[Arg8]-Vasotocin-Gly-Lys

Membrane fusion under mildly acidic pH occurs naturally during viral infection in cells and has been exploited in the field of nanoparticle-mediated drug delivery to circumvent endosomal entrapment of the cargo. Herein, we aimed to confer virus-like fusogenic activity to HDL in the form of a ca. 10-nm disc comprising a discoidal lipid bilayer and two copies of a lipid-binding protein at the edge. A series of HDL mutants were prepared with a mixture of three lipids and a cell-penetrating peptide (TAT, penetratin, or Arg8) fused to the protein. In a lipid-mixing assay with anionic liposomes at pH 5.5, one HDL mutant showed the fusogenic activity higher than known fusogenic liposomes. In live mammalian cells, this HDL mutant showed high plasma membrane-binding activity in the presence of serum independent of pH. In the absence of serum, a mildly acidic pH dependency for binding to the plasma membrane and the subsequent lipid mixing between them was observed for this mutant. We propose a novel strategy to develop HDL-based drug carriers by taking advantage of the HDL lipid/protein composite structure.

The excitatory effect of [Arg8]-vasopressin and its potential contribution to the circadian cycle of electrical activity in the suprachiasmatic nucleus of the rat was investigated using extracellular recordings from hypothalamic slices from virgin female rats. The majority of neurons tested for their responses to vasopressin and [Arg8]-vasotocin displayed coincident, dose-dependent excitation by both peptides, although the relative efficacy varied between neurons, with some showing a highly preferential excitation by vasotocin. Perifusion with the vasopressin receptor antagonist d(CH2)5[Tyr(OEt)2,Val4,Cit8]-vasopressin was able to block the majority of responses to vasopressin or vasotocin (20/25), and similar excitation could be induced by the selective agonist [Phe2,Orn8]-vasotocin, indicating a mainly V1 receptor-mediated effect. Few neurons (3/27; 11%) responded to the oxytocin-specific agonist, [Thr4,Gly7]-oxytocin, suggesting a low occurrence of oxytocin receptors. In addition to blocking the action of exogenous vasopressin, the V1 antagonist caused a reversible suppression of spontaneous basal activity in 7/25 cases, consistent with the presence of an endogenous excitatory vasopressin tone. In agreement with previous reports, the activity of suprachiasmatic nucleus neurons showed a significant correlation between spontaneous activity and the light-dark cycle, with activity decreasing during the subjective dark phase. When neurons were divided on the basis of their response to vasopressin and/or vasotocin, the peptide-sensitive neurons continued to show a strong correlation (r = 0.513, P < 0.01) while the insensitive neurons showed no correlation (r = 0.136, P > 0.05). These data confirm the presence of V1 type receptors in the suprachiasmatic nucleus and also indicate a small number of neurons possessing additional classes of receptor selective for either oxytocin or vasotocin. Contrary to previous reports, they also demonstrate that endogenous vasopressin tonically excites suprachiasmatic nucleus neurons. The fact that vasopressin-sensitive (but not vasopressin-insensitive) neurons show a level of basal activity correlated with time, suggests that this tone may contribute to the circadian cycle of electrical activity in the suprachiasmatic nucleus.

[Arg8]Vasotocin (AVT) is considered to be the most primitive known vertebrate neurohypophyseal peptide of the vasopressin/oxytocin hormone family and may thus be ancestral to all the other vertebrate peptide hormones. The molecular evolution of the corresponding receptor family has now been studied by cloning an AVT receptor, consisting of 435 amino acid residues, from the teleost fish, the white sucker Catostomus commersoni. Frog oocytes injected with the AVT receptor-encoding cRNA respond to the application of AVT, but not to its structural and functional counterpart isotocin, by an induction of membrane chloride currents indicating the coupling of the AVT receptor to the inositol phosphate/calcium pathway. The pharmacological properties of the expressed AVT receptor show that it represents, or is closely related to, an ancestral nonapeptide receptor: oxytocin, aspargtocin, mesotocin, and vasopressin activated the receptor, but other members of the vasopressin/oxytocin family tested showed little or no potency; antagonists of the mammalian vasopressin V1 and oxytocin receptors blocked the AVT response. Comparison of AVT receptor sequences spanning transmembrane domains two to five, deduced by cloning cDNAs from the Pacific salmon Oncorhynchus kisutch, the cave-dwelling fish Astyanax fasciatus, and the anuran Xenopus laevis, with those of their mammalian counterparts emphasizes amino acid residues that are involved in hormone binding. The presence of a 5.0-kb transcript in various teleost tissues (pituitary, liver, gills, swim bladder, and lateral line) points to a physiological role for the fish AVT receptor in metabolic, osmoregulatory, and sensory processes.