binding sites in canine renal plasma membranes on the surface of LLC-PK1 kidney cells. Dissociation constants of 15 nM and 202 nM were calculated from the results of competition binding assays conducted with membranes and cells, respectively. B-MLVP stimulated adenylate cyclase activity and elevated cellular 3',5',cyclic-AMP (cAMP) content in a manner similar to AVP, indicating it is an agonist of VP action in renal tissue. These observations indicate that B-MLVP is an agonist of VP action and may be used to study renal VP receptors by employing avidin coupled to various reporter groups.     

Functional architecture of vasopressin/oxytocin receptors

Three-dimensional models of G protein-coupled receptors (GPCR) have been defined using most experimental data available and protein modeling techniques. The endogenous ligand binding sites have been qualitatively described and putative receptor activation mechanisms have been proposed. The model has been recently refined to take into account recent crystallographic data. Most experimental results published are in excellent qualitative agreement with the initial model. We have undertaken to study more systematically by site directed mutagenesis the vasopressin/oxytocin receptor binding domain as a prototype of neuropeptide receptors. The experimental results are in very good agreement with the models. The residues responsible for the neuropeptide binding have been identified and confirm the predicted localization of the neuromediator in the transmembrane domain of the receptors. The side chain of the 8th residue of vasopressin interacts with a non-conserved receptor residue located in the first extracellular loop. As predicted from the model, this interaction is completely responsible for the selectivity of the ligand-receptor interaction. Finally, aromatic residues which allow the modulation of the efficacy of agonists have been identified.     

Antagonistic properties of tetra-substituted analog of vasopressin with selective antidiuretic effects]

Biological properties of a novel vasopressin analogue were investigated. It was found that this analogue has no vasopressor and oxytocic activities but it exhibits a selective antidiuretic effect which is weaker than that of adiuretin (DDAVP). Novel analogue inhibits vasopressor, oxytocic and antidiuretic effects caused by arginine--vasopressin. The usefulness of novel compound as a pharmacological tool--vasopressin antagonist is suggested.     

Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes

The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response.Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E₂ (PGE₂) by human granulocytes, release of PGE₂ and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.     

No improvement in ethanol-induced memory deficits after administration of a vasopressin analog

Acute ingestion of ethanol impairs memory, an effect which might be related to ethanol-induced inhibition of vasopressin release. This was studied using tests of memory and cognitive function in 26 normal subjects before and after ethanol ingestion. Equal numbers of subjects received randomly, by double-blind intranasal administration, placebo or 1-desamino-8-D-arginine vasopressin prior to ethanol ingestion. Administration of the vasopressin analog did not reverse the ethanol-induced deficits in memory and cognitive function.     

Sentence memory affected by vasopressin analog (DDAVP) in cross-over experiment

DDAVP has been shown to facilitate memory, especially retrieval, in humans. Healthy young male adult subjects received DDAVP (60 micrograms) in a cross-over design with a one-week interval between sessions. Results indicated that DDAVP improved immediate memory during the first but not the second testing session, particularly for low-verbal subjects. Treatment with DDAVP also facilitated delayed (one-week) recall in the opposite group, a cross-over interaction that suggests a retrieval locus for the DDAVP effect. Furthermore, since DDAVP improved immediate memory more for low-verbal subjects and delayed memory more for high-verbal subjects, it appears that individual difference factors will be important in understanding the effects of vasopressin on memory.     

Effect of a vasopressin analog on the chemoreactive properties of sensorimotor cortex neurons

Subcutaneous injection of 10 micrograms desglycilargininvasopressin (DG-AVP) does not alter the mean frequency of background unit activity of sensorimotor cortical neurons. However, the pattern of impulse activity is essentially changed. At the same time the reactions of sensorimotor cortical neurons to microiontophoretic administration of acetylcholine and noradrenaline experience definite changes. It is suggested that the DG-AVP-induced changes in chemoreactive properties of neurons underlie the effect of this peptide on the learning and memory.     

CRAMP analog having potent antibiotic activity without hemolytic activity

CRAMP-18 is an 18-residue functional region, corresponding to residues 16-33 of a mouse-derived antibiotic peptide CRAMP. To develop novel antibiotic peptides possessing strong antibiotic activity against bacterial, fungal and tumor cells without hemolytic activity, three analogs of CRAMP-18 were synthesized containing either Leu- or Lys-substitution. Leu-substitution ([L(1, 8)]-CRAMP-18) in the hydrophobic helix face of CRAMP-18 induced a dramatic increase in antibiotic activity without a significant increase in hemolytic activity. Lys-substitution ([K(2, 13)]-CRAMP-18 or [K(9, 16)]-CRAMP-18) in the hydrophilic helix face produced a smaller response. Therefore, [L(1, 8)]-CRAMP-18 may be an attractive candidate for developing novel peptide antibiotics.     

A chlorodiazirine analog of pentamidine with anti-trypanosomal activity

A chlorodiazirine derivative of pentamidine was synthesized and tested for anti-trypanosomal activity using EATRO stock 164 trypanosomes in cell culture. Anti-trypanosomal activity was measured as a decrease in [3H]hypoxanthine incorporation by the organisms. The derivative, 3,3'-[1,5-pentanediylbis(oxy-4,1-phenylene)]bis(3-chloro-3H-diazir ine), at a treatment level of 0.1 microM inhibited isotope incorporation by 40-50% compared to nontreated controls. At this concentration, pentamidine inhibited incorporation only 10-15%. The derivative is a nonionic molecule with much different solubility properties than the parent compound and should readily cross the blood-brain barrier.     

Morphine-like activity of the enkephalin analog Tyr-D-Ala-Gly-Phe-NH2

The analgetic activity of the tetrapeptide enkephalin analog, its influence on the interneuronal transmission of excitation in various areas of the central nervous system and on opiate receptors of vas deferens were studied. The tetrapeptide was found to have a marked analgetic effect during intravenous injection to mice but to be less active than morphine. The tetrapeptide as well as morphine inhibited the impulse summation in rabbits and both spontaneous and bradykinin-induced neuronal activity in the rat sensory motor cortex. The tetrapeptide inhibited the contractions of isolated vas deferens in mice. The opiate antagonist naloxone eliminated both analgetic effect of the tetrapeptide and its inhibitory effect on the impulse summation, neuronal activity and contractions of vas deferens.     

Stimulation of testicular ornithine decarboxylase activity by arginine vasopressin

Intratesticular injection with arginine vasopressin caused stimulation of ornithine decarboxylase activity in the testes of immature rats. The increase in ornithine decarboxylase activity in response to arginine vasopressin was dose and time dependent. Maximal stimulation of ornithine decarboxylase activity occurred at 2 h after injection with 0.1 micrograms of arginine vasopressin. It was observed that stimulation of ornithine decarboxylase activity occurred in seminiferous tubules and in Leydig cells of the testis in response to arginine vasopressin.     

Dynamics of changes in the excitability of Helix lucorum neurons in response to an analog of vasopressin

Application of desglycine-arginine-vasopressin to spontaneously nonactive command neurones of the snail's defensive reflex in the process of low-frequency (2-4 min intervals) intracellular stimulation led under certain conditions to an increase of excitability that was expressed in the shortening of latency of action potential generation, increase of the number of action potentials in response to a stimulus of fixed value, increase of membrane resistance, lowering of critical level of membrane depolarization and amplifying of pacemaker activity. However in spite of unidirectional changes of all the recorded parameters, the increases of values, opposite to the latency, did not correlate with the increases of membrane resistance and correlated well with the changes of depolarization critical level. If desglycine arginine-vasopressin was added to the medium during worsening of the neurones' excitability was probably caused by the influence of desglycine-arginine-vasopressin on the membrane active properties.     

Improvement of the selective perception and learning with the analog of C-terminal fragment of vasopressin in rats

New analogue of the AVP(6-9), the D-MPRG was administered intranasally in different doses. The administration improved the process of conditioning. The most efficient dose turned out to be 0/01 mcg/kg. Tetrapeptide administered 1 hour before each learning session and immediately after it accelerated the avoidance conditioning. The peptide effect was more obvious in conditioning with a negative reinforcement. The data obtained suggest that the D-MPRG mostly affects the perception of experimental environment as well as improves the consolidation of memory traces. The peptide was also shown to delay extinction of previously learned skills.     

Immunomodulatory activity of an anti-HSV-1 synthetic stigmastane analog

Many viral infections are associated with the development of immunopathologies and autoimmune diseases, which are of difficult treatment and for which no vaccines are yet available. Obtaining compounds that conjugate both antiviral and immunomodulatory activities in the same molecule would be very useful for the prevention and/or treatment of these immunopathologies.The compound (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) displays anti-Herpes simplex virus type 1 activity in vitro and reduces the incidence of herpetic stromal keratitis (HSK) in mice, a chronic inflammatory syndrome induced by ocular HSV-1 infection.In the present study, compound 1 showed opposite immunomodulatory properties in vitro. It induced the release of pro-inflammatory cytokines in HSV-1-infected epithelial cells of ocular origin, and significantly reduced the production of these cytokines in LPS-activated macrophages. RNA microarrays revealed various overexpressed and repressed genes in compound 1 treated infected epithelial cells and activated macrophages, many of which are associated with innate immune responses and inflammatory processes. These immunomodulatory properties of compound 1, together with its previously reported antiviral activity, make it a potential drug for the treatment of HSK and many other immunopathologies of viral and non-viral origin.     

Neonatal administrations of a vasopressin analog (DDAVP) and hypertonic saline enhance learning behavior in rats

Groups of newborn Wistar rats received daily 1-desamino-8-D-arginine-vasopressin (DDAVP), oxytocin (OXT), hypertonic saline or normal saline for 14 days from day 1 to day 14 of life. One or three months later they were trained in a maze for brightness discrimination (BD). A group of untreated adult male rats received posttrial DDAVP or normal saline for brightness discrimination. Subsequently all the retentions of BD were tested after one month. We found that the neonatal treatments with both DDAVP and hypertonic saline facilitated acquisition and subsequent maintenance of brightness discrimination in immature and mature rats, and also that posttreatment with DDAVP enhanced retention of BD in adult rats. Oxytocin and normal saline had no effect on these parameters. The results are interpreted as showing that endogenous AVP and its synthetic analog enhance the development and adult function of central neural substrates involved in learning behaviors.