The effects of repeated physical stress or fasting on catccholamine storage and release in the rainbow trout, Oncorhynchus mykiss

Rainbow trout, Oncorhynchus mykiss, were subjected to either physical stress (twice daily chasing to exhaustion for 5 days) or a period of 2 months of fasting. Following these treatments, the levels of catecholamines, adrenaline and noradrenaline, stored within the kidney and posterior cardinal vein (PCV) were determined. The ability of the catecholamine-storing chromaffin cells to release catecholamines in response to cholinergic stimulation was measured using an in situ saline-perfused PCV preparation. In the physically stressed fish, the concentration (μg catecholamine g^?1 tissue) of noradrenaline within the anterior and middle thirds of the kidney increased; the concentration of adrenaline was unchanged in all tissues. The content (μg) of noradrenaline or adrenaline, within the various tissues, was similar in both groups of fish with the exception of a higher noradrenaline content in the middle third of the kidney in the physically stressed fish. The total catecholamine content (μg catecholamine) of these tissues (kidney+PCV) was unaffected by physical stress. With the exception of a lower concentration of adrenaline in the middle third of the kidney, the concentrations of catecholamines were unaffected by fasting. There was a trend towards a greater content (μg) of noradrenaline within all of the tissue regions of the fed fish, however, a significant difference was only observed in the anterior third of the kidney. The content of adrenaline in the fed fish was greater in all regions of the kidney as well as the middle third of the PCV. The total catecholamine content (kidney + PCV) was lower in the fasted fish owing to significantly lower PCV and kidney masses. Prolonged physical stress caused a decrease in the responsiveness of the chromaffin cells to the cholinoceptor agonist carbachol (10^?8 to 10^?4mol). The ED₅₀ (the dose of carbachol required to elicit a half maximal response) for catecholamine release was 0·96 ± 10^?6mol carbachol in the physically stressed fish and 0·84 ± 10^?7 in the control fish. Fasting did not alter the pattern of catecholamine release. The ED₅₀ values were 0·96 ± 10^?7 and 1·24 ± 10^?7 mol for fasted and fed fish, respectively. Thus, a physical stress affected both catecholamine storage and release whereas fasting affected only storage and not the release process.     

Cholinoceptor-mediated control of catecholamine release from chromaffin cells in the American eel,Anguilla rostrata

The cholinergic agonist-induced secretion of catecholamines from chromaffin cells in the American eel, Anguilla rostrata, was assessed using a salineperfused posterior cardinal vein preparation. Direct membrane depolarization with 60 mmol·l^-1 K⁺ caused a significant release of catecholamines (adrenaline + noradrenaline) into the perfusate which was unaffected by pre-treatment with the ganglion blocker, hexamethonium (final concentration = 10^-3 mol · l^-1). The nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, evoked catecholamine release in response to several doses exceeding 10^-7 mol; at 10^-5 mol the response was abolished by pre-treatment with the ganglion blocker, hexamethonium (final concentration = 10^-3 mol · l^-1). The muscarinic receptor agonist, pilocarpine, did not elicit catecholamine release in response to any of the doses administered (10^-8–10^-4 mol). A single injection of the mixed nicotinic/muscarinic cholinoceptor agonist, carbachol (10^-5 mol), caused the release of catecholamines which was abolished by pre-treatment with hexamethonium but which was unaffected by pre-treatment with the muscarinic receptor antagonist atropine (final concentration = 10^-5 mol · l^-1). The results of this study indicate that the process of cholinergic agonist-induced catecholamine secretion from the chromaffin cells in the American eel is mediated exclusively by activation of nicotinic receptors with no involvement of the muscarinic receptor.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium iodide - MS222 ethylaminobenzoate - HPLC high-performance liquid chromatography - PCV posterior cardinal vein - SEM standard error of the mean     

Immunohistochemical localization of bioactive peptides and amines associated with the chromaffin tissue of five species of fish

Biogenic peptides and amines associated with the chromaffin tissue in Atlantic cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla), spiny dogfish (Squalus acanthias) and Atlantic hagfish (Myxine glutinosa) were identified utilizing immunohistochemical techniques. Within the posterior cardinal vein (PCV) in cod, trout and eel, a subpopulation of chromaffin cells displayed immunoreactivity to tyrosine hydroxylase (TH) and dopamine--hydroxylase (DH) but not to phenylethanolamine-N-methyltransferase (PNMT). TH-like immunorectivity was observed within cells in hagfish hearts. Nerve fibres displaying vasoactive intestinal peptide (VIP) immunoreactivity and pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity innervated cod, trout and ell chromaffin cells. In eel, neuropeptide Y (NPY)-like and peptide YY (PYY)-like immunoreactivity was located within cells in the PCV, including chromaffin cells. Serotonin-like immunoreactivity was observed within eel and cod chromaffin cells and in hagfish hearts. In the dogfish axillary bodies, nerves displaying TH-like, VIP-like, PACAP-like, substance P-like and galanin-like immunoreactivity were observed. These results are compared with those of other vertebrates, and potential roles for these substances in the control of catecholamine release are suggested.     

Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that endogenously produced hydrogen sulfide (H(2)S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water Po(2) = 35 mmHg), plasma H(2)S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H(2)S in vitro when incubated with l-cysteine. In both tissues, the production of H(2)S was eliminated by adding the cystathionine beta-synthase inhibitor, aminooxyacetate. Cystathionine beta-synthase and cystathionine gamma-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H(2)S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x 10(-6) M) or depolarizing levels of KCl (1 x 10(-2) M) caused secretion of catecholamines without altering H(2)S output, suggesting that neuronal excitation is required for H(2)S release. Addition of H(2)S (at concentrations exceeding 5 x 10(-7) M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca(2+)-free perfusate was used. These data, together with the finding that H(2)S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H(2)S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca(2+)-mediated exocytosis.     

The control of teleost ACTH cells

Summary Adrenocorticotropic hormone was spontaneously released from goldfish pituitaries in vitro, most of the activity (79%) coming from the anterior half of the gland. It is distinct from melanophore stimulating hormone, 86% of which was released from the posterior half of the gland.Cortisol treatment of whole animals increased the adrenocorticotropic hormone content of the pituitaries put had no effect on the thyrotropic hormone content. In organ cultured pituitaries cortisol (5 /ml) inhibited the release of adrenocorticotropic hormone from the glands by 65% while hypothalamic homogenates increased the release by 32%. The spontaneous activity of adrenocorticotropic hormone cells may be controlled by inhibition by cortisol and stimulation by the hypothalamus possibly acting via a releasing factor.The technical assistance of Miss P. Robins is gratefully acknowledged.     

The influence of chronic anaemia on catecholamine secretion in the rainbow trout (Oncorhynchus mykiss)

The effect of long-term (7 day) anaemia on catecholamine release was examined in rainbow trout (Oncorhynchus mykiss) in vivo during acute exposure to hypoxia and in situ using a perfused post-cardinal vein preparation. The first goal was to distinguish among reductions in blood O₂ partial pressure, O₂ concentration and haemoglobin percentage saturation as potential stimuli for, or correlates of, catecholamine secretion during hypoxia. The second goal was to elucidate the role of these factors in promoting enhanced chromaffin cell responsiveness in trout subjected to chronic hypoxia (Montpetit and Perry 1998). Anaemic fish (haematocrit lowered from 28.4±2.4% to 11.9±1.6%) displayed a marked reduction in haemoglobin-O₂ binding affinity [P ₅₀ (P _aO₂ at 50% Hb-O₂ saturation) was increased from 14.7 mm Hg to 24.3 mm Hg]. Upon exposure to hypoxia, the anaemic fish released catecholamines into their circulation at higher values of arterial O₂ partial pressure (∼52 mm Hg versus ∼18 mm Hg) and haemoglobin O₂ saturation (<70% versus <55%) than did control fish. In addition, anaemic fish achieved significantly greater circulating levels of total catecholamines (noradrenaline plus adrenaline) during acute hypoxia (294.8±67.3 versus 107.0±35.6 nmol l⁻¹). These results do not support the view that catecholamine release is triggered by a reduction in haemoglobin O₂ saturation or arterial PO₂, per se. Nor are they consistent with the idea that catecholamine release occurs at a threshold value of arterial PO₂ corresponding to a critical reduction in blood O₂ concentration. The effects of the non-selective cholinergic receptor carbachol on catecholamine secretion from chromaffin tissue were assessed using perfused posterior cardinal vein preparations derived from control or anaemic fish. For adrenaline secretion, there was no statistically significant change in the ED₅₀ (dose eliciting 50% response). For noradrenaline secretion however, preparations originating from anaemic fish displayed an enhanced responsiveness to carbachol as indicated by a significant 4.5-fold reduction in the carbachol ED₅₀ value from 2.53 × 10⁻⁶ mol kg⁻¹ to 5.67 × 10⁻⁷ mol kg⁻¹. These results demonstrate that anaemia-induced hypoxaemia, in the absence of any lowering of PO₂, is able to modulate the responsiveness of chromaffin cells to cholinergic stimulation. Accepted: 21 April 1999     

The effect of exogenous catecholamines on the ventilatory and cardiac responses of normoxic and hyperoxic rainbow trout,Oncorhynchus mykiss

Ventilation frequency, opercular pressure amplitude, heart rate, dorsal aortic pressure, arterial pH, arterial O₂ tension, and plasma catecholamine levels were recorded in rainbow trout, Oncorhynchus mykiss, during normoxia (19.7 kPa, 148 mmHg) or hyperoxia (51.2 kPa, 384 mmHg) after injection of various concentrations of catecholamines. In normoxic fish, adrenaline injection resulted in a depression of arterial O₂ tension, hypoventilation due to a drop in ventilation frequency, and a drop in heart rate, while dorsal aortic pressure increased. Noradrenaline depressed ventilation frequency, but opercular pressure amplitude increased to a far greater extent, and dorsal aortic pressure increased. During hyperoxia, adrenaline injection lowered ventilation frequency, opercular amplitude and heart rate, but dorsal aortic pressure increased. The stimulatory effects of noradrenaline on ventilation were abolished during hyperoxia, but the cardiac responses were similar to those seen during normoxia. These results indicate that catecholamines can modify the ventilatory output from the respiratory centre, and modification of ventilation frequency can occur independently of opercular pressure amplitude.Abbreviations f _g ventilation frequency - HPLC high performance liquid chromatography - P _op opercular pressure amplitude - f _h heart rate - P _DA dorsal aortic pressure - pH_a arterial pH - P _aO₂ arterial oxygen tension - PO₂ oxygen tension     

Bacterial lipopolysaccharide (LPS) and interleukin 1 (IL-1) exert multiple physiological effects in the tilapia Oreochromis mossambicus (Teleostei)

To gain insight in immuno-endocrine communication in teleosts the physiological effects of interleukin 1 and bacterial lipopolysaccharide in teleosts were investigated. Tilapia (Oreochromis mossambicus) were treated with murine interleukin 1 and E. coli lipopolysaccharide in vivo, and lipopolysaccharide was administered to pituitary lobes and head kidneys in vitro. The integument of the fish appeared to be a sensitive target for the preparations tested, since proliferation of chloride cells and of epidermal mucous cells was observed as well as an increase in epidermal thickness. These effects may relate to an acute phase-like reaction caused by the treatments. Lipopolysaccharide administration furthermore resulted in an increase in plasma free fatty acids levels. Lipopolysaccharide, but not interleukin 1, stimulated the interrenal axis of the fish, as judged by the increase in cortisol production measured in superfusion of head kidneys. In addition to these in vivo effects, lipopolysaccharide also displayed several effects in vitro. Pituitary adrenocorticotropic hormone, as well as -melanocyte stimulating hormone, release was inhibited, and the head kidney responsiveness to adrenocorticotropic hormone was inhibited after pretreatment of the tissue with the E. coli product. This latter effect coincided with the release of an unidentified -melanocyte stimulating hormone immunoreactive fraction by the head kidneys which could be stimulated by lipopolysaccharide. The data strongly support the notion that the immune system is involved in adaptive regulations in teleosts, and that immuno-endocrine interactions are phylogenetically old mechanisms.Abbreviations ACTH adrenocorticotropic hormone - AUC area under the curve - FFA free fatty acids - HPLC high-performance liquid chromatography - IL-1 interleukin 1 - LPS lipopolysaccharide/endotoxin - -MSH alpha melanocyte stimulating hormone - NIL neurointermediate lobe - POMC proopiomelanocortin - RIA radioimmunoassay - RPD rostral pars distalis     

Compliance and smooth muscle reactivity of rainbow trout (Oncorhynchus mykiss) vessels in vitro

Systemic veins have a profound influence on cardiac output in mammals. Venoregulatory mechanisms have not been adequately studied in fish and their existence has been questioned. In the present study, two characteristics of vascular mechanics, compliance and agonist-induced tension development, were investigated in rainbow trout vessels in vitro. Rapid compliance in the anterior cardinal vein and efferent branchial artery was calculated from step-wise changes in the volume-pressure curve of isolated vessel segments. Agonist-induced tension development was examined in four veins; anterior and posterior cardinals, intestinal and duct of Cuvier. Venous compliance was not altered in response to epinephrine, norepinephrine or angiotensin II, while efferent branchial artery compliance was decreased by 10^-6 mol·l^-1 epinephrine and norepinephrine but not angiotensin II. The ratios of venous to arterial compliance in vessels from two rainbow trout strains were similar (21:1 and 32:1) and consistent with the ratio reported for mammalian viens (24:1). Trout veins contracted in response to agonists in both an, agonist- and vesselspecific manner. The greatest tension per vessel wet weight was produced in anterior cardinal vein. The response pattern of anterior cardinal vein and duct of Cuvier were similar; acetylcholine, arginine vasotocin, epinephrine and norepinephrine, and the thromboxane A₂ agonist, U-44,069, produced approximately identical contractions, whereas angiotensin II was virtually ineffective. Conversely, angiotensin II was more potent than epinephrine in posterior cardinal vein. In cumulative dose-response experiments, epinephrine was equipotent in anterior cardinal vein and duct of Cuvier, whereas the latter was less sensitive to acetylcholine. Both atrial natriuretic peptide and sodium nitroprusside relaxed precontracted veins. This is the first study to determine compliance in fish vessels and the contractile nature of different rainbow trout veins. These findings suggest that venous tone and therefore cardiac output in fish may be regulated by neural or humoral mechanisms.Abbreviations ACH acetylcholine - ACV anterior cardinal vein - ANG II salmon asn¹-val⁵ angiotensin II - ANP rat atrial natriuretic peptide - AVT arginine vasotocin - DNR Department of Natural Resources - DOC duct of Cuvier - EBA efferent branchial artery - EC₅ threshold dose producing 5% maximal contraction - EC₅₀ dose producing 50% maximal contraction - EPI epinephrine - HI K⁺ 80 mmol·l^-1 - KCl IV, intestinal vein - NEPI norepinephrine - PBS phosphate buffered saline - PCV posterior cardinal vein - SNP sodium nitroprusside - U-44,069 thromboxane A₂ agonist     

Chromaffin cells and interrenal tissue in the head kidney of the grouper,Epinephilus tauvina (Teleostei,Serranidae): a morphological (optical and ultrastructural) study

This work analyses the distribution, histology and ultrastructure of chromaffin cells (CCs) and interrenal tissue (It) in the head kidney of Epinephilus tauvina. Histological examination revealed that chromaffin cells are found in small groups under the endothelium of the posterior cardinal vein (PCV) and are mostly closely associated with the interrenal tissue. Ultrastructure examination confirmed the existence of two main chromaffin cell types, distinguished by different types of secretory granules. The first type was characterized by the presence of vesicles with round, strongly electron dense core granules, which were eccentrically located. Such cells were interpreted as being noradrenaline cells. Meanwhile, cells with vesicles that were completely electron lucent or that contained small less dense eccentric granules were identified as adrenaline cells. Nerve endings were invaginated into the chromaffin cells through synaptic junctions. Interrenal tissue consisted of nests, cords, or strands of cells in contact with the posterior cardinal vein (PCV) and interposed with haematopoietic tissue. Ultrastructure analysis revealed only one interrenal cell type, which contained abundant smooth endoplasmic reticulum (sER) and numerous mitochondria with tubulo‐vesicular cristae, characteristics of steroid‐producing cells. The interrenal tissue cells have different cytological aspects that can be linked to a steroidogenic cell cycle allowing a periodical renewal of organelles.     

Intra-adrenal interactions in fish: catecholamine stimulated cortisol release in sea bass (Dicentrarchus labrax L.)

The effect of the catecholamines, adrenaline and noradrenaline, on sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system technique. Increasing concentrations of catecholamines (10(-6), 10(-8) and 10(-10) M) stimulated cortisol production in a dose-dependent manner, in sea bass only. The increase in cortisol production stimulated by adrenaline (10(-6) M) and noradrenaline (10(-6) M) was inhibited by sotalol (2 x 10(-5) M), but not by prazosin suggesting that catecholamines stimulate cortisol release through the beta-receptor subtype. To evaluate catecholamine-induced signal transduction in head kidney cells, measurements of cAMP production and [H3]myo-inositol incorporation were determined in head kidney cell suspensions. Adrenaline and noradrenaline (10(-6) M) increased cAMP production, but had no effect on total inositol phosphate accumulation. These results indicate that catecholamines released from the chromaffin cells within the interrenal tissue may act as a paracrine factor to stimulate interrenal steroidogenesis in the sea bass.     

Substrate mobilization and hormonal changes in rainbow trout (Oncorhynchus mykiss,L.) and common carp (Cyprinus carpio,L.) during deep hypoxia and subsequent recovery

Common carp (at 20°C) and rainbow trout (at 15°C) were fitted with an indwelling cannula in the dorsal aorta. The fish were exposed to a controlled decline of waterpO₂ followed by 90 min deep hypoxia at 0.3 kPa (carp) or 4.8 kPa (trout). Thereafter, normoxic recovery was monitored in both species for 48 h. At regular intervals blood samples were analysed for glucose, lactate, free fatty acids, adrenaline, noradrenaline and cortisol. The oxygen restriction was maximal in both species and resulted in a significant increase of plasma lactate levels. In carp, adrenaline, noradrenaline and cortisol levels increased to 2, 50, and 753 ng·ml^-1 respectively during anoxia, whereas in trout these hormones increased to 12, 8 and 735 ng·ml^-1 respectively during hypoxia. In hypoxic trout, the plasma levels of glucose (3 mol·l^-1) were increased modestly whereas levels of free fatty acids (0.25 mmol·l^-1) were decreased to 0.15 mmol·l^-1. In carp, however, a marked and prolonged hyperglycaemia (from 5 to 10 mmol·l^-1) and a significant continuous depression of plasma levels of free fatty acids (from 0.4 to 0.2 mmol·l^-1) were observed indicating a difference in metabolic organization. It is suggested that hyperglycaemia is likely to be the result of hepatic glycogenolysis, stimulated by circulating catecholamines and a stimulation of gluconeogenesis by cortisol during recovery. The mechanism for the decline of plasma levels of free fatty acids is most probably a reduction of lipolytic activity, which appears to be an adaptation to hypoxia.     

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca²⁺]_i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca²⁺]_i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K⁺ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.     

An investigation of the role of circulating catecholamines in the control of ventilation during acute moderate hypoxia in rainbow trout (Oncorhynchus mykiss)

Summary Gill ventilation volume ( w), arterial blood oxygen tension (PaO₂) and arterial blood oxygen content (CaO₂) of rainbow trout (Oncorhyncus mykiss) were monitored during normoxia [waterPO₂ (PwO₂) 155 Torr], hypoxia (PwO₂=72±5.8 Torr), or hyperoxia (PwO₂=643±32 Torr). Fish hyperventilated during acute (30 min) hypoxia and hypoventilagted during acute hyperoxia. Plasma catecholamine levels were unchanged after 30 min of hypoxia or hyperoxia. In addition, selective adrenoceptor blockade with either propranolol (-adrenoceptor antagonist) or phentolamine (-adrenoceptor antagonist) did not modify the hyperventilatory response during hypoxia. These results indicate that circulating catecholamines are not involved in the control of ventilation in moderately hypoxic rainbow trout. In the summer, intra-arterial infusion of catecholamine in normoxic trout caused transient (adrenaline) or persistent (noradrenaline) hypoventilation. These observations also do not support a role for catecholamines in the stimulation of ventilation.During hypoxia,PwO₂,PaO₂ andCaO₂ were depressed whereas during hyperoxia, onlyPwO₂ andPaO₂ was elevated significantly. Thus, it is suggested that the hypoventilatory response to hyperoxia is mediated by a direct effect of elevatedPwO₂/PaO₂, whereas the hyperventilatory response to hypoxia is mediated by changes inPwO₂/PaO₂, and/orCaO₂.     

Blood Concentrations of Adrenaline in Dogs after Intravenous Administration of 5-Hydroxytryptamine

THE release of catecholamines from the adrenal medulla of the cat by close intra-arterial injection of 5-hydroxytryptamine (5-HT) has been demonstrated by Reid¹. We have shown a similar release of catecholamines following intravenous injections of 5-HT in the dog, identified the catecholamine as adrenaline and demonstrated an enhancement of the release by treatment of the dog with hexamethonium.     

ACTH, alpha-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.     

Effects of chronic dietary salt loading on the renin angiotensin and adrenergic systems of rainbow trout (Oncorhynchus mykiss)

Previous studies have demonstrated that chronic dietary salt loading causes hypertension and a decreased sensitivity of the systemic vasculature to α-adrenergic stimulation and other hypertensive stimuli (e.g. hypercapnia) in rainbow trout (Oncorhynchus mykiss). This reduced sensitivity to hypertensive stimuli is consistent with a possible blunting of homeostatic responses normally aimed at raising blood pressure. To test this idea, we examined the consequences of long-term salt feeding and the associated hypertension on the interactive capacities of the renin angiotensin system (RAS) and adrenergic systems to elevate blood pressure in trout. Secretion of catecholamines in response to a range of doses of homologous ANG II in vivo and in situ (using a perfused posterior cardinal vein preparation) was reduced in the salt-fed fish. The reduced sensitivity to ANG II could not be explained by alterations in stored catecholamine (adrenaline or noradrenaline) levels or the general responsiveness of the chromaffin cells to depolarizing stimuli (60 mmol/l KCl). Despite the decreased responsiveness of the chromaffin cells to ANG II, plasma catecholamines were increased to a greater extent in the salt-fed fish during acute hypoxia (a condition that activates the RAS). Interestingly, the pressor effects of ANG II in vivo were actually heightened in the salt-fed fish. The increased pressor response to exogenous ANG II was likely attributable to its direct interaction with vascular ANG II receptors because the effect persisted even after blockade of α-adrenergic receptors. Treating fish with the vascular smooth muscle relaxant papaverine caused similar reductions in blood pressure and increases in plasma ANG II levels regardless of diet. Similarly, inhibition of angiotensin converting enzyme with lisinopril reduced blood pressure equally in control and salt-fed fish. These results indicate that, while long-term dietary salt loading blunts the response of trout chromaffin cells to ANG II, the RAS itself appears to be unaffected. Indeed, the capacity of ANG II to elevate blood pressure is not compromised nor do fish exhibit a reduced capacity to mount an acute humoral adrenergic stress response during acute hypoxia.     

Effect of acute and chronic epinephrine administration on clearance and metabolism of [3H]-epinephrine in trout (Oncorhynchus mykiss)

Several studies have measured the rate of catecholamine clearance, metabolism, and tissue accumulation in fish. However, no information is available on the effect of repeated stress or high circulating catecholamine levels on catecholamine clearance and metabolism. We measured the clearance and metabolism of [³H]-epinephrine (approximately 0.1 g·kg^-1) in SW-acclimated rainbow trout subjected to acute (five injections in 1 day) and chronic (4 days; five injections per day) administration of 4.0 g·kg^-1 epinephrine or saline. In addition, a saturation experiment, where 4.0 g·kg^-1 of unlabelled epinephrine was injected concurrently with [³H]-epinephrine, investigated whether catecholamine clearance and metabolism are affected by high circulatin levels. Neither the rate constants for catecholamine clearance, nor the post-injection proportions of unmetabolised [³H]-epinephrine, deaminated [³H]-epinephrine and O-methylated [³H]-epinephrine were affected by the acute or chronic injection protocols. The concurrent injection of [³H]-epinephrine and 4.0 g·kg^-1 of unlabelled epinephrine resulted in an elevated postinjection ³H:¹⁴C ratio, but increased proportions of O-methylated [³H]-epinephrine and reduced proportions of unmetabolised [³H]-epinephrine. We conclude that in fish (1) catecholamine clearance and metabolism are unlikely to be compromised by repeated exposure to acute stressors; (2) catecholamine extraction and/or metabolism is enhanced when circulating levels are high; and (3) there is a marked capacity to rapidly (minutes) clear and inactivate catecholamines that are released in response to stressful stimuli.Abbreviations CA catecholamines - dpm disintegrations per minute - MAO monoamine oxidase - COMT catecholamine-O-methyltransferase - MOPEG 3-methoxy-4-hydroxyphenylglycol - VMA 3-methoxy-4-hydroxymendelic acid - SW seawater - HPLC high-performance liquid chromatography     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

Cardiac force-interval relationship,adrenaline and sarcoplasmic reticulum in rainbow trout

The force-interval relationship was examined at 20 and 10 °C in electrically paced atrial and ventricular tissue of rainbow trout,Oncorhynchus mykiss, regarding dependence on the sarcoplasmic reticulum and influence of adrenaline. In both tissues, adrenaline (10^-6 mol·l^-1) doubled control force developed at 0.5 Hz. In atrial but not in ventricular tissue it also shortened the diastolic interval needed for recovery of a given fraction of the control force. In atrial tissue and in ventricular tissue at 20 °C, the fraction of force recovered in the presence of adrenaline was diminished by 10 mol·l^-1 of ryanodine, a specific inhibitor of the sarcoplasmic reticulum. In atrial tissue not exposed to adrenaline and in ventricular tissue at 10 °C irrespective of adrenaline, ryanodine did not affect recovery. In atrial but not in ventricular tissue it also diminished control force. In conclusion, the cardiac sarcoplasmic reticulum of trout seems to support force development during adrenaline dependent increases in heart rate, and in atrial tissue also the force at steady state.Abbreviations E-C coupling excitation-contraction coupling - P-R potential - SR sarcoplasmic reticulum - SE standard error of the mean