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.     

Interaction of atrial natriuretic factor and osmoregulatory hormones in the Pekin duck

This study examined the influence of plasma atrial natriuretic factor (ANF) on the osmoregulatory hormones arginine vasotocin (AVT), angiotensin II (AII), aldosterone, and corticosterone in the blood of conscious Pekin ducks. Synthetic chicken ANF was iv infused at a nonhypotensive, natriuretic, and diuretic dose of 100 ng.kg-1.min-1 in normally hydrated ducks and birds in which the circulating levels of AVT, AII, aldosterone, and corticosterone were elevated by 24-hr dehydration. In normally hydrated animals the effect of ANF was limited to a reduction (P less than 0.05) in the basal concentration of aldosterone; in dehydrated birds both AII and aldosterone levels were reduced (P less than 0.05). ANF infusion was consistently without effect on plasma AVT or corticosterone concentrations. The iv infusions of AII with or without simultaneous infusion of ANF showed that ANF inhibited (P less than 0.05) the aldosterone response to AII. Autoradiographic and membrane binding techniques indicated that there were both AII and ANF receptors in the subcapsular zone of the duck adrenal gland and that ANF had no affinity for the AII binding sites. The results demonstrate that ANF is capable of modulating the duck angiotensin-aldosterone system and suggest a role for this peptide in avian salt and fluid homeostasis.     

Arginine Vasotocin Gene Expression and Secretion during Osmotic Stimulation and Hemorrhagic Hypotension in Hens

In chickens, hyperosmolality stimulates the secretion of vasotocin (AVT) and up-regulates hypothalamic AVT gene expression. Hemorrhage, on the other hand, has not been considered an effective stimulus for AVT release in this species. The effects of acute osmotic stress and prolonged hemorrhagic hypotension on AVT gene expression and secretion were studied in White Leghorn hens. Conscious hens were osmotically stimulated by administering a single ip injection of 3MNaCl (5 ml/kg). Urethane-anesthetized hens were bled to a mean arterial pressure of 80–90 mm Hg and the pressure was maintained within this range by additional bleeding. A total of about 30% of the estimated blood volume was removed. Both experiments were terminated after 1 hr of stimulation. Plasma AVT levels in the hyperosmotic and hypovolemic hens were 4- and 2-fold higher, respectively, compared to controls. Hypothalamic AVT mRNA levels, detected by Northern blot analysis, were 2.5- and 2-fold higher in the osmotically stimulated and hypotensive groups, respectively, compared to control groups. As determined byin situhybridization, both osmotic stimulation and hypovolemia resulted in an increase in the number of AVT mRNA-containing neurons in the supraoptic and paraventricular nuclei. Our results indicate that, under the conditions used, hypotension and hyperosmolality are equally effective in stimulating AVT gene expression and secretion of AVT.     

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.     

Radioimmunoassayable avt and avp in adult mammalian brain tissue: comparison of normal and brattleboro rats.

Arginine vasopressin (AVP) and vasotocin (AVT) were measured by radioimmunoassay in extracts of cerebral cortex, cerebellum, brain stem, pineal, hypothalamus, and neurohypophysis from normal Long-Evans rats. AVP was present in expected concentrations in pituitary and hypothalamus and there was no evidence of its accumulation elsewhere. AVT was not detectable in these tissues (within the limits imposed on our assay by the presence of excessive amounts of AVP) but was easily detectable in pineal tissue with a concentration of 22.4 plus or minus 6.6 muU/gland. Extracts of neurohypophysis and pineal glands from homozygous Brattleboro rats (rats with hereditary hypothalamic diabetes insipidus) revealed the total absence of AVP and AVT. We conclude that the Brattleboro rat is incapabel of synthesizing biologically active neurohypophyseal peptides which contain arginine in position 8.     

Metabolic mapping of functional activity in rat brain and pituitary after water deprivation

Glucose utilization in rat brain and pituitary was measured in control and water-deprived rats by autoradiographic assessment of the metabolic trapping of radioactivity from [1-14C]glucose. Two days of water deprivation resulted in significant increases in hematocrit, plasma osmolality and vasopressin levels, indicating a functional activation of magnocellular vasopressin neurons. The uptake and retention of radioactivity from [1-14C]glucose in the dehydrated rats, compared to controls, was 103% greater in the magnocellular portion of the paraventricular nucleus and 74% greater in the supraoptic nucleus. Water deprivation also resulted in significant increases in glucose utilization (30-40%) in the lateral and anterior hypothalamic areas, somatosensory cortex and cingulate cortex. No change in glucose utilization after 2 days of water deprivation was apparent in the parvocellular paraventricular nucleus, periventricular nucleus of the hypothalamus, corpus callosum, organum vasculosum of the lamina terminalis (OVLT) or the subfornical organ (SFO). In the pituitary, glucose utilization was increased in the neural lobe but was unchanged in the anterior and intermediate lobes after water deprivation. Under the conditions of the present study, no increase in metabolic activity was apparent in 2 brain regions thought to be possible sources of osmoreception, the OVLT and SFO. These results do not support, but do not exclude, functional involvement of the OVLT and SFO in regulating the activity of magnocellular neurons of the paraventricular nucleus and supraoptic nucleus during chronic water deprivation.     

Seasonal changes in hypothalamic-pituitary-adrenal axis sensitivity in free-living house sparrows (Passer domesticus)

Recent evidence indicates that house sparrows (Passer domesticus) seasonally regulate corticosterone responses to capture, handling, and restraint. Responses during molt and in the fall are lower than responses in the winter and while breeding. This study tested whether changes in either adrenal tissue responsiveness to adrenocorticotropin (ACTH) or pituitary responsiveness to corticotropin releasing factor (CRF) or arginine vasotocin (AVT) could provide the mechanism regulating these seasonal changes. House sparrows were captured at two sites (Massachusetts and New Mexico, USA) and during the above four seasons and injected with exogenous ACTH, CRF, and AVT. ACTH stimulated further corticosterone release in all birds except Massachusetts birds in the winter, suggesting that reduced adrenal sensitivity to ACTH cannot explain reduced corticosterone release during fall and molt. However, exogenous ACTH was less effective during molt at both sites, implying that adrenal sensitivity does change. Pituitary sensitivity also changed seasonally, but these pituitary changes did not match the seasonal changes in corticosterone release. CRF and AVT only succeeded in elevating corticosterone in the spring in Massachusetts birds and in the winter in New Mexico birds, whereas CRF alone also stimulated corticosterone release in New Mexico birds in the fall. Taken together, these data indicate that house sparrows can alter the amount of corticosterone released from adrenal tissue, the amount of ACTH released from the pituitary, and the amount of CRF and AVT released from the hypothalamus, but that none of these changes correlate with seasonal changes in corticosterone release. Consequently, seasonal modulation of corticosterone release in house sparrows appear to result from a complicated mix of adrenal, pituitary, and hypothalamic changes that also vary seasonally.     

Plasma aldosterone, angiotensin II, and arginine vasotocin concentrations in the toad, Bufo marinus, following osmotic treatments

To clarify the physiological roles of the renin-angiotensin-aldosterone system (RAAS) and arginine vasotocin (AVT) on body fluid regulation in amphibians, we measured plasma concentrations of aldosterone (ALDO), angiotensin II (ANG II), and AVT after various osmotic challenges in the marine toad, Bufo marinus (Bufonidae). Hematocrit value (Ht) as an indicator of plasma volume, plasma osmolality and concentrations of plasma components (Na(+), Cl(-), K(+), and urea) were also measured. The toads were maintained under various osmotic treatments for 7 days. In dehydrated toads, plasma concentrations of ALDO, ANG II, AVT, and all plasma components measured were increased. In toads maintained in 300 mosmol/kg H(2)O NaCl solution, plasma osmolality, Na(+), Cl(-), urea, and plasma AVT concentrations were significantly increased, and Ht and plasma concentrations of ALDO and ANG II were significantly decreased. In toads maintained in tap water, plasma osmolality, and concentrations of Na(+) and ALDO were significantly decreased. We also estimated total body water (TBW), plasma volume (PV) using Evans Blue dye and Ht in the toads under various osmotic treatments. In dehydrated toads, TBW and PV were significantly decreased and Ht was significantly increased in comparison with those of control. In toads maintained in 300 mosmol/kg H(2)O NaCl solution, TBW and PV were significantly increased and Ht was significantly decreased in comparison with those of control. There was a significant negative correlation between Ht and PV or TBW. These results show that dehydration, which induces hypovolemic and hyperosmotic conditions, stimulates increases of plasma ALDO, ANG II, and AVT concentrations, while hypervolemic treatment induces decreases of plasma ALDO and ANG II concentrations. There were significant correlations between plasma osmolality and AVT concentration, between Ht and concentrations of RAAS hormones, and between plasma concentrations of ALDO and ANG II. These results suggest that volumetric and osmometric systems regulated by RAAS hormones and AVT are present in B. marinus.     

Reduction of exogenous vasopressin RNA poly(A) tail length increases its effectiveness in transiently correcting diabetes insipidus in the Brattleboro rat.

Magnocellular hypothalamic neurons in Brattleboro rats can accumulate, transport, and translate exogenous [Arg8]vasopressin (AVP) mRNA after injection in the hypothalamo-hypophysial tract in amounts sufficient to reverse transiently the animals' characteristic diabetes insipidus. In the present study, different preparations of hypothalamic RNA extracted from normal rats or synthetic AVP RNA were injected into the lateral hypothalamus of Brattleboro rats. Poly(A)- RNA and poly(A)+ RNA from which tails were removed by RNase H digestion were much more effective than poly(A)+ RNA in expressing AVP in the magnocellular hypothalamic neurons and in raising urine osmolarity. Synthetic AVP RNA lacking a poly(A) tail also produced a very potent dose-dependent diabetes insipidus reversal. Our results suggest that a short or absent poly(A) tail may facilitate the accumulation, transport, or expression of exogenous AVP mRNA by magnocellular neurons.     

Effect of hypernatraemia and the neurohypophysial peptide, arginine vasotocin (AVT) on behavioural thermoregulation in the agamid lizard, Ctenophorus ornatus

Hypernatraemia induced by chronic injections of sodium chloride provokes thermal depression in the agamid lizard, Ctenophorus (formerly Amphibolurus) ornatus, with a fall of two degrees Celsius in the mean body temperature selected behaviourally in a photo-thermal gradient. The placement of an electrolytic lesion in the base of the hypothalamus, designed to eliminate secretion of the neuropeptide arginine vasotocin (AVT), did not affect the lizards' thermoregulatory behaviour and their Preferred Body Temperature (PBT) was not significantly different from that of unoperated controls. Saline loading, however, did not induce thermal depression in these tract-operated individuals and their PBT was significantly higher than that of salt-loaded intact individuals. When AVT was injected into operated, salt-loaded, animals, however, thermal depression was observed, supporting the hypothesis that thermal depression brought about by hypernatraemia is mediated through the action of AVT. AVT similarly significantly depressed the PBT of injected intact individuals by 3.2 degrees C when compared with hydrated controls. Immunostaining for AVT confirmed that the lesions placed in the region of the median eminence virtually eliminated AVT located in the neurohypophysial tract, and the pars nervosa. This is the first report of an effect of this peptide on behavioural thermoregulation in a lizard.     

Diel variations in arginine vasotocin content of goldfish brain and pituitary: effects of photoperiod and pinealectomy

Arginine vasotocin (AVT) content was determined in the telencephalon, the hypothalamus, and the pituitary gland of sham-operated and pinealectomized goldfish subjected to 20 degrees and 12/12LD or 24DD photoperiods in winter or spring. The tissues collected at 1000 or 2200 hr were homogenized and extracted in acetic acid. Hypothalamic and telencephalic AVT content, determined by radioimmunoassay, fluctuated throughout the light-dark cycle; AVT content was higher at 2200 than at 1000 hr under the 12/12LD photoperiod. No fluctuations were detected in pituitary AVT content. Telencephalic AVT did not fluctuate under constant darkness, and hypothalamic AVT increased and pituitary AVT decreased compared to the AVT content detected in the 12/12LD groups. No significant effects of pinealectomy or season on AVT levels in the telencephalon, hypothalamus, and the pituitary were evident. The results indicate that AVT content within the preoptico-neurohypophyseal system of the goldfish fluctuate within 24-hr periods and that photoperiod has an effect on the pattern of these fluctuations. The photoperiodic influences do not seem to be mediated by the pineal organ.     

cDNA cloning of chicken orexin receptor and tissue distribution: sexually dimorphic expression in chicken gonads

Orexin-A and -B are known to stimulate food intake in mammals. However, the critical roles of orexins in birds are not fully understood, since orexins have no stimulatory effect on food intake in the chicken. To understand the physiological role(s) of orexins in birds, we have cloned chicken orexin receptor (cOXR) cDNA by RT-PCR, and analysed the tIssue distribution of OXR mRNA in the chicken. The cOXR cDNA is 1869 bp long and encodes 501 amino acids. The cloned cDNA for cOXR corresponds to the type 2 OXR in mammals, and shows approximately 80% similarity to those of mammals at the amino acid level. Expression analysis by RNase protection assay revealed OXR mRNA was distributed widely in brain regions, and expression in the cerebrum, hypothalamus and optic tectum were abundant. In peripheral tIssues, OXR mRNA was expressed in the pituitary gland, adrenal gland and testis, but no mRNA expression was observed in other tIssues examined. Furthermore, we found that the amount of cOXR mRNA was different between testis and ovary, while prepro-orexin mRNA is equally expressed in the gonads of both sexes in the chicken. These data indicate that the orexins have neuroendocrine actions in chickens, which are mediated through hypothalamic receptors as has been observed in mammals. In addition, orexin may have specific role(s) in the regulation of gonadal function in which sex-dependent mechanisms could be involved.     

Volumetric control of arginine vasotocin and mesotocin release in the frog (Rana ridibunda)

Adult male frogs (Rana ridibunda) were subjected to several volumetric and osmometric stimuli and the influence on circulating concentrations of arginine vasotocin (AVT) and mesotocin was studied by the use of highly specific radioimmunoassays. During progressive blood withdrawal (haemorrhage group) urine flow decreased to zero, whereas no change occurred in the plasma and urine osmolality. Control levels of 34.3 +/- 7.3 pmol AVT/1 gradually increased up to 638.3 +/- 179.1 pmol/1 (P less than 0.001) after a blood loss of up to 50-60% of the blood volume. Plasma mesotocin concentrations also increased from 42.4 +/- 9.2 to 70.8 +/- 12.0 pmol/1 (n = 7). Hypervolaemia, produced by the repeated intravenous injection of isotonic Ringer solution, increased the urine flow and osmolality compared to controls but had no influence on the plasma levels of AVT and mesotocin. Hypernatraemia without volume change profoundly increased the urine osmolality but the urine flow was not affected; the plasma concentrations of AVT and mesotocin remained at the control level. Finally, during a 1-h immobilization stress a pronounced antidiuresis occurred in the presence of a constant plasma and urine osmolality and control plasma levels of AVT and mesotocin. It is concluded that the release of AVT and, to a smaller extent, of mesotocin is under volumetric control.     

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.