Effect of acute restraint on hypothalamic pro-vasotocin mRNA expression in flounder, Platichthys flesus.

Arginine vasotocin (AVT) stimulates release of adenocorticotrophin hormone (ACTH) in trout. However, AVT's role in fish hypothalamic-pituitary-interrenal-axis (HPIA) is not fully understood. Here, we examined distribution of AVT and glucocorticoid receptor (GR) in the magnocellular preoptic nucleus (PM) and the AVT/cortisol response to acute restraint in flounder. The GR/AVT distribution in the PM was determined using double immunohistochemistry (IHC). Flounder were confined in nets, immersed in water for 30m, with plasma and tissue samples taken prior to, 3, 24 and 48h post-confinement. Plasma osmolality, Na(+), Cl(-) and cortisol were taken as indicators of HPIA activation. Plasma AVT was measured proVT mRNA expression in the PM was detected using in situ hybridisation (ISH) with a S35 labelled oligoprobe for homologous flounder proVT. Double IHC showed the presence of GR in AVT synthesising neurones of the PM. Plasma Na(+), Cl(-), osmolality and cortisol (1.0+/-0.9 to 183.6+/-3.1mM; p<0.001) increased significantly 3h post-restraint: recovering to control levels after 48h. Plasma AVT levels did not change. However, a concomitant increase in proVT mRNA expression in the magnocellular (PMm) and gigantocellular (PMg) neurones of the PM was observed (11.1+/-1.8 to 55.2+/-9.1% 24h post-restraint; p<0.001) and levels still remained significantly elevated at 48h (p<0.01). This suggests that PMm and PMg AVT neurones are associated with HPIA activation following acute restraint, including potential cortisol negative feedback. The extended elevation of hypothalamus proAVT mRNA expression following a single acute stressor affords a possible mechanism to moderate sensitivity of the HPIA to subsequent challenges.

Arginine vasotocin a key hormone in fish physiology and behaviour: a review with insights from mammalian models.

The arginine vasotocin (AVT) neuroendocrine system clearly provides integrative regulation of many aspects of fish physiology and behaviour, including circadian and seasonal biology, responses to stress, metabolism, reproduction, cardiovascular function, and osmoregulation. These are all considered here providing an important context for the design of experiments and interpretation of results for investigations of specific aspects of AVT function. Salt and water balance is a consistent function from fish to mammals and is examined in more detail. Both AVT and AVP secretion is sensitive to hyperosmotic stimuli and associated cellular dehydration, while hypovolaemia would appear less important. AVT and AVP both mediate renal water conservation, though actions involve different receptors and precise targets in fish (V1) and mammals (V2). The actions of AVT to promote gill NaCl extrusion in fish are conserved in the AVP-induced natriuresis in mammalian kidney to support restoration of plasma osmolality. The AVT/AVP regulatory mechanisms involve both altered neurohypophysial peptide secretion and changes in target-tissue receptor expression/modulation of action. Both mechanisms importantly afford integration with the actions of other related hormone systems.

Altered plasma and pituitary arginine vasotocin and hypothalamic provasotocin expression in flounder (Platichthys flesus) following hypertonic challenge and distribution of vasotocin receptors within the kidney.

Plasma AVT concentration, pituitary AVT content, hypothalamic provasotocin mRNA expression and other osmoregulatory parameters were measured in euryhaline flounder 4, 8, and 24 h after the hypertonic challenge of transfer from fresh water (FW) to seawater (SW). Osmolality and the concentration of major plasma ions, sodium and chloride, were significantly higher in fish transferred to SW by comparison with time matched controls, an effect evident within 4 h. By comparison with time matched controls, pituitary store of AVT was lower while plasma AVT concentration was higher 8 and 24 h after transfer to SW. Higher provasotocin mRNA expression in the hypothalamus was also seen at 4 and 8 h in flounder transferred from FW to SW compared with time matched controls. The lower pituitary store and higher circulating levels imply substantial AVT secretion occurs in the early phase response to this hypertonic challenge. Changes in the regulation of AVT synthesis and secretion appeared quickly following movement to SW, consistent with the rapid osmoregulatory response, including reduced urine production that fish require to accommodate the dehydrative water losses and salt loading on exposure to the new hyperosmotic environment. qPCR measures of whole kidney vasotocin receptor mRNA expression indicated similar levels in SW and FW. Immunohistochemistry for the vasotocin receptor in flounder kidney showed localisation on the afferent and efferent arterioles of the glomerulus and on the capillary bed that extends from the efferent arteriole to the smooth muscle surrounding the collecting duct. Localisation of the vasotocin receptor was comparable in SW and FW fish.

Renal morphology of the euryhaline flounder (Platichthys flesus): distribution of arginine vasotocin receptor.

The current study characterized tubular segmentation of the European flounder nephron and localized the vasotocin receptor expression by immunohistochemistry. Flounder nephron was shown to comprise a prominent renal corpuscle, short neck segment, proximal tubule I, proximal tubule II, collecting tubule, and collecting duct. Using specific antibodies raised against flounder vasotocin receptor, specific V(1) receptor staining was detected within the glomeruli, the endothelial surface of the afferent and efferent arterioles, and the capillaries surrounding the collecting duct system. Immunostaining for the receptor was exclusively vascular and there did not appear to be a tubular component.

Neurohypophysial hormones and renal function in fish and mammals.

The two major basic neurohypophysial peptides, arginine vasopressin (AVP) of mammals and arginine vasotocin (AVT) of all non-mammalian vertebrates, share common structure and major roles in regulating renal function. In this review the complexity of AVP actions within the mammalian kidney is discussed and comparisons are made with the emerging picture of AVT's renal effects in fish. It has become apparent that the antidiuretic action of the neurohypophysial hormones is an ancient phylogenetic phenomenon, although this is based upon reduced glomerular filtration in fish by comparison with predominant tubular effects in mammals. Nonetheless, there appears to be retention of AVP effects upon the functional heterogeneity of nephron populations in mammals. Preliminary evidence for the possible existence of V(2)-type (tubular) neurohypophysial hormone receptors in fish, implies possible AVT actions which parallel those in mammals on tubular ion transport. Further insight from recent mammalian tubule microperfusion studies suggests that in teleost fish both apical (tubular lumen) and basolateral (blood borne) AVT have the potential to modulate renal function, though this remains to be examined.

Plasma concentrations of arginine vasotocin and urotensin II are reduced following transfer of the euryhaline flounder (Platichthys flesus) from seawater to fresh water.

Plasma concentrations and stored levels of the neuroendocrine peptides arginine vasotocin (AVT) and urotensin II (UII) were measured in the euryhaline flounder (Platichthys flesus) following the acute hypo-osmotic challenge of direct seawater (SW) to fresh water (FW) transfer. Hormone measures, plasma osmolality, and ion concentrations and tissue water content were determined 1, 4, 8, 24, 72, and 144 h after transfer. Plasma AVT concentration fell initially following FW transfer but then returned toward pretransfer levels by day 6. Plasma UII concentration decreased while urophysial UII content was increased following hypo-osmotic challenge relative to SW time-matched controls, suggesting down regulation of the UII system during the initial stages after FW transfer. These changes in neuroendocrine activity were associated with a significant fall in plasma osmolality and major plasma ions. Positive correlations were observed between plasma AVT and osmolality and Cl- and Mg2+ concentrations, suggesting functional association of these plasma parameters with AVT action and/or control of AVT secretion. The initial response to hypotonic challenge involves reduced plasma AVT and UII levels consistent with the proposed role for these hormones, supporting flounder osmoregulation in hypertonic media.

Day-night variations in plasma melatonin and arginine vasotocin concentrations in chronically cannulated flounder (Platichthys flesus).

Chronically catheterised, free swimming flounder (Platichthys flesus) have been used in experiments examining the day-night variations in circulating levels of melatonin (Mel) and arginine vasotocin (AVT). Under normal photoperiod (16 h light/8 h dark) serial blood samples taken from individual fish demonstrated a Mel rhythm with daytime levels at 09.00 and 15.00 h (238+/-14 and 179+/-12 fmol x ml(-1), respectively) lower than those at 23.00 h (1920+/-128 fmol x ml(-1)). Maintenance of fish in 24-h light abolished the light/dark Mel rhythm and circulating levels were comparable to those measured during the day in fish under normal photoperiod illumination. In fish maintained under 24 h dark, although a daily rhythm was still apparent, at the time when it would be normally dark, plasma Mel concentration was reduced and at times when it would be normally light, levels were higher than in fish maintained under normal light/dark illumination. Plasma AVT concentrations were higher in fish during the day (4.4+/-0.8 fmol x ml(-1)) than those at night (1.5+/-0.4 fmol x ml(-1)), the opposite to that seen with Mel. During acute study infusion of AVT resulted in reduced levels of plasma Mel, although this did not achieve statistical significance. Infusion of Mel did not alter circulating AVT concentration.

Do circulating plasma AVT and/or cortisol levels control pulsatile urea excretion in the gulf toadfish (Opsanus beta)?

Previous work has shown that pulsatile urea excretion at the gills of the gulf toadfish is due to periodic activation of a facilitated diffusion transport system with molecular and pharmacological similarity to the UT-A transport system of the mammalian kidney. In mammals, AVP and glucocorticoids are two important endocrine regulators of this system. The present study focused on the potential role of circulating AVT (the teleost homologue of AVP) and cortisol levels as possible triggers for urea pulses. Long-term (34-84 h) monitoring of plasma levels by repetitive sampling at 2-h intervals from chronic cannulae in individual toadfish demonstrated that circulating AVT concentrations are low (10(-12)-10(-11) M), and show no relationship to the occurrence of natural urea pulses. In contrast, plasma cortisol levels decline greatly prior to natural pulses and rise rapidly thereafter. AVT injections into the caudal artery or ventral aorta elicited pulse events, but these were extremely small (1-10%) relative to natural pulses, and occurred only at unphysiological dose levels (10(-9) M in the plasma). AVP was a partial agonist, but isotocin, insulin-like growth factor-1, and atrial natriuretic peptide were without effect at the same concentration. Artificially raising plasma cortisol levels by cortisol injection tended to reduce responsiveness to AVT. Pharmacological reduction of plasma cortisol levels by metyrapone injection elicited small pulses similar to those caused by AVT. Following such pulse events, AVT was ineffective in inducing pulses. We conclude that decreases in circulating cortisol play an important permissive role in urea pulsing, but that circulating AVT levels are not involved.

Cloning and characterization of an arginine vasotocin receptor from the euryhaline flounder Platichthys flesus.

A sequence coding for an arginine vasotocin (AVT) receptor has been identified by the screening of a hepatic cDNA library from the teleost Platichthys flesus. The 2701-bp receptor sequence is predicted to yield a 384-amino acid peptide, analysis of which indicates a seven-transmembrane spanning sequence typical of G-protein-coupled receptors with the N terminus on the outer surface of the cell membrane. Sequence analysis showed this sequence to have a high homology with the Catostomus commersoni AVT receptor (76%) and mammalian vasopressin V(1)-type receptor (62%), but only 55% homology with the C. commersoni isotocin receptor. A two-electrode voltage clamp was used to characterize the receptor expressed in Xenopus laevis oocytes. AVT induced an inward current which was dose dependent over the range 16.7 fmol to 5 pmol; isotocin was without effect over the same dose range. The mammalian vasopressin V(1)-type receptor agonist ([Phe(2), Orn(8)] oxytocin)() induced an inward current but was less potent than AVT, whereas the mammalian vasopressin V(2)-type receptor agonist ([Deamino(1), Val(4), D-Arg(8)] AVP) was without effect. Injection of oocytes with heparin or BAPTA suppressed the response to AVT, indicating receptor linkage to the phospholipase C-phosphatidylinositol pathway. Northern analysis demonstrated the presence of this AVT receptor mRNA in the brain, kidney, and gill of flounder.

Cloning of pro-vasotocin and pro-isotocin cDNAs from the flounder Platichthys flesus; levels of hypothalamic mRNA following acute osmotic challenge.

Sequences coding for pro-vasotocin and pro-isotocin have been identified by screening a flounder (Platichthys flesus) hypothalamic cDNA library. The 1074-bp proVT and 727-bp proIT sequences contain a signal peptide and hormone, connected to a neurophysin by a Gly-Lys-Arg sequence. Both sequences also have an elongated carboxyl-terminal with a leucine-rich core resembling copeptin but lacking the amino terminal Arg residue. The levels of pro-vasotocin and pro-isotocin mRNA in the hypothalamus were measured concomitantly with pituitary AVT content and plasma AVT concentration following acute transfer of fish between freshwater and seawater. Three days after transfer from seawater to freshwater there appears to be a down regulation of the AVT hormone system with a fall in hypothalamic pro-vasotocin mRNA levels, an increase in pituitary AVT content, and a fall in plasma levels, but these changes did not achieve statistical significance compared to controls. No change in the AVT system was detected 3 days following the transfer of fish from freshwater to seawater. Hypothalamic isotocin mRNA levels did not change following hypo- or hyperosmotic challenge.

Changes in plasma arginine vasotocin (AVT) concentration and dorsal aortic blood pressure following AVT injection in the teleost Platichthys flesus.

Dorsal aortic blood pressure and plasma arginine vasotocin (AVT) concentrations have been assessed in free swimming, chronically cannulated flounder following AVT injection. Intraarterial AVT at doses of 4.76 x 10(-12) mol.kg-1 and greater caused a biphasic change in blood pressure (an initial fall followed by a sustained pressor response). Doses above 4.76 x 10(-12) mol.kg-1 were associated with plasma AVT concentrations (20 min after injection) 2-3 orders of magnitude greater than the physiological range and must be considered pharmacological. Injection of the lowest pressor AVT dose (4.76 x 10(-12) mol.kg-1), 20 min after injection, increased plasma AVT concentrations to 23.4 +/- 6.1 fmol x ml-1. This increase is close to plasma AVT concentrations recently reported in untreated fish; however, in the initial period after injection plasma levels were calculated to be considerably higher than the physiological range. These results confirm that AVT is pressor in flounder but suggest that the pressor response may occur only at circulating AVT levels above the normal physiological range. The biphasic response to AVT and the differing responses to mammalian V1 and V2 type receptor agonists in the current work suggests that AVT may contribute to regional blood flow distribution in teleosts rather than blood pressure regulation.

Effect of acute manipulation of blood volume and osmolality on plasma [AVT] in seawater flounder.

Chronically cannulated seawater (SW)-adapted flounder (Platichthys flesus) were used unanesthetized and unrestrained in an experimental series that acutely manipulated blood volume and plasma osmolality to determine their influence on plasma arginine vasotocin (AVT) concentrations. Immunoreactive AVT was measured using a radioimmunoassay validated for flounder and other teleosts. After hemorrhage-induced hypovolemia or hypervolemia produced by saline infusion, no major changes in plasma AVT concentrations were detected. Raising plasma osmolality by intraperitoneal injection of 1 M NaCl compared with control 150 mM NaCl-injected fish (329.4 +/- 3.4 vs. 320.4 +/- 3.0 mosmol/kgH2O, P < 0.05) produced an increase in plasma AVT concentration (6.7 +/- 1.2 vs. 4.2 +/- 0.2 fmol/ml, P < 0.05). In a separate study, plasma composition in a large number of uncannulated SW-adapted flounder was determined. This demonstrated a positive linear relationship between the natural variation in plasma AVT concentrations and plasma osmolality and Na+ and Cl- concentrations observed between fish. These data indicate that AVT secretion in SW-adapted flounder is closely related and perhaps directly sensitive to changes in plasma tonicity.

A radioimmunoassay for the determination of arginine vasotocin (AVT): plasma and pituitary concentrations in fresh- and seawater fish.

A specific radioimmunoassay was developed and characterized for the measurement of arginine vasotocin (AVT) in teleost fish. Specificity of the antibody for AVT was demonstrated by parallelism of a series of AVT standards with serially diluted pituitary and plasma extracts. Crossreactivity of the antibody with the other teleost neurohypophysial peptide, isotocin, was less than 1% and the sensitivity of the assay was 0.24 fmol/assay tube. AVT was extracted from plasma by reverse-phase liquid chromatography [efficiency of 87.6 +/- 9.3% (n = 5)] and demonstrated as an effective procedure for plasma volumes ranging from 0.4 to 1.2 ml. Plasma AVT concentrations measured in a range of euryhaline and stenohaline teleost fish were between 10(-12) and 2 x 10(-11) M (1-20 pg/ml). There were no consistent differences between plasma AVT levels in euryhaline fish (flounder, trout, and eel) adapted to fresh water (FW) and sea water (SW). In flounder, pituitary AVT levels in FW- and SW-adapted fish were also similar.

Arginine vasotocin and fish osmoregulation.

Pituitary arginine vasotocin (AVT) secretion is sensitive to the osmotic challenge associated with transfer of euryhaline teleosts between sea water (SW) and fresh water (FW). Pituitary AVT content in FW-adapted flounders greatly exceeds that in SW-adapted fish. Plasma AVT concentrations are in the range 10(-12)-10(-11) M (1-100 pg/ml). In euryhaline species, like the eel, flounder and trout, there were no consistent, marked differences in plasma AVT concentrations between FW- and SW-adapted fish. In SW- but not FW-adapted flounders plasma AVT and sodium concentrations are correlated. During the initial period of acclimation from FW to SW eels, show a transitory rise in plasma AVT concentration which is associated with a transitory increase in plasma Angiotensin II. In view of the range of plasma AVT concentration observed in SW- and FW-adapted fish, it is evident that of the described dose-dependent effects of AVT on urine production, only the antidiuretic responses are likely to be of physiological significance. In addition to the presence of a V1-type vascular receptor for AVT, the nephron also possesses a V2-type receptor, coupled to adenylate cyclase. In the gill tissue AVT receptors are also present, but in this tissue receptor occupancy leads to inhibition of cAMP production rather than the stimulation observed in renal tissue. The functional significance of the gill AVT receptor remains to be established.