Effect of parental mate choice and semi-natural early rearing environment on the growth performance and seawater tolerance of Chinook salmon Oncorhynchus tshawytscha.

To assess whether parental mate choice and early rearing in a semi-natural spawning channel may benefit the culture of Chinook salmon Oncorhynchus tshawytscha, 90 day growth trials were conducted using hatchery O. tshawytscha (hatchery), mate choice O. tshawytscha (i.e. the offspring of parents allowed to choose their own mate) that spent 6 months in a spawning channel prior to hatchery rearing (channel) and mate choice O. tshawytscha transferred to the hatchery as fertilized eggs (transfer). During the growth trials, all O. tshawytscha stocks were reared separately or in either mixed channel and hatchery or transfer and hatchery groups for comparison of performance to traditional practices. After 60 days in fresh water, all O. tshawytscha were transferred to seawater for an additional 30 days. Reared separately, all stocks grew c. 4.5 fold over 90 days but specific growth rate (G) and food conversion efficiency were higher in fresh water than after seawater transfer on day 60. In contrast, hatchery O. tshawytscha from mixed hatchery and channel and hatchery and transfer growth trials had a larger mass and length gain than their counterparts on day 60, but reduced G in seawater. In general, plasma levels of growth hormone, insulin-like growth factor I and cortisol did not differ among any O. tshawytscha groups in either the separate or mixed growth trials. Despite some differences in gill Na(+),K(+)-ATPase activity, all O. tshawytscha had a high degree of seawater tolerance and experienced virtually no perturbation in plasma chloride following seawater transfer. Overall, all O. tshawytscha exhibited similar growth and seawater performance under traditional hatchery conditions and any benefit derived from either parental mate choice or semi-natural early rearing environment was only observed in the presence of mutual competition with hatchery O. tshawytscha.

Inhibition of glutamine synthetase during ammonia exposure in rainbow trout indicates a high reserve capacity to prevent brain ammonia toxicity.

Glutamine synthetase (GSase), the enzyme that catalyses the conversion of glutamate and ammonia to glutamine, is present at high levels in vertebrate brain tissue and is thought to protect the brain from elevated ammonia concentrations. We tested the hypothesis that high brain GSase activity is critical in preventing accumulation of brain ammonia and glutamate during ammonia loading in the ammonia-intolerant rainbow trout. Trout pre-injected with saline or the GSase inhibitor methionine sulfoximine (MSOX, 6 mg kg(-1)), were exposed to 0, 670 or 1000 micromol l(-1) NH(4)Cl in the water for 24 and 96 h. Brain ammonia levels were 3- to 6-fold higher in ammonia-exposed fish relative to control fish and MSOX treatment did not alter this. Brain GSase activity was unaffected by ammonia exposure, while MSOX inhibited GSase activity by approximately 75%. Brain glutamate levels were lower and glutamine levels were higher in fish exposed to ammonia relative to controls. While MSOX treatment had little impact on brain glutamate, glutamine levels were significantly reduced by 96 h. With ammonia treatment, significant changes in the concentration of multiple other brain amino acids occurred and these changes were mostly reversed or eliminated with MSOX. Overall the changes in amino acid levels suggest that multiple enzymatic pathways can supply glutamate for the production of glutamine via GSase during ammonia exposure and that alternative transaminase pathways can be recruited for ammonia detoxification. Plasma cortisol levels increased 7- to 15-fold at 24 h in response to ammonia and MSOX did not exacerbate this stress response. These findings indicate that rainbow trout possess a relatively large reserve capacity for ammonia detoxification and for preventing glutamate accumulation during hyperammonaemic conditions.

Distribution and regional stressor-induced regulation of corticotrophin-releasing factor binding protein in rainbow trout (Oncorhynchus mykiss).

The corticotrophin-releasing factor (CRF) system plays a key role in the co-ordination of the physiological response to stress in vertebrates. Although the binding protein (BP) for CRF-related peptides, CRF-BP, is an important player in the many functions of the CRF system, the distribution of CRF-BP and the impact of stressors on its expression in fish are poorly understood. In the present study, we describe the distribution of CRF-BP in the brain and peripheral tissues of rainbow trout (Oncorhynchus mykiss) using a combination of real-time reverse transcriptase-polymerase chain reaction, in situ hybridisation and immunohistochemistry. Our results indicate a widespread and highly localised distribution of CRF-BP in the central nervous system, but do not support a significant peripheral production of the protein. Major expression sites in the brain include the area ventralis telencephali, nucleus preopticus, anterior and lateral tuberal nuclei, and the posterior region of the pituitary pars distalis. We further characterise changes in CRF-BP gene expression in three discrete brain regions after exposure to 8 h and 24 h of social stress or hypoxia. The plasma cortisol concentration in subordinate fish was much higher than in dominant fish and controls, and was indicative of a relatively severe stressor. By contrast, the increase in plasma cortisol concentration in fish exposed to hypoxia was characteristic of the response to a mild stressor. Changes in CRF-BP gene expression were only observed after 24 h of either stressor, and were region-specific. CRF-BP mRNA in the telencephalon increased in both subordinate fish and fish exposed to hypoxia, but CRF-BP in the preoptic area only increased after 24 h of hypoxia exposure. In the hypothalamus, CRF-BP mRNA levels decreased in dominant fish relative to controls after 24 h. Taken together, our results support a diverse role for CRF-BP in the central actions of the fish CRF system, but a negligible role in the peripheral functions of circulating CRF-related peptides. Furthermore, the differential changes in forebrain CRF-BP mRNA appear to occur independently of the hypothalamic-pituitary-inter-renal axis.

Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia.

The key strategy for coping with elevated brain ammonia levels in vertebrates is the synthesis of glutamine from ammonia and glutamate, catalyzed by glutamine synthetase (GSase). We hypothesized that all four GSase isoforms (Onmy-GS01-GS04) are expressed in the brain of the ammonia-intolerant rainbow trout Oncorhynchus mykiss and that cerebral GSase is induced during ammonia stress. We measured GSase activity and the mRNA expression of Onmy-GS01-GS04 in fore-, mid- and hindbrain and liver, as well as ammonia concentrations in plasma, liver and brain of fish exposed to 9 or 48 h of 0 (control) or 670 micromol l(-1) NH(4)Cl (75% of the 96 h-LC(50) value). The mRNA of all four GSase isoforms were detected in brain (not liver). After 9 h of NH(4)Cl exposure, brain, liver and plasma ammonia content were elevated by two- to fourfold over control values. Midbrain, hindbrain and liver GSase activities were 1.3- to 1.5-fold higher in ammonia-exposed fish relative to control fish. Onmy-GS01-GS04 mRNA levels in brain (not liver) of ammonia-exposed fish (9 h) were significantly elevated by two- to fourfold over control values. After 48 h of the NH(4)Cl treatment, ammonia content and GSase activity, but not mRNA levels, in all tissues examined remained elevated compared to control fish. Taken together, these findings indicate that all four GSase isoforms are constitutively expressed in trout brain and are inducible under high external ammonia conditions. Moreover, elevation of GSase activities in fore-, mid- and hindbrain in response to environmental ammonia underlines the importance of brain GSase in the ammonia-stress response.

Neuropeptides and the control of food intake in fish.

The brain, particularly the hypothalamus, integrates input from factors that stimulate (orexigenic) and inhibit (anorexigenic) food intake. In fish, the identification of appetite regulators has been achieved by the use of both peptide injections followed by measurements of food intake, and by molecular cloning combined with gene expression studies. Neuropeptide Y (NPY) is the most potent orexigenic factor in fish. Other orexigenic peptides, orexin A and B and galanin, have been found to interact with NPY in the control of food intake in an interdependent and coordinated manner. On the other hand cholecystokinin (CCK), cocaine and amphetamine-regulated transcript (CART), and corticotropin-releasing factor (CRF) are potent anorexigenic factors in fish, the latter being involved in stress-related anorexia. CCK and CART have synergistic effects on food intake and modulate the actions of NPY and orexins. Although leptin has not yet been identified in fish, administration of mammalian leptin inhibits food intake in goldfish. Moreover, leptin induces CCK gene expression in the hypothalamus and its actions are mediated at least in part by CCK. Other orexigenic factors have been identified in teleost fish, including the agouti-related protein (AgRP) and ghrelin. Additional anorexigenic factors include bombesin (or gastrin-releasing peptide), alpha-melanocyte-stimulating hormone (alpha-MSH), tachykinins, and urotensin I. In goldfish, nutritional status can modify the expression of mRNAs encoding a number of these peptides, which provides further evidence for their roles as appetite regulators: (1) brain mRNA expression of CCK, CART, tachykinins, galanin, ghrelin, and NPY undergo peri-prandial variations; and (2) fasting increases the brain mRNA expression of NPY, AgRP, and ghrelin as well as serum ghrelin levels, and decreases the brain mRNA expression of tachykinins, CART, and CCK. This review will provide an overview of recent findings in this field.

Limited extracellular but complete intracellular acid-base regulation during short-term environmental hypercapnia in the armoured catfish, Liposarcus pardalis.

Environmental hypercapnia induces a respiratory acidosis that is usually compensated within 24-96 h in freshwater fish. Water ionic composition has a large influence on both the rate and degree of pH recovery during hypercapnia. Waters of the Amazon are characteristically dilute in ions, which may have consequences for acid-base regulation during environmental hypercapnia in endemic fishes. The armoured catfish Liposarcus pardalis, from the Amazon, was exposed to a water P(CO(2)) of 7, 14 or 42 mmHg in soft water (in micromol l(-1): Na(+), 15, Cl(-), 16, K(+), 9, Ca(2+), 9, Mg(2+), 2). Blood pH fell within 2 h from a normocapnic value of 7.90+/-0.03 to 7.56+/-0.04, 7.34+/-0.05 and 6.99+/-0.02, respectively. Only minor extracellular pH (pH(e)) recovery was observed in the subsequent 24-96 h. Despite the pronounced extracellular acidosis, intracellular pH (pH(i)) of the heart, liver and white muscle was tightly regulated within 6 h (the earliest time at which these parameters were measured) via a rapid accumulation of intracellular HCO(3)(-). While most fish regulate pH(i) during exposure to environmental hypercapnia, the time course for this is usually similar to that for pH(e) regulation. The degree of extracellular acidosis tolerated by L. pardalis, and the ability to regulate pH(i) in the face of an extracellular acidosis, are the greatest reported to date in a teleost fish. The preferential regulation of pH(i) in the face of a largely uncompensated extracellular acidosis in L. pardalis is rare among vertebrates, and it is not known whether this is associated with the ability to air-breathe and tolerate aerial exposure, or living in water dilute in counter ions, or with other environmental or evolutionary selective pressures. The ubiquity of this strategy among Amazonian fishes and the mechanisms employed by L. pardalis are clearly worthy of further study.

Transition in organ function during the evolution of air-breathing; insights from Arapaima gigas, an obligate air-breathing teleost from the Amazon.

The transition from aquatic to aerial respiration is associated with dramatic physiological changes in relation to gas exchange, ion regulation, acid-base balance and nitrogenous waste excretion. Arapaima gigas is one of the most obligate extant air-breathing fishes, representing a remarkable model system to investigate (1) how the transition from aquatic to aerial respiration affects gill design and (2) the relocation of physiological processes from the gills to the kidney during the evolution of air-breathing. Arapaima gigas undergoes a transition from water- to air-breathing during development, resulting in striking changes in gill morphology. In small fish (10 g), the gills are qualitatively similar in appearance to another closely related water-breathing fish (Osteoglossum bicirrhosum); however, as fish grow (100-1000 g), the inter-lamellar spaces become filled with cells, including mitochondria-rich (MR) cells, leaving only column-shaped filaments. At this stage, there is a high density of MR cells and strong immunolocalization of Na(+)/K(+)-ATPase along the outer cell layer of the gill filament. Despite the greatly reduced overall gill surface area, which is typical of obligate air-breathing fish, the gills may remain an important site for ionoregulation and acid-base regulation. The kidney is greatly enlarged in A. gigas relative to that in O. bicirrhosum and may comprise a significant pathway for nitrogenous waste excretion. Quantification of the physiological role of the gill and the kidney in A. gigas during development and in adults will yield important insights into developmental physiology and the evolution of air-breathing.

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.

The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish.

Although environmental, social and physical stressors have been shown to inhibit food intake and feeding behavior in fish, little is known about the mechanisms that mediate the appetite-suppressing effects of stress. Since the hypothalamic-pituitary-interrenal (HPI) axis is activated in response to most forms of stress in fish, components of this axis may be involved in mediating the food intake reductions elicited by stress. Recent investigations into the brain regulation of food intake in fish have identified several signals with orexigenic and anorexigenic properties. Among these appetite-regulating signals are related neuropeptides that can activate the HPI axis, namely corticotropin-releasing factor (CRF) and urotensin I (UI). Central injections of CRF or UI, or treatments that result in an increase in hypothalamic CRF and UI gene expression, can elicit dose-dependent decreases in food intake that can be reversed by pre-treatment with a CRF-receptor antagonist. Evidence also suggests that cortisol, the end product of HPI activation in most fishes (i.e. Osteichthyes), may be involved in the regulation of food intake. Overall, while elements of the HPI axis may mediate some of the appetite-suppressing effects of stress, it is undetermined how either CRF-related peptides, cortisol, or other elements of the stress response interact with the complex circuitry of the hypothalamic feeding center.

Appetite-suppressing effects of urotensin I and corticotropin-releasing hormone in goldfish (Carassius auratus).

Fish urotensin I (UI), a member of the corticotropin-releasing hormone (CRH) family of peptides, is a potent inhibitor of food intake in mammals, yet the role of UI in the control of food intake in fish is not known. Therefore, to determine the acute effects of UI on appetite relative to those of CRH, goldfish were given intracerebroventricular (i.c.v.) injections of carp/goldfish UI and rat/human CRH (0.2-200 ng/g) and food intake was assessed for a 2-hour period after the injection. UI and CRH both suppressed food intake in a dose-related manner and UI (ED50 = 3.8 ng/g) was significantly more potent than CRH (ED50 = 43.1 ng/g). Pretreatment with the CRH receptor antagonist, alpha-helical CRH(9-41), reversed the reduction in food intake induced by i.c.v. UI and CRH. To assess whether endogenous UI and CRH modulate fish appetite, goldfish were given intraperitoneal implants of the glucocorticoid receptor antagonist, RU-486 (50 and 100 microg/g), or the cortisol synthesis inhibitor, metyrapone (100 and 200 microg/g), and food intake was monitored over the following 72 h. Fish treated with either RU-486 or metyrapone were characterized by a sustained and dose-dependent reduction in food intake. Pretreatment with i.c.v. implants of alpha-helical CRH(9-41) partially reversed the appetite-suppressing effects of RU-486 and metyrapone. In a parallel experiment, the effects of RU-486 (100 microg/g) and metyrapone (200 microg/g) intraperitoneal implants on brain UI and CRH gene expression were assessed. Relative to sham-implanted controls, fish treated with RU-486 or metyrapone had elevated UI mRNA levels in the hypothalamus and CRH mRNA levels in the telencephalon-preoptic brain region. Together, these results suggest that UI is a potent anorectic peptide in the brain of goldfish and that endogenous CRH-related peptides can play a physiological role in the control of fish appetite.

Brain regulation of feeding behavior and food intake in fish.

In mammals, the orexigenic and anorexigenic neuronal systems are morphologically and functionally connected, forming an interconnected network in the hypothalamus to govern food intake and body weight. However, there are relatively few studies on the brain control of feeding behavior in fish. Recent studies using mammalian neuropeptides or fish homologs of mammalian neuropeptides indicate that brain orexigenic signal molecules include neuropeptide Y, orexins, galanin and beta-endorphin, whereas brain anorexigenic signal molecules include cholecystokinin, bombesin, corticotropin-releasing factor, cocaine- and amphetamine-regulated transcript, and serotonin. Tachykinins may also have an anorectic action in fish. The brain hypothalamic area is associated with regulation of food intake, while sites outside the hypothalamus are also involved in this function. There is correlation between short-term changes in serum growth hormone levels and feeding behavior, although possible mechanisms integrating these functions remain to be defined.

Differential expression of corticotropin-releasing factor (CRF) and urotensin I precursor genes, and evidence of CRF gene expression regulated by cortisol in goldfish brain.

Corticotropin-releasing factor (CRF) and urotensin I (UI) precursor cDNAs were cloned and sequenced from a goldfish brain cDNA library in order to investigate the distribution of CRF and UI mRNAs in goldfish brain and the regulation of CRF and UI gene expression. The CRF (966-bp) and UI (769-bp) cDNAs encode 163- and 146-amino acid precursors, respectively, and consist of a signal peptide sequence, a cryptic region, and a 41-amino acid mature peptide at the carboxy terminal. The deduced amino acid sequences of the CRF and UI peptides exhibit a sequence identity of 54%. Northern blot analysis revealed a single size of CRF (1.3 kb) and UI (2.0 kb) mRNAs, which are expressed in the telencephalon-preoptic, hypothalamic, optic tectum-thalamus, and posterior brain regions, but not in the pituitary. In addition, while the CRF gene is strongly expressed in the olfactory bulbs, the UI gene is not. In brain regions in which both genes are expressed, the mRNA levels of CRF were three- to sevenfold higher that those of UI. While the low expression levels of the UI gene prevented further analysis of its regulation, the regulation of CRF gene expression by cortisol was examined. In response to intraperitoneal implants of cortisol (300 microg/g BW) the level of CRF mRNA in the telencephalon-preoptic region decreased to 69% of control values at 6 and 24 h posttreatment. In sham-treated fish, in parallel with a transient injection stress-elicited increase in plasma cortisol, CRF mRNA levels declined to 72% of control value at 6 h postinjection and recovered after 24 h. Injection of the glucocorticoid antagonist, RU-486 (100 microg/g BW), prevented the reduction in CRF gene expression associated with the injection stress at 6 h and increased CRF mRNA levels to 145% of control value after 24 h. In contrast, the various implants had no effect on CRF mRNA levels in either the hypothalamus or the optic tectum-thalamus region. These results provide evidence of differential expression of the CRF and UI genes in hypothalamic and extrahypothalamic regions of goldfish brain. Furthermore, they demonstrate that stress levels of plasma cortisol can lead to a decrease in CRF gene expression that is mediated by glucocorticoid receptors in the telencephalon-preoptic region and give an indication of the regional specificity of the regulation of CRF gene expression by cortisol.

Cardiovascular control via angiotensin II and circulating catecholamines in the spiny dogfish, Squalus acanthias.

The contributions of circulating angiotensin II (Ang II) and catecholamines to cardiovascular control in the spiny dogfish were investigated by monitoring the effects of exogenous and endogenous dogfish [Asn1, Pro3, Ile5]-Ang II (dfAng II) on plasma catecholamine levels and blood pressure regulation. Bolus intravenous injections of dfAng II (30-1200 pmol kg-1) elicited dose-dependent increases in plasma adrenaline and noradrenaline concentrations, caudal artery pressure (PCA), and systemic vascular resistance (RS), and a decrease in cardiac output (Q). Similar injections of Ang II in dogfish pre-treated with the alpha-adrenoceptor antagonist yohimbine (4 mg kg-1) also elicited dose-dependent increases in plasma catecholamine levels yet the cardiovascular effects were abolished. Dogfish treated with yohimbine were hypotensive and had elevated levels of plasma Ang II and catecholamines. Intravenous injection of the smooth muscle relaxant papaverine (10 mg kg-1) elicited a transient decrease in PCA and RS, and increases in plasma Ang II and catecholamine levels. In dogfish first treated with lisinopril (10(-4) mol kg-1), an angiotensin converting enzyme inhibitor, papaverine treatment caused a more prolonged and greater decrease in PCA and RS, an attenuated increase in plasma catecholamines, and no change in plasma Ang II. By itself, lisinopril treatment had little effect on PCA, and no effect on RS, plasma Ang II or catecholamines. In yohimbine-treated dogfish, papaverine treatment elicited marked decreases in PCA, RS, and Q, and increases in plasma Ang II and catecholamines. Among the three papaverine treatments, there was a positive linear relationship between plasma Ang II and catecholamine concentrations, and the cardiovascular and hormonal changes were most pronounced in the yohimbine + papaverine treatment. Therefore, under resting normotensive conditions, while Ang II does not appear to be involved in cardiovascular control, catecholamines play an important role. However, during a hypotensive stress elicited by vascular smooth muscle relaxation. Ang II indirectly contributes to cardiovascular control by dose-dependently stimulating catecholamine release.

Mediation of humoral catecholamine secretion by the renin-angiotensin system in hypotensive rainbow trout (Oncorhynchus mykiss).

The individual contributions of, and potential interactions between, the renin-angiotensin system (RAS) and the humoral adrenergic stress response to blood pressure regulation were examined in rainbow trout. Intravenous injection of the smooth muscle relaxant, papaverine (10 mg/kg), elicited a transient decrease in dorsal aortic blood pressure (PDA) and systemic vascular resistance (RS), and significant increases in plasma angiotensin II (Ang II) and catecholamine concentrations. Blockade of alpha-adrenoceptors before papaverine treatment prevented PDA and RS recovery, had no effect on the increase in plasma catecholamines, and resulted in greater plasma Ang II concentrations. Administration of the angiotensin-converting enzyme inhibitor, lisinopril (10(-4) mol/kg), before papaverine treatment attenuated the increases in the plasma concentrations of Ang II, adrenaline, and noradrenaline by 90, 79, and 40%, respectively and also prevented PDA and RS recovery. By itself, lisinopril treatment caused a gradual and sustained decrease in PDA and RS, and reductions in basal plasma Ang II and adrenaline concentrations. Bolus injection of a catecholamine cocktail (4 nmol/kg noradrenaline plus 40 nmol/kg adrenaline) in the lisinopril+papaverine-treated trout, to supplement their circulating catecholamine concentrations and mimic those observed in fish treated only with papaverine, resulted in a temporary recovery in PDA and RS. These results indicate that the RAS and the acute humoral adrenergic response are both recruited during an acute hypotensive stress, and have important roles in the compensatory response to hypotension in rainbow trout. However, whereas the contribution of the RAS to PDA recovery is largely indirect and relies on an Ang II-mediated secretion of catecholamines, the contribution from the adrenergic system is direct and relies at least in part on plasma catecholamines.

Does gill boundary layer carbonic anhydrase contribute to carbon dioxide excretion: a comparison between dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss).

In vivo experiments were conducted on spiny dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss) in sea water to determine the potential role of externally oriented or gill boundary layer carbonic anhydrase in carbon dioxide excretion. This was accomplished by assessing pH changes in expired water using a stopped-flow apparatus. In dogfish, expired water was in acid-base disequilibrium as indicated by a pronounced acidification (delta pH=-0.11+/-0.01; N=22; mean +/- s.e.m.) during the period of stopped flow; inspired water, however, was in acid-base equilibrium (delta pH=-0.002+/-0.01; N=22). The acid-base disequilibrium in expired water was abolished (delta pH=-0.005+/-0.01; N=6) by the addition of bovine carbonic anhydrase (5 mg l-1) to the external medium. Addition of the carbonic anhydrase inhibitor acetazolamide (1 mmol l-1) to the water significantly reduced the magnitude of the pH disequilibrium (from -0.133+/-0.03 to -0.063+/-0.02; N=4). However, after correcting for the increased buffering capacity of the water caused by acetazolamide, the acid-base disequilibrium during stopped flow was unaffected by this treatment (control delta [H+]=99.8+/-22.8 micromol l-1; acetazolamide delta [H+]=81.3+/-21.5 micromol l-1). In rainbow trout, expired water displayed an acid-base disequilibrium (delta pH=0.09+/-0.01; N=6) that also was abolished by the application of external carbonic anhydrase (delta pH=0.02+/-0.01). The origin of the expired water acid-base disequilibrium was investigated further in dogfish. Intravascular injection of acetazolamide (40 mg kg-1) to inhibit internal carbonic anhydrase activity non-specifically and thus CO2 excretion significantly diminished the extent of the expired water disequilibrium pH after 30 min (from -0.123+/-0.01 to -0.065+/-0.01; N=6). Selective inhibition of extracellular carbonic anhydrase activity using a low intravascular dose (1.3 mg kg-1) of the inhibitor benzolamide caused a significant reduction in the acid-base disequilibrium after 5 min (from -0.11+/-0.01 to -0.07+/-0. 01; N=14). These results demonstrate that the expired water acid-base disequilibrium originates, at least in part, from excretory CO2 and that extracellular carbonic anhydrase in dogfish may have a significant role in carbon dioxide excretion. However, externally oriented carbonic anhydrase (if present in dogfish) plays no role in catalysing the hydration of the excretory CO2 in water flowing over the gills and thus is unlikely to facilitate CO2 excretion.

Cardiovascular effects of angiotensin-II-mediated adrenaline release in rainbow trout Oncorhynchus mykiss.

To determine the contribution of plasma catecholamines to the cardiovascular effects of elevated levels of angiotensin II (Ang II) in trout, this study investigated (1) the stimulatory effects of [Asn1-Val5]-Ang II on plasma catecholamine levels, (2) the cardiovascular effects of Ang II with and without alpha-adrenoceptor blockade and (3) the relationship between plasma adrenaline concentrations and their cardiovascular effects. Bolus intravascular injections of Ang II (25-1200 pmol kg-1) elicited dose-dependent (between 75 and 1200 pmol kg-1) increases in plasma adrenaline levels; mean plasma noradrenaline levels only increased in response to a dose of 1200 pmol kg-1. Ang-II-elicited increases in plasma adrenaline levels ranged from 3.3+/-0.3 nmol l-1 for 75 pmol kg-1 Ang II to 125.1+/-40.0 nmol l-1 for 1200 pmol kg-1 Ang II. Injections of Ang II (25-1200 pmol kg-1) also elicited dose-dependent increases in dorsal aortic pressure (PDA), systemic resistance (RS), cardiac output (Q) and stroke volume (Vs). In fish first treated with the alpha -adrenoceptor blocker phenoxybenzamine, Ang II injections elicited a decrease in q_dot and Vs, and the increases in PDA and RS following administration of the 600 and 1200 pmol kg-1 Ang II doses were significantly reduced. Bolus injections of adrenaline (1.8x10(-10) to 1.4x10(-8) mol kg-1) elicited dose-dependent increases in PDA at a plasma adrenaline concentration of 16.5 nmol l-1 and in RS at a plasma adrenaline concentration of 50.5 nmol l-1. Adrenaline injections also elicited increases in Q and Vs at plasma adrenaline concentrations of 50.5 nmol l-1; however, higher plasma adrenaline concentrations were not associated with further increases in either Q or Vs. These results demonstrate that, in vivo, Ang II can act as a potent non-cholinergic secretagogue of humoral adrenaline in trout and that some of the cardiovascular effects of exogenous Ang II can be attributed to increased levels of plasma adrenaline. Our data also indicate that the cardiovascular effects of Ang-II-mediated humoral catecholamines are recruited in a dose-dependent manner and, as such, may require an acute stimulation of the renin-angiotensin system to contribute significantly to the pressor activity of endogenous angiotensins.

The adrenergic stress response in fish: control of catecholamine storage and release.

In fish, the catecholamine hormones adrenaline and noradrenaline are released into the circulation, from chromaffin cells, during numerous 'stressful' situations. The physiological and biochemical actions of these hormones (the efferent adrenergic response) have been the focus of numerous investigations over the past several decades. However, until recently, few studies have examined aspects involved in controlling/modulating catecholamine storage and release in fish. This review provides a detailed account of the afferent limb of the adrenergic response in fish, from the biosynthesis of catecholamines to the exocytotic release of these hormones from the chromaffin cells. The emphasis is on three particular topics: (1) catecholamine biosynthesis and storage within the chromaffin cells including the different types of chromaffin cells and their varying arrangement amongst species; (2) situations eliciting the secretion of catecholamines (e.g. hypoxia, hypercapnia, chasing); (3) cholinergic and non-cholinergic (i.e. serotonin, adrenocorticotropic hormone, angiotensin, adenosine) control of catecholamine secretion. As such, this review will demonstrate that the control of catecholamine storage and release in fish chromaffin cells is a complex processes involving regulation via numerous hormones, neurotransmitters and second messenger systems.

Angiotensins stimulate catecholamine release from the chromaffin tissue of the rainbow trout.

Immunohistochemical and pharmacological techniques were utilized to investigate the relationships between angiotensins and catecholamine release from the chromaffin tissue of rainbow trout (Oncorhynchus mykiss). Double labeling with [Asp1, Ile5]angiotensin II-fluorescein isothiocyanate (ANG II-FITC) and anti-dopamine beta-hydroxylase revealed specific ANG II binding sites on chromaffin cells. Injection (1 nmol/kg body wt) of either ANG II-FITC, [Asn1, Val5, Asn9]ANG I, [Asp1, Ile5, His9]ANG I, [Asn1, Val5]ANG II, [Asp1, Val5]ANG II, or [Asp1, Ile5]ANG II elicited catecholamine release from in situ perfusion preparations of the head kidney. Catecholamine release elicited by [Asn1, Val5]ANG II (10(-13) to 10(-7) mol/kg body wt) was dose dependent, and the secretion of epinephrine (Epi) was greater than that of norepinephrine (NE). Relative to the results obtained with the [Asn1, Val5]ANG II treatment (1 nmol/kg body wt), Epi release was 72 and 82% lower in response to injections (1 nmol/kg body wt) of [Asn1, Val5]ANG I [amino acid (AA) positions 1-7] and [Asn1, Val5]ANG I (AA 1-6), respectively. Pretreatment with either losartan (10(-5) M), PD-123319 (10(-5) M), or hexamethonium (10(-3) M) had no effect on [Asn1, Val5]ANG II-elicited catecholamine release. Pretreatment with captopril (10(-4) M) significantly reduced [Asn1, Val5, Asn9]ANG I-elicited Epi and NE release and decreased basal catecholamine release. These results provide direct evidence that angiotensins can elicit catecholamine release from the chromaffin tissue via specific ANG II binding sites and indicate that the synthesis of ANG II may be either local or systemic.