Biological half-life and organ distribution of [3H]8-arginine vasopressin following administration of vasopressin receptor antagonist OPC-31260

The effects of the antidiuretic (V(2)) non-peptide receptor antagonist OPC-31260 on the plasma vasopressin level and the biological half-life and organ distribution of radiochemically pure, biologically active [(3)H]8-arginine vasopressin [spec. act.: 15.9 mCi/mmol (588 GBq/mmol)] were studied in Wistar rats. The plasma vasopressin level increased significantly throughout the whole experimental period (24 h). There was no change in the fast phase of the curves of total radioactivity disappearance from the plasma after the administration of [(3)H]arginine vasopressin (control: 1.51+/-0.17 min, OPC-31260-treated: 1.42+/-0.12 min, n=10). The fast phase of the disappearance curves of intact [(3)H]arginine vasopressin did not change either following the administration of OPC-31260 in a dose of 30 mg/kg p.o. (control: 1.06+/-0.19 min, OPC-31260-treated: 1.00+/-0.15 min, n=6). The slow phase of the biological half-life, which is characteristic for the examined compound, proved to be significantly longer (total radioactivity control: 9.29+/-0.61 min, OPC-31260-treated: 12.33+/-0.42 min, P<0.05, n=10; [(3)H]arginine vasopressin radioactivity: control: 5.96+/-0.58 min, OPC-31260-treated: 8.90+/-0.37 min, P<0.05, n=6). In the control rats, the radioactivity was accumulated to the greatest extent in the neurohypophysis, adenohypophysis and kidney. Following OPC-31260 administration, significantly more radioactive compounds accumulated in the kidney (control: 0.30+/-0.052 total radioactivity %/100 mg organ weight, OPC-31260-treated: 0.50+/-0.133 total radioactivity %/100 mg organ weight, P<0.05, n=10) and neurohypophysis (control: 0.37+/-0.053 total radioactivity %/100 mg organ weight, OPC-31260-treated: 0.52+/-0.076 total radioactivity %/100 mg organ weight, P<0.05, n=10). Our results permit the conclusion that the antidiuretic antagonist OPC-31260 not only blocks the V(2) receptors, but also increases the biological half-life of vasopressin. The longer biological half-life of vasopressin following OPC-31260 administration may play a role in the elevation of the plasma vasopressin level.     

Reversal of copper-induced inhibition of vasopressin responsiveness by reducing agents

Copper inhibits the hydro-osmotic response to vasopressin in the urinary bladder of Bufo marinus at a site proximal to cyclic AMP production. This effect is not reversed by washing in Cu2+ -free Ringer's solution but is overcome by serosal addition of reducing agents, suggesting that vasopressin responsiveness in this tissue is modulated by either the redox potential or the sulfhydryl content of the serosal membrane.     

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.     

Effects of methoxsalen from Poncirus trifoliata on acetylcholinesterase and trimethyltin-induced learning and memory impairment

Previously, we identified methoxsalen (8-methoxy-2',3',6,7-furocoumarin) as the bioactive compound probably responsible for acetylcholinesterase (AchE) inhibition achieved by feeding crude extract of Poncirus trifoliate. To confirm the activity of methoxsalen, Institute of Cancer Research (ICR) mice were fed a control or a methoxsalen-supplemented diet for 4 weeks, and then learning and memory enhancing effects with respect to trimethyltin (TMT)-induced neurotoxicity were evaluated. The brain tissues of ICR mice were dissected after completion of the behavioral tests for biochemical analysis. Methoxsalen effectively reversed TMT-induced memory impairment on both Y-maze and passive avoidance tests. Brain AchE activity was inhibited by the oral consumption of all concentrations of methoxsalen. Moreover, the level of oxidative stress was significantly ameliorated in the groups on methodsalen containing diets. This is the first in vivo study conducted with methoxsalen in the field of AD research, and it indicates that further investigation of methoxsalen is warranted.     

Effects of peripherally injected vasopressin and des-glycinamide vasopressin on the extinction of a spatial learning task in rats

An elevated eight-arm radial maze was employed to study the effects of neuropeptide administration on the spatial learning abilities of food-deprived rats. Following 18 days of reinforced training, each animal was briefly exposed to the maze with no food available in any of the eight food-cups. Immediately after this preliminary trial, animals were injected with a single subcutaneous dose of either saline, arginine vasopressin (AVP: 1.0 or 5.0 micrograms/kg), or an AVP analog with only weak endocrinological activity, des-gly-arginine vasopressin (DG-AVP: 1.0, 5.0 or 10.0 micrograms/kg). Additional extinction trials were conducted at 2, 4, 6 and 8 h post-injection. These tests consisted of individually placing an animal on the empty maze and recording the number of arms chosen in a 5-min period. In this situation, animals learn that food is no longer present in the maze and, consequently, extinguish responding. Vasopressin potentiated this radial maze extinction behavior while DG-AVP produced behavioral results directionally opposite to those predicted by a memory facilitation hypothesis. In a subsequent experiment, vasopressin had no effects on unconditioned locomotor activity measured 2 and 4 h post-injection. These results suggest that: vasopressin improved the learning that occurred during extinction of conditioned appetitive behaviors, these vasopressin effects on conditioned behavior were independent of any unconditioned, sedative or non-specific actions of the peptide, and peripheral endocrinological responses may be necessary to demonstrate memory-enhancing effects following peripherally administered AVP.     

Reversal of angiotensin converting enzyme (ACE) inhibitor induced vasodilatation by vasopressin in hindquarters of rat

The effect of exogenously administered vasopressin was observed on captopril induced vasodilatation in hindquarters of anaesthetised rats. Drops of perfusate were counted for 6 min and mean of the outflow was expressed as drops per min (dpm). In the control group (n = 6) the rate of flow was 9.5 +/- 1.04 dpm which increased to 12.33 +/- 1.36 dpm following captopril (200 micrograms/ml) infusion. In test group (n = 6) pretreated with vasopressin (4 I.U./kg 1 hr before) the rate of flow was 9.16 +/- 0.98 dpm which was reduced to 5.5 +/- 1.04 dpm following infusion with captopril. It is concluded that vasopressin reverses the vasodilatory effect of captopril.     

Light microscopic autoradiographic visualization of [3H]-arginine vasopressin binding sites in rat brain

Specific [³H]-arginine vasopressin ([³H]-AVP) binding sites were identified in the rat brain by light microscopic autoradiography. Discrete intrahypothalamic nuclei were densely labelled by [³H]-AVP. High specific binding was observed in the paraventricular and supraoptic nuclei. These binding sites may represent specific receptors for AVP, postulated to exist in the mammalian central nervous system.     

Reversal of fungitoxicity of 8-quinolinols and their copper(II) bischelates. II. Reversal of the action of 8-quinolinol by DL-alpha-lipoic acid

The effect of amino acids and derivatives, Krebs cycle acids and related compounds, fatty acids, and vitamins and related compounds on the toxicity of 8-quinolinol and bis(8-quinolinolato)copper(II) to Aspergillus oryzae (ATCC 1011) was studied. Only aliphatic thiol-containing compounds (cysteine, glutathione, dithioerythritol, and dithiothreitol) and DL-alpha-lipoic acid protected against 8-quinolinol but not its copper(II) bischelate. It is suggested that 8-quinolinol inhibits lipoic acid biosynthesis, and the mode of fungitoxicity of 8-quinolinol is different from that of bis(8-quinolinolato)copper(II).     

The composition of crystalline complexes of neurophysin-M with [8-arginine]-vasopressin and oxytocin

Neurophysin-M, a methionine-containing protein that is the major constituent of neurophysin, has been crystallized as complexes with [8-arginine]-vasopressin. Three moles of vasopressin alone or 2 moles of vasopressin together with 1 mole of oxytocin are bound/mole of protein. An amorphous complex of the protein with oxytocin alone contains 2 moles of the hormone/mole of protein. Deamino-[8-arginine]-vasopressin, a highly active basic analogue of vasopressin, is not bound by neurophysin. The primary amino group of both vasopressin and oxytocin is necessary for binding with neurophysin.     

1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin: a new photoaffinity label for hydroosmotic hormone receptors. Characterization of the ligand and irreversible stimulation of hydroosmotic water flow in toad bladder by photoaffinity labeling

The photoreactive analog of vasopressin [1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin [( Asu1,6, Phe (p-N3)3]AVP) has been synthesized. This analog retains a high binding affinity for the vasopressin receptor in plasma membranes from bovine kidney inner medulla (apparent dissociation constant, KD = 8.5 X 10(-9) M). [Asu1,6, Phe (p-N3)3] AVP was found to be biologically active in triggering the characteristic increase in toad bladder permeability to water. Photolysis of the analog in the presence of the toad bladder results in a hydroosmotic response which persists, in spite of repeated washings, for more than 18 h. The irreversible stimulation of the bladder is inhibited when photolysis is carried out in the presence of vasopressin. Our findings indicate that with photoactivation [Asu1,6, Phe(p-N3)3]AVP binds covalently to hormonal receptors and forms an active hormone-receptor complex. This analog, therefore, is a suitable tool for studies of hydroosmotic receptor function and for receptor isolation.     

Metabolic effects of vasopressin infusion in the starved rat. Reversal of ketonaemia.

The effects of vasopressin on the metabolism of starved rats were investigated by using a constant-infusion regimen (50 pmol/kg body wt. per min, after an initial loading dose of 150 pmol/kg body wt.). 2. Blood ketone bodies decreased by 50% in 10 min, and this was accompanied by a 60% decrease in the plasma non-esterified fatty acids. 3. Blood glucose increased by 0.9 mM within 5 min and decreased to control values over the 40 min infusion. Small increases in lactate and pyruvate also occurred. 4. Plasma insulin was not increased by vasopressin infusion. 5. The net decrease in blood ketone bodies caused by vasopressin was similar when somatostatin was infused simultaneously (1 nmol/kg body wt. per min). 6. Hepatic ketone bodies were significantly decreased by vasopressin, as was the 3-hydroxybutyrate/acetoacetate ratio. A small increase in the hepatic concentration of several glycolytic intermediates also occurred. 7. Vasopressin did not decrease the ketonaemia produced by infusions of octanoate or long-chain triacylglycerol in rats that had been pre-treated with the anti-lipolytic agent 3,5-dimethylpyrazole. 8. In comparison with vasopressin, the infusion of adrenaline or glucose had much smaller effects in decreasing the ketonaemia of starvation, despite the 4-fold increase in plasma insulin, at 10 min, with the glucose infusion. 9. The primary metabolic effect of vasopressin in the starved rat appears to be that of decreased supply of non-esterified fatty acid to the liver. It is suggested that vasopressin has a direct anti-lipolytic effect in adipose tissue.     

Reversal of diabetic nephropathy by a ketogenic diet

Intensive insulin therapy and protein restriction delay the development of nephropathy in a variety of conditions, but few interventions are known to reverse nephropathy. Having recently observed that the ketone 3-beta-hydroxybutyric acid (3-OHB) reduces molecular responses to glucose, we hypothesized that a ketogenic diet, which produces prolonged elevation of 3-OHB, may reverse pathological processes caused by diabetes. To address this hypothesis, we assessed if prolonged maintenance on a ketogenic diet would reverse nephropathy produced by diabetes. In mouse models for both Type 1 (Akita) and Type 2 (db/db) diabetes, diabetic nephropathy (as indicated by albuminuria) was allowed to develop, then half the mice were switched to a ketogenic diet. After 8 weeks on the diet, mice were sacrificed to assess gene expression and histology. Diabetic nephropathy, as indicated by albumin/creatinine ratios as well as expression of stress-induced genes, was completely reversed by 2 months maintenance on a ketogenic diet. However, histological evidence of nephropathy was only partly reversed. These studies demonstrate that diabetic nephropathy can be reversed by a relatively simple dietary intervention. Whether reduced glucose metabolism mediates the protective effects of the ketogenic diet remains to be determined.     

Reversal of learning deficits in a Tsc2+/- mouse model of tuberous sclerosis

Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) are commonly affected with specific neuropsychological problems, including long-term and working memory deficits. Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene (Tsc2(+/-) mice) show deficits in learning and memory. Cognitive deficits in Tsc2(+/-) mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved. We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder.