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Adrenaline and noradrenaline, released as hormones and/or neurotransmitters, exert diverse physiological functions in vertebrates, and teleost fishes are widely used as model organisms to study adrenergic regulation; however, such investigations often rely on receptor subtype-specific pharmacological agents (agonists and antagonists; see Glossary) developed and validated in mammals. Meanwhile, evolutionary (phylogenetic and comparative genomic) studies have begun to unravel the diversification of adrenergic receptors (ARs) and reveal that whole-genome duplications and pseudogenization events in fishes results in notable distinctions from mammals in their genomic repertoire of ARs, while lineage-specific gene losses within teleosts have generated significant interspecific variability. In this Review, we visit the evolutionary history of ARs (including α1-, α2- and β-ARs) to highlight the prominent interspecific differences in teleosts, as well as between teleosts and other vertebrates. We also show that structural modelling of teleost ARs predicts differences in ligand binding affinity compared with mammalian orthologs. To emphasize the difficulty of studying the roles of different AR subtypes in fish, we collate examples from the literature of fish ARs behaving atypically compared with standard mammalian pharmacology. Thereafter, we focus on specific case studies of the liver, heart and red blood cells, where our understanding of AR expression has benefited from combining pharmacological approaches with molecular genetics. Finally, we briefly discuss the ongoing advances in ‘omics’ technologies that, alongside classical pharmacology, will provide abundant opportunities to further explore adrenergic signalling in teleosts.

The study of breathing in fishes has featured prominently in Journal of Experimental Biology (JEB), particularly during the latter half of the past century. Indeed, many of the seminal discoveries in this important sub-field of comparative respiratory physiology were reported first in JEB. The period spanning 1960–1990 (the ‘golden age of comparative respiratory physiology’) witnessed intense innovation in the development of methods to study the control of breathing. Many of the guiding principles of piscine ventilatory control originated during this period, including our understanding of the dominance of O2 as the driver of ventilation in fish. However, a critical issue – the identity of the peripheral O2 chemoreceptors – remained unanswered until methods for cell isolation, culture and patch-clamp recording established that gill neuroepithelial cells (NECs) respond to hypoxia in vitro. Yet, the role of the NECs and other putative peripheral or central chemoreceptors in the control of ventilation in vivo remains poorly understood. Further progress will be driven by the implementation of genetic tools, most of which can be used in zebrafish (Danio rerio). These tools include CRISPR/Cas9 for selective gene knockout, and Tol2 systems for transgenesis, the latter of which enables optogenetic stimulation of cellular pathways, cellular ablation and in vivo cell-specific biosensing. Using these methods, the next period of discovery will see the identification of the peripheral sensory pathways that initiate ventilatory responses, and will elucidate the nature of their integration within the central nervous system and their link to the efferent motor neurons that control breathing.

The hypoxic ventilatory response (HVR) in fish is an important reflex that aids O2 uptake when low environmental O2 levels constrain diffusion. In developing zebrafish (Danio rerio), the acute HVR is multiphasic, consisting of a rapid increase in ventilation frequency (fV) during hypoxia onset, followed by a decline to a stable plateau phase above fV under normoxic conditions. In this study, we examined the potential role of catecholamines in contributing to each of these phases of the dynamic HVR in zebrafish larvae. We showed that adrenaline elicits a dose-dependent β-adrenoreceptor (AR)-mediated increase in fV that does not require expression of β1-ARs, as the hyperventilatory response to β-AR stimulation was unaltered in adrb1−/− mutants, generated by CRISPR/Cas9 knockout. In response to hypoxia and propranolol co-treatment, the magnitude of the rapidly occurring peak increase in fV during hypoxia onset was attenuated (112±14 breaths min−1 without propranolol to 68±17 breaths min−1 with propranolol), whereas the increased fV during the stable phase of the HVR was prevented in both wild type and adrb1−/− mutants. Thus, β1-AR is not required for the HVR and other β-ARs, although not required for initiation of the HVR, are involved in setting the maximal increase in fV and in maintaining hyperventilation during continued hypoxia. This adrenergic modulation of the HVR may arise from centrally released catecholamines because adrenaline exposure failed to activate (based on intracellular Ca2+ levels) cranial nerves IX and X, which transmit O2 signals from the pharyngeal arch to the central nervous system.

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus-abundant transcript 1 (DrHiat1), also found in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues 4 days post-fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole-body ammonia excretion rates compared with control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify DrHiat1b/DrHIAT1 in particular as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom.

In vertebrates, parathyroid hormone (PTH) is important for skeletogenesis and Ca2+homeostasis. However, little is known about the molecular mechanisms by which PTH regulates skeleton formation and Ca2+balance during early development. Using larval zebrafish as an in vivo model system, we determined that PTH1 regulates the differentiation of epithelial cells and the development of craniofacial cartilage. We demonstrated that translational gene knockdown of PTH1 decreased Ca2+uptake at 4 days after fertilization. We also observed that PTH1-deficient fish exhibited reduced numbers of epithelial Ca2+channel (ecac)-expressing cells, Na+/K+-ATPase-rich cells, and H+-ATPase-rich cells. Additionally, the density of epidermal stem cells was decreased substantially in the fish experiencing PTH1 knockdown. Knockdown of PTH1 caused a shortening of the jaw and impeded the development of branchial arches. Results from in situ hybridization suggested that the expression of collagen 2a1a (marker for proliferating chondrocytes) was substantially reduced in the cartilage that forms the jaw and branchial aches. Disorganization of chondrocytes in craniofacial cartilage also was observed in PTH1-deficient fish. The results of real-time PCR demonstrated that PTH1 morphants failed to express the transcription factor glial cell missing 2 (gcm2). Coinjection of PTH1 morpholino with gcm2capped RNA rescued the phenotypes observed in the PTH1 morphants, suggesting that the defects in PTH1-deficient fish were caused, at least in part, by the suppression of gcm2. Taken together, the results of the present study reveal critical roles for PTH1 in promoting the differentiation of epidermal stem cells into mature ionocytes and cartilage formation during development.

The gill is both a site of gas transfer and an important location of chemoreception or gas sensing in fish. While often considered separately, these two processes are clearly intricately related because the gases that are transferred between the ventilatory water and blood at the gill are simultaneously sensed by chemoreceptors on, and within, the gill. Modulation of chemoreceptor discharge in response to changes in O2 and CO2 levels, in turn, is believed to initiate a series of coordinated cardiorespiratory reflexes aimed at optimising branchial gas transfer. The past decade has yielded numerous advances in terms of our understanding of gas transfer and gas sensing at the fish gill, particularly concerning the transfer and sensing of carbon dioxide. In addition, recent research has moved from striving to construct a single model that covers all fish species, to recognition of the considerable inter-specific variation that exists with respect to the mechanics of gas transfer and the cardiorespiratory responses of fish to changes in O2 and CO2 levels. The following review attempts to integrate gas transfer and gas sensing at the fish gill by exploring recent advances in these areas. © 2002 Wiley-Liss, Inc.

The gill is both a site of gas transfer and an important location of chemoreception or gas sensing in fish. While often considered separately, these two processes are clearly intricately related because the gases that are transferred between the ventilatory water and blood at the gill are simultaneously sensed by chemoreceptors on, and within, the gill. Modulation of chemoreceptor discharge in response to changes in O2 and CO2 levels, in turn, is believed to initiate a series of coordinated cardiorespiratory reflexes aimed at optimising branchial gas transfer. The past decade has yielded numerous advances in terms of our understanding of gas transfer and gas sensing at the fish gill, particularly concerning the transfer and sensing of carbon dioxide. In addition, recent research has moved from striving to construct a single model that covers all fish species, to recognition of the considerable inter-specific variation that exists with respect to the mechanics of gas transfer and the cardiorespiratory responses of fish to changes in O2 and CO2 levels. The following review attempts to integrate gas transfer and gas sensing at the fish gill by exploring recent advances in these areas. © 2002 Wiley-Liss, Inc.

The hypoxia-inducible factor (HIF) pathway is a key regulator of cellular O2 homeostasis and an important orchestrator of the physiological responses to hypoxia (low O2) in vertebrates. Fish can be exposed to significant and frequent changes in environmental O2, and increases in Hif-α (the hypoxia-sensitive subunit of the transcription factor Hif) have been documented in a number of species as a result of a decrease in O2. Here, we discuss the impact of the Hif pathway on the hypoxic response and the contribution to hypoxia tolerance, particularly in fishes of the cyprinid lineage, which includes the zebrafish (Danio rerio). The cyprinids are of specific interest because, unlike in most other fishes, duplicated paralogs of the Hif-α isoforms arising from a teleost-specific genome duplication event have been retained. Positive selection has acted on the duplicated paralogs of the Hif-α isoforms in some cyprinid sub-families, pointing to adaptive evolutionary change in the paralogs. Thus, cyprinids are valuable models for exploring the evolutionary significance and physiological impact of the Hif pathway on the hypoxic response. Knockout in zebrafish of either paralog of Hif-1α greatly reduces hypoxia tolerance, indicating the importance of both paralogs to the hypoxic response. Here, with an emphasis on the cardiorespiratory system, we focus on the role of Hif-1α in the hypoxic ventilatory response and the regulation of cardiac function. We explore the effects of the duration of the hypoxic exposure (acute, sustained or intermittent) on the impact of Hif-1α on cardiorespiratory function and compare relevant data with those from mammalian systems.

This is the first description of the ventilatory response of the Atlantic cod (Gadus morhua) to hypoxia. Using a pharmacological approach, we tested the hypotheses that during hypoxia (1) elevation of circulating catecholamines, arising from chromaffin tissue or peripheral adrenergic neurones, contributes to hyperventilation and that (2) the hyperventilatory response can be modulated by the hypoxia-induced hypertension. The ventilatory response to hypoxia (final water PO₂ = 46 mmHg, reached in ≤25 min) was not significantly altered by pretreatment offish with either α-or β-adrenoceptor antagonists (phentolamine or sotalol, respectively) or with an inhibitor of catecholamine release from peripheral neurones (bretylium), despite a significant increase in plasma catecholamine levels in all treatment groups. Since the utilized regime did not induce metabolic acidosis (a potential ventilatory stimulant), we conclude that the hyperventilation was caused solely by a depression of the oxygen status. Although changes in the internal as well as the external oxygen status may have potentially contributed to the hyperventilation, we suggest, on the basis of the rapid ventilatory response after only small depressions of water PO₂, that the initial stimulus is external. During hypoxia, a doubling of mean ventral aortic blood pressure was observed. This elevation of blood pressure was due to an increased adrenergic nervous and humoral activity. The lack of modification in the ventilatory response to hypoxia after transient or persistent abolishment of the hypertension by pharmacological agents (bretylium or phentolamine, respectively) indicated that during hypoxia there was no relationship between blood pressure and ventilation in this species.

Rainbow trout (Oncorhynchus mykiss) and American eels (Anguilla rostrata) were infused intra-arterially with NaHCO₃ (∼800 μmol/kg/h) to assess their acid-base regulatory mechanisms during metabolic alkalosis. In both species, plasma pH and [HCO₃ ⁻] increased during infusion, although the changes were more pronounced in the eel. In trout, pH and [HCO₃⁻] were returned to preinfusion values by 12 h after infusion was stopped, while in the eel there was only incomplete clearance of accumulated [HCO₃⁻] during the 24-h postinfusion period. In each case, approximately 20 mmol/L of HCO₃⁻ was removed from the plasma during postinfusion. The predominant mechanism employed by both trout and eel to regulate blood acid-base status during alkalosis was manipulation of branchial net Cl⁻ flux (\documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage{wasysym} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document}$$J_{{\mathrm{net}}}^{{\mathrm{Cl-}}}$$\end{document}), although this was accomplished with markedly different strategies. 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In contrast, the eel relied almost exclusively on reduction of \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage{wasysym} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document}$$J_{{\mathrm{out}}}^{{\mathrm{Cl-}}}$$\end{document}. Manipulation of CC surface area to increase \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage{wasysym} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document}$$J_{{\mathrm{in}}}^{{\mathrm{Cl-}}}$$\end{document}or the use of intracellular buffering appeared to be unimportant for regulating metabolic alkalosis in eel.

An extracorporeal circulation of rainbow trout (Oncorhynchus mykiss) was utilized to continuously monitor the rapid and progressive effects of endogenous or exogenous catecholamines on blood respiratory/acid-base status, and to provide in vivo evidence for adrenergic retention of carbon dioxide (CO2) in fish blood (cf. Wood and Perry 1985). Exposure of fish to severe aquatic hypoxia (final PwO2=40–60 torr; reached within 10–20 min) elicited an initial respiratory alkalosis resulting from hypoxia-induced hyperventilation. However, at a critical arterial oxygen tension (PaO2) between 15 and 25 torr, fish became agitated for approximately 5 s and a marked (0.2–0.4 pH unit) but transient arterial blood acidosis ensued. This response is characteristic of abrupt catecholamine mobilization into the circulation and subsequent adrenergic activation of red blood cell (RBC) Na+/H+ exchange (Fievet et al. 1987). Within approximately 1–2 min after the activation of RBC Na+/H+ exchange by endogenous catecholamines, there was a significant rise in arterial PCO2 (PaCO2) whereas arterial PO2 was unaltered; the elevation of PaCO2 could not be explained by changes in gill ventilation. Pre-treatment of fish with the a-adrenoceptor antagonist phentolamine did not prevent the apparent catecholamine-mediated increase of PaCO2. Conversely, pre-treatment with the ß-adrenoceptor antagonist sotalol abolished both the activation of the RBC Na+/H+ antiporter and the associated rise in PaCO2, suggesting a causal relationship between the stimulation of RBC Na+/H+ exchange and the elevation of PaCO2. To more clearly establish that elevation of plasma catecholamine levels during severe hypoxia was indeed responsible for causing the elevation of PaCO2, fish were exposed to moderate hypoxia (final PwO2=60–80 torr) and then injected intraarterially with a bolus of adrenaline to elicit an estimated circulating level of 400 nmol·l-1 immediately after the injection. This protocol activated RBC Na+/H+ exchange as indicated by abrupt changes in arterial pH (pHa). In all fish examined, PaCO2 increased after injection of exogenous adrenaline. The effects on PaO2 were inconsistent, although a reduction in this variable was the most frequent response. Gill ventilation frequency and amplitude were unaffected by exogenous adrenaline. Therefore, it is unlikely that ventilatory changes contributed to the consistently observed rise in PaCO2. Pretreatment of fish with sotalol did not alter the ventilatory response to adrenaline injection but did prevent the stimulation of RBC Na+/H+ exchange and the accompanying increases and decreases in PaCO2 and PaO2, respectively. These results suggest that adrenergic elevation of PaCO2, in addition to the frequently observed reduction of PaO2 are linked to activation of RBC Na+/H+ exchange. The physiological significance and the potential mechanisms underlying the changes in blood respiratory status after addition of endogenous or exogenous catecholamines to the circulation of hypoxic rainbow trout are discussed.

Summary Gill ventilation volume ( $$\dot V$$ 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 (ß-adrenoceptor antagonist) or phentolamine (a-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.

An extracorporeal circulation and stopflow technique were used to characterize the acid—base disequilibrium in the arterial (post‐branchial) blood of rainbow trout, Oncorhynchus mykiss, after alteration of the blood pH through acid, base, or catecholamine infusion, or by inducing endogenous catecholamine release. Arterial blood was routed from the coeliac artery through an external circuit in which pH and the partial pressures of oxygen and carbon dioxide were monitored continuously. The stopflow condition was imposed by turning off the pump which drove the external loop.The results of this study support the prediction that blood acid—base status is one factor determining the size of the disequilibrium. Under control conditions, the arterial blood exhibited a statistically significant positive disequilibrium (increase in pH upon stopflow) of 0.02 ± 0.01 to 0.04 ± 0.01 units. Decreasing blood pH by the infusion of a fixed acid load ((NH4)2SO4) or by adrenergic activation of the red blood cell Na+/H+ antiporter increased the magnitude of the positive disequilibrium (0.11 ± 0.01 to 0.17 ± 0.02 units). © 1996 Wiley‐Liss, Inc.

Rainbow trout (Oncorhynchus mykiss) and American eels (Anguilla rostrata) were exposed (15 min) to two levels of external hypoxia (water Po2 [Pwo2] = 5.3 and 4.0 kPa for trout; 4.0 and 2.7 kPa for eels). Arterial Po2 (Pao2) fell to 1.7-2.0 kPa in trout and 1.1-1.3 kPa in eel. Plasma catecholamines were elevated in trout during both levels of hypoxia but were unaltered in hypoxic eels. The goals of this study were to elucidate the mechanisms for the differences in these plasma catecholamine levels. Levels of stored catecholamines were measured in the kidney, posterior cardinal vein (PCV), and the heart. In both species the anterior region of the PCV was the primary storage site, with levels being greater in trout than in eels. The ability of the chromafin cells to respond to two chemical stimuli, 60 mmol L?¹ K? or carbachol (10?8 to 10?5 mol), was assessed with an in situ saline-perfused PCV preparation. Trout released greater quantities of catecholamines in response to both stimuli than did eels. Differences in the cholinergic stimulation of the chromafin cells may account for the differences in plasma levels during hypoxia. In trout, carbachol selectively caused the release of adrenaline over noradrenaline, which suggests the presence of two populations of chromaffin cells, those containingpredominantly adrenaline and others containing predominantly noradrenaline.

Whole-body unidirectional and net Na+and Cl–fluxes were determined in rainbow trout Oncorhynchus mykissexposed to chloramine-T (9 mg/L) alone or in combination with 17 mmol NaCl/L (0.1 % weight per volume, w/v) after acclimation to either city of Ottawa tap water (TW: Na+0.121 ± 0.004 mmol/L; K+0.015 ± <0.001 mmol/L; Ca2+0.351 ± 0.004 mmol/L; Cl–0.141 ± 0.002 mmol/L) or artificial soft water (ASW: Na+0.043 ± 0.003 mmol/L; K+0.005 ± <0.001 mmol/L; Ca2+0.063 ± 0.002 mmol/L; Cl–0.026 ± 0.002 mmol/L). Preexposure fluxes revealed that ASW-acclimated fish had reduced rates of Na+uptake and efflux compared with TW-acclimated fish. However, rates of Cl–uptake among ASW-acclimated fish were comparable with those of TW-acclimated fish. Exposure to chloramine-T in TW resulted in significant increases in the Cl–efflux, giving rise to a significant and negative net flux. Exposure of ASW-acclimated fish to chloramine-T caused a significant negative increase in the Na+efflux and net flux but only a negative increase in the Cl–net flux. The addition of 17 mmol NaCl/L (0.1% w/v) eliminated the negative net ionic fluxes, resulting in significant increases in influx. The combined use of chloramine-T with NaCl is a potential method of remediating ionic disturbances that may be caused by treating trout with chloramine-T.

Rainbow trout (Oncorhynchus mykiss) were exposed to ion-poor (soft) water to test the hypothesis that naturally induced proliferation of branchial chloride cells causes a thickening of the blood-to-water diffusion barrier. This was achieved by using a combination of scanning and transmission electron-microscopic techniques. Fish were exposed to soft-water conditions ([Na⁺]= 0.055 mmol l-1, [Cl^-]0.029 mmol 1^-1, [Ca²⁺] 0.059 mmol 1^-1, and [K⁺]0.007 mmol 1^-1) for 1, 2, and 4 weeks. Marked chloride cell proliferation was evident at all sampling times with an approximate doubling of the gill epithelial surface area covered by chloride cells exposed to the water (”chloride cell fractional area"). The increases in chloride cell fractional area resulted from both increased numbers of cells and expanded apical surfaces of exposed individual cells. As a result of chloride cell proliferation, soft-water exposure was associated with a doubling of the lamellar blood-to-water diffusion distance from 3.260.08 m to 6.580.43 m as determined from transmission electron micrographs. These data demonstrated a positive correlation between chloride cell fractional area and blood-to-water diffusion distance. We conclude that, in trout, chloride cell proliferation during soft-water exposure, while presumably benefiting ionic regulation, may impair gas transfer owing to the associated thickening of the blood-to-water diffusion barrier.

A new in vitro assay was developed and critically characterized to measure the rate of CO2excretion by trout red blood cells (RBCs) from HCO3- in their natural plasma under normal in vivo conditions of acid-base status. The assay is based on the addition of [14C]bicarbonate to the whole blood and collection of the resultant 14CO2in the overlying gas phase. The assay simulates the exposure of blood passing through the gills, and measured CO2excretion rates are representative of those occurring in vivo. Rates are linear over the 3 min time course of the assay, related to haematocrit in a non-linear fashion, elevated by the addition of carbonic anhydrase, reduced by blockade with acetazolamide, and sensitive to variations of equilibration . Large variations in plasma [HCO3-] have only a small effect on CO2excretion rates when the blood is chronically equilibrated at these levels. Acute elevations in [HCO3-], however, create a non-equilibrium situation, resulting in large increases in CO2excretion. When the blood is acidified, to duplicate typical post-exercise metabolic acidosis, adrenaline causes a marked inhibition of RBC CO2excretion. The response is transient, reaching a peak 5–8 min after addition of adrenaline and disappearing by 30–60 min. The magnitude of the adrenergic inhibition is correlated with the magnitude of the RBC pHi regulatory response, expressed as the RBC transmembrane pH difference (pHe—pHi). These results support the ‘CO2retention theory’ explaining observed increases in blood vivo after exhaustive exercise and catecholamine infusions in fish.

We have characterized the elevation of circulating catecholamines in the intact Atlantic cod (Gadus morhua) during graded acute (30 min) hypoxia. The potential mechanisms contributing to the mobilization of catecholamines during hypoxia were then assessed in vivo using nerve sectioning and pharmacological techniques and in situ using a perfused head kidney preparation. Pre-branchial plasma adrenaline concentrations were significantly elevated at all levels of aquatic hypoxia utilised [water PO2 (PwO2) = 10 kPa (75 mmHg), 7.3kPa (55 mmHg) or 5.3 kPa (40mmHg)], whereas noradrenaline levels did not increase significantly in these particular experiments in which PwO2 was lowered gradually over a 30 min period. All subsequent experiments were performed using a more rapid induction of hypoxia to reach a final PwO2 of 5.3 kPa within the first 5-10 min of exposure. Blood withdrawn from pre-branchial (ventral aortic) and post-branchial (dorsal aortic) cannulae after 30 min revealed pronounced reductions in PO2 and O2 content (CO2) as well as elevated pH. These data support the notion that blood acidosis is not a prerequisite for catecholamine mobilization during hypoxia. Bilateral sectioning of spinal nerves 1–4 innervating the head kidney prevented the elevation of noradrenaline during rapidly induced hypoxia, but had no effect on the rise in plasma adrenaline concentration. After each experiment, fish were exposed to air for 3 min to induce severe stress. Plasma catecholamine levels were significantly reduced during stress, suggesting that the sectioning of the spinal nerves to the head kidney was indeed effective. These results indicated that mechanisms other than neural stimulation of head kidney chromaffin tissue were contributing to the rise in plasma adrenaline level during hypoxia. Neuronal overflow into the circulation, however, was an unlikely possibility since the increase of adrenaline could not be prevented by treating denervated fish with bretylium (an inhibitor of catecholamine release from adrenergic nerve terminals). These data suggested a local direct stimulatory effect of blood hypoxaemia on adrenaline release from chromaffin tissue. This hypothesis was confirmed using a blood-perfused head kidney preparation in which hypoxaemia markedly stimulated adrenaline overflow into the effluent blood. Further experiments using a Ringer-perfused head kidney preparation were designed to test the hypothesis that blood catecholamine levels in vivo are, in part, controlled by the concentration of catecholamines in the blood entering the head kidney. The results show conclusively that overflow of a particular catecholamine during cholinergic stimulation of the head kidney is controlled independently by the inflowing concentration of that catecholamine. We suggest that this mechanism of ‘auto-inhibition’ of catecholamine overflow is a functional negative feedback mechanism involved in the control of plasma catecholamine levels in the cod.

The effects of serotonin (5-hydroxytryptamine; 5-HT) on catecholamine release from chromaffin tissue were investigated in the rainbow trout (Oncorhynchus mykiss) in vivo and in situ. Intra-arterial injections of serotonin in vivo caused dose-dependent (50–250nmolkg−1) increases in both plasma noradrenaline and adrenaline levels. Pre-treatment of fish with the serotonergic receptor antagonist methysergide did not abolish these increases. An in situ saline-perfused head kidney preparation was developed and validated to study the potential direct effect of serotonin on catecholamine release. The chromaffin cells in the preparation showed a dose-dependent release of catecholamines in response to bolus injections of the cholinergic receptor agonist carbachol (10−7–10−4 molkg-1). The carbachol-induced release of noradrenaline, but not of adrenaline, was reduced significantly when the nicotinic receptor antagonist hexamethonium (10−4 mol l−1) was present in the perfusion fluid. The removal of calcium from the perfusion fluid prevented the usual release of catecholamines evoked by carbachol. Bolus injections of serotonin (250nmolkg−1) into the inflowing perfusion fluid resulted in significantly increased levels of adrenaline and noradrenaline in the outflowing perfusate. Addition of hexamethonium to the perfusion fluid did not abolish this serotonin-induced release of catecholamines. The serotonin-induced release of adrenaline, however, was abolished totally by the addition of methysergide. Serotonin is present in high concentrations (44.61±5.96 μgg−1 tissue) in the anterior region of the posterior cardinal vein within the head kidney. Carbachol (10−5 molkg−1) did not elicit release of the stored serotonin from the perfused head kidney preparation. We conclude that the chromaffin cells in the perfused trout head kidney preparation display characteristics similar to those of other vertebrates and that this preparation is a useful tool for studying the control of catecholamine release in fish. The results demonstrate that serotonin has a direct impact on the chromaffin cells by interacting with methysergide-sensitive receptors to initiate the release of adrenaline. The potential physiological role of serotonin on catecholamine release in trout is discussed.

Gas transfer and blood acid-base status in the blood-perfused trout head preparation (in vitro) were compared with intact trout (in vivo) fitted with oral membranes, dorsal aortic, ventral aortic, and opercular cannulae. Gas transfer rates were calculated from both arterial-venous blood differences and inspired-expired water differences using the Fick method. Oxygen uptake and carbon dioxide excretion were lower, while ammonia excretion was higher, in the blood-perfused head relative toin vivo rates. Gas transfer rates calculated from water were consistently greater than rates calculated from blood, the difference being greaterin vitro compared toin vivo. We conclude that the inadequacy of O2 and CO2 transfer in the blood-perfused head was not due to abnormal gill diffusive conductance, but was more likely related to the reduced magnitude of the blood-to-water O2 and CO2 diffusion gradients, low hematocrit, and decreased perfusion flow rate . Under the conditions of the present study, the blood-perfused trout head is not a suitable preparation for the study of oxygen transfer. We conclude this preparation is useful for evaluating branchial carbon dioxide or ammonia transfer only when comparable measurements or manipulations cannot be made on intact fish.

Carbon dioxide excreted across fish gills is hydrated catalytically to form HCO3- and H+ ions in water near the gill surface. We tested the possibility that CO2 excretion is functionally linked to ammonia excretion through chemical reactions in the gill-water boundary layer. A bloodperfused trout head preparation was utilized in which the convective and diffusive components of branchial gas transfer were controlled. Pre-incubation of blood perfusate with the carbonic anhydrase inhibitor, acetazolamide, reduced both carbon dioxide and ammonia excretion in the blood-perfused preparation. Increasing the buffering capacity of inspired ventilatory water significantly reduced ammonia excretion, but carbon dioxide excretion was unaffected. Each of these experimental treatments significantly reduced the acidification of ventilatory water flowing over the gills. It is proposed that the catalysed conversion of excreted CO2 to form HCO3- and H+ ions provides a continual supply of H+ ions need for the removal of NH3 as NH4+. We suggest, therefore, that acidification of boundary layer water by CO2 enhances blood-to-water NH3 diffusion gradients and facilitates ammonia excretion.

Gulf toadfish, Opsanus beta, are facultatively ureotelic and can excrete the majority of their nitrogenous waste as urea. Urea excretion occurs in “pulses.” The hypothesis that pulsatile urea excretion reflects sudden, transient, generalized increases in the branchial conductance was investigated by the simultaneous monitoring of cardiorespiratory variables, oxygen uptake, and whole‐body urea, ammonia, and/or 3H2O effluxes. The direct monitoring of both expired branchial water and water exiting a respirometer demonstrated that urea pulses were derived from the gills. No significant changes in ventilation or cardiac frequency, oxygen uptake, or ammonia efflux were observed during natural urea pulses, refuting the hypothesis that pulsatile urea excretion reflects pulsatile increases in the generalized diffusive properties of the gill for solute transfer. An alternative model for pulsatile urea excretion postulates that the gill urea permeability is increased periodically by the insertion and/or activation of specific urea transporters into gill cell membranes. Pulsatile urea excretion was abolished by pretreatment with the cytoskeletal‐disrupting agent colchicine; colchicine may block trafficking of urea transporter–containing vesicles. Exocytosis of water following the fusion of vesicles with gill cell membranes could explain the significantly elevated 3H2O efflux observed during urea pulses.

We have investigated the effects of chronic hypoxia on the acute adrenergic stress response of adult rainbow trout (Oncorhynchus mykiss). The goal of this study was to determine whether a prior 5‐d exposure of fish to lowered environmental oxygen levels (60 or 80 Torr) would influence the nature of catecholamine secretion from chromaffin tissue in situ. Using a saline‐perfused posterior cardinal vein preparation, it was demonstrated that the basal (unstimulated) secretion of noradrenaline and adrenaline was increased at 60‐Torr hypoxia. In response to cholinergic (carbachol‐elicited) stimulation, noradrenaline and adrenaline secretion were significantly affected by prior exposure to hypoxia. The construction of dose response curves revealed that noradrenaline secretion was enhanced at the lowest doses of carbachol (1 ‐ 5 X 1007 mol kg01) and that this was reflected by an approximate 10‐fold reduction in the ED50 (the dose of carbachol eliciting halfmaximal noradrenaline secretion). The effect of chronic hypoxia on in situ carbachol‐evoked adrenaline secretion was similar but less pronounced. The results of this study suggest that during chronic moderate hypoxia, increased basal catecholamine secretion and enhanced responsiveness of chromaffin cells to cholinergic stimulation, as well aiding the ongoing stress, may assist the physiological adaptations to subsequent bouts of more severe acute stress.

Carbonic anhydrase activity in the extracellular fluid of lower vertebrates is considered to be minimal, either because of the absence of carbonic anhydrase or because of the presence of naturally occurring inhibitors. The presence of carbonic anhydrase activity and circulating inhibitors was measured in plasma and subcellular fractions of gill tissue in elasmobranchs and teleosts. Plasma carbonic anhydrase activity was confirmed in the former but in extremely low amounts, especially compared with activity in red cells. The activity was correlated with plasma iron concentration and red cell hemolysis, which suggests that it is a byproduct of endogenous hemolysis during red cell turnover. A subcellular fraction of dogfish gills rich in microsomes contained significantly higher carbonic anhydrase activity than previously found in teleosts, making elasmobranchs the only aquatic lower vertebrates to possess putative basolateral membrane‐associated carbonic anhydrase in the gill vasculature. It is suggested that branchial membrane‐associated carbonic anhydrase is correlated more with a pH and/or CO2‐sensitive ventilatory drive than with the maintenance of resting CO2 excretion. The occurrence and effectiveness of plasma carbonic anhydrase inhibitors were highly species‐specific, with the salmonids having the most potent inhibitor. Cross‐reactivity of inhibitor to red cell carbonic anhydrase appeared to be related to phylogenetic proximity. Selection for the presence of carbonic anhydrase inhibitors in fish plasma appears to be the result of multiple physiological pressures, including preservation of red cell intracellular pH, ventilatory control, and red cell fragility.

We have investigated the influence of cortisol on the β-adrenoreceptor population of rainbow trout [Oncorhynchus mykiss (Walbaum)] erythrocytes and determined what impact it has on the adrenergic responsiveness of erythrocytes in vitro to exogenous catecholamines. To do so, the erythrocyte β-adrenoreceptors were characterized in fish with chronically elevated plasma cortisol levels (118±5.9ngml−1, >10 days) and compared with shams, using radioreceptor assay techniques. The number of ‘internalized’, low-affinity receptors was increased when cortisol levels were raised, but the number of high-affinity, ‘surface’ receptors was not altered. The physiological significance of this response was ascertained by assessing the in vitro sensitivity (or responsiveness) of erythrocytes to adrenaline and noradrenaline (10–1000 nmol I−1) under normoxic or hypoxic conditions. Erythrocyte sensitivity to catecholamines, as determined by changes in both whole-blood pH (ΔpHe) and intracellular cyclic AMP content, was greater in hypoxic than in normoxic blood. Although cortisol further enhanced the responsiveness of erythrocytes to catecholamines, this amplification in sensitivity was observed only during hypoxia. When the radioreceptor assay was conducted using erythrocytes from the catecholamine sensitivity experiments, results were consistent with initial receptor density data. An increase in surface receptor density was associated with hypoxia in vitro. This hypoxia-specific increase in surface β-adrenoreceptors was significantly enhanced in the cortisol-treated erythrocytes, showing that cortisol had a significant impact on erythrocyte β-adrenoreceptor dynamics in addition to the direct influence of hypoxia. This study has shown (1) that, by itself, in vitro hypoxia simultaneously initiates the movement of internal receptors to the cell surface, where they become physiologically active, and the replenishment of the internal receptor pool,(2) that cortisol increases receptor availability by increasing the internal pool of low-affinity receptors in the absence of any stimulus for receptor mobilization, and (3) that the sensitivity of erythrocytes to catecholamines is directly proportional to the number of high-affinity receptors present at the erythrocyte surface. Thus, we suggest that, under conditions of chronic stress, cortisol may pre-adapt the erythrocytes to receive additional physiological inputs that can ultimately enhance respiratory performance beyond that which would be possible in the absence of chronically elevated levels of cortisol.

Although many studies have characterized these receptors according to pharmacological criteria, this work represents only the second direct characterization of the rainbow trout β-adrenergic receptors. Radioligand binding assays using (±)-4-(3-t-butylamino-2-hydroxy-propoxy)-[5,7-3H]benzimidazol-2-one ([3H]CGP 12177) and l-[4,6-propyl 3H]dihydroalprenolol ([3H]DHA) were conducted to determine equilibrium binding times, ligand-receptor dissociation constants (KD) and binding capacities (Bmax). Furthermore, we assessed the influence of erythrocyte handling, suspension medium and endogenous catechol amines on Bmax and KD. Maximal binding was obtained when erythrocytes were handled minimally and maintained suspended in plasma rather than physiological saline. Washing and resuspending the erythrocytes, as well as the transfer of the erythrocytes into saline, significantly impaired apparent radioligand affinity and receptor density. Endogenous catecholamines, at levels considered normal for non-stressed animals, did not interfere with the radioligand binding assays, and thus eliminated the need to wash and resuspend erythrocytes. Based on the binding characteristics after intentional lysis of erythrocytes, it is shown that the total receptor population of trout erythrocytes can be estimated by propranolol-displaceable DHA binding, the density of high-affinity surface receptors can best be determined by isoproterenol-displaceable CGP binding, and the number of receptors located within the erythrocytes can be calculated from the difference between the total receptor density and the number of isoproterenol-displaceable DHA binding sites. Each of these components must be considered when performing radioreceptor assays using these radioligands and this has significant implications for the interpretation of erythrocyte β-adrenoreceptor localization and mobilization.

Metabolic responses (rates of CO2 production from 14C-labelled glucose or lactate, and total O2 consumption) of red blood cells were monitored in rainbow trout (Oncorhynchus mykiss) at rest and during 12 h of recovery from exhaustive exercise. Extracellular acid-base status, red blood cell intracellular pH (pHi), and plasma.metabolite and catecholamine levels were recorded simultaneously. Despite a post-exercise rise in plasma glucose level, glucose oxidation was depressed, at least partly because of a rise in plasma lactate level. However, lactate oxidation was stimulated markedly, especially at 0–2 h post-exercise. Subsequent multifactorial experiments in vitro demonstrated that augmentation of lactate oxidation was due partly to increased plasma lactate, and partly to separate stimulatory effects of elevated and catecholamine levels. Changes in pH and HCO3− level were not directly involved, but the stimulatory effects of catecholamines occurred only under acidotic conditions. Total red cell O2 consumption remained generally stable after exercise. Similar multifactorial experiments in vitro demonstrated that respiratory, metabolic and mixed acidoses all inhibited , an effect largely attributable to the lowered pH. This inhibition was reversed by typical post-exercise levels of epinephrine and norepinephrine; again, catecholamines had no effect under control conditions. Red cell pHi regulation was achieved without an increase in above resting levels. Our results indicate a complex sensitivity of red cell metabolism to acid-base status and a shift in substrate preference for oxidation after strenuous exercise. The mobilization of catecholamines plays an important coordinating role and helps sustain normal rates of oxidative metabolism by red cells in the face of post-exercise blood acidosis.

Red blood cell metabolism was studied in vitro using whole blood obtained by catheter from resting rainbow trout (Oncorhynchus mykiss). Preparations were viable as shown by stable NTP, metabolite and catecholamine levels and acid-base status, all of which remained at in vivo levels over the 2h incubation period. Enzymes diagnostic of glycolysis, the tricarboxylic acid (TCA) cycle and phosphagen metabolism were all present-in significant amounts in red blood cells. In direct comparisons of 14C-labelled substrates at normal resting plasma concentrations, rates of CO2 production were in the order: glucose>lactate>ala-nine>oleate. Total CO2 production rates from these four oxidative substrates did not equal directly measured O2 consumption rates, indicating that other substrates may also be important in vivo. Oxidative pathway Km values for glucose (8.4mmoll−1), lactate (3.3mmoll−1) and alanine (0.8mmoll−1) were well within the normal physiological ranges of plasma concentrations. Glucose concentration did not affect lactate oxidation rates, but there was some inhibition (27%) of glucose oxidation by high lactate concentrations (20mmol l−1). The observed Km values and competitive interactions suggest that changes in plasma concentrations associated with environmental stresses can considerably alter the relative rates of oxidation of glucose and lactate in vivo. Considerable pentose-phosphate shunt activity was detected in red cells, as indicated by high activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and high CO2 production rates from (l-14C)-labelled glucose. Even in the presence of normal O2 levels, a significant percentage (28%) of glucose metabolism was directed to lactate production. Taken together, these results demonstrate that rainbow trout whole blood incubated in vitro constitutes a dynamic and viable system for metabolic studies at the pathway level.

Summary Daily intramuscular injection of cortisol (4 mg kg-1 body weight) in rainbow trout,Salmo gairdneri, for 10 days caused significant increases in the number and individual apical surface area of gill chloride cells per mm2 of filament epithelium. Concomitantly, whole body influxes of sodium (Na+) and chloride (Cl-) increased. Acute (3 h) intra-arterial infusion of cortisol did not affect whole body Na+ or Cl- influx. A significant correlation was observed between both Na+ and Cl- influxes and the fractional apical surface area of filament chloride cells in control, sham (saline-injected) and experimental (cortisol-injected) fish. The chloride cells displayed similar ultrastructural modifications in trout undergoing cortisol treatment as in trout transferred to ion-deficient water. These findings suggest the existence of structure/function relationships in which branchial chloride cell morphology is an important determinant of Na+ and Cl- transport capacity. We conclude that chronic cortisol treatment enhances whole body Na+ and Cl- influxes by promoting proliferation of branchial chloride cells. The results of correlation analysis indicate that the chloride cell is an important site of NaCl uptake in freshwater rainbow trout.

Exposure of adult brown bullheads Ictalurus nebulosus (120–450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (JnetNa+) and net chloride flux (JnetCl-) resulting largely from changes in whole body sodium influx (JinNa+) and chloride influx (JinCl-). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in JinCl-during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in JinNa+did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the post-hypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl-/HCO3-exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.

Whole-body ionic fluxes and gill chloride cell (CC) morphology were monitored in rainbow trout (Salmo gairdneri) exposed acutely or chronically to natural fresh water (NFW; [Na+]=0·120 mmol l−1; [Cl−]=0·164 mmol l−1) or artificially prepared fresh water with reduced [NaCl] (AFW; [Na+]=0·017 mmoll−1; [Cl−]=0·014mmoll−1). Net fluxes of Na+ and Cl− became extremely negative (indicating net NaCl loss to the environment) upon immediate exposure to AFW exclusively as a result of reduced NaCl influx ( and ) and were gradually restored to control rates during prolonged (30 days) exposure to AFW. The restoration of in AFW was due both to increased and to reduced Cl− efflux , whereas the primary response contributing to the restoration of was an increase of . The total apical surface area of branchial CCs exposed to the external environment increased markedly after 24 h in AFW and remained elevated for 1 month as a consequence of enlargement of individual CCs and, to a lesser extent, increased CC density. and were correlated significantly with total CC apical surface area. Plasma cortisol levels rose transiently in fish exposed to AFW. Treatment of NFW-adapted fish with cortisol for 10 days (a protocol known to cause CC proliferation) caused pronounced increases in and as measured in both NFW and AFW. These results suggest that an important adaptational response of rainbow trout to low environmental [NaCl] is cortisol-mediated enlargement of branchial epithelial CCs which, in turn, enhances the NaCl-transporting capacity of the gill as a result of the proliferation of Na+ and Cl− transport sites.

This study tests the hypothesis that catecholamines regulate glucose availability during hypoxia in the rainbow trout by activating glycogen phosphorylase (GPase) while inhibiting pyruvate kinase (PK) in the liver. The net result would be an increase in liver glycogenolysis and a reduction of glycolysis and/or enhancement of gluconeogenesis. We used the criteria of Stalmans & Hers (1975) and report much lower resting percent GPase a (active) values (20–30%) than those previously published. Dorsal aortic injections of epinephrine or norepinephrine increased plasma glucose (16–46%), had no effect on liver or muscle glycogen levels, decreased the activity of PK, and increased total and percent GPase a activities. Pre-treatment with the β-adrenoreceptor antagonist propranolol eliminated these effects. During moderate hypoxia, plasma glucose remained unchanged, while lactate levels increased fourfold. When fish were pre-treated with propranolol, hypoxia depressed plasma glucose levels (−26%), total and percent GPase a, and increased PK activity, suggesting that hypoxia mediated the dephosphorylation of these enzymes. We conclude that catecholamines stimulate hepatic β-adrenoreceptors during hypoxia and sustain plasma glucose levels by nullifying the deleterious effects of hypoxia on metabolic function. The specific metabolic consequences of these catecholamine-mediated effects are an increase in the activity of the active form of GPase and a reduction in PK activity, which suggests an activation of glycogenolysis and an inhibition of glycolysis and/or activation of gluconeogenesis, respectively.

The effects of severe experimental anaemia on red blood cell HCO3- dehydrationin vitro were examined in rainbow trout,Oncorhynchus mykiss. After 5 days of anaemia (haematocrit=4.9±1.1%) induced by intraperitoneal injection of phenylhydrazine hydrochloride, fish displayed elevated arterial CO2 tensions (anaemic PaCO2=3.19±0.42 torrvs. control PaCO2=1.35±0.17 torr) and a significant acidosis (anaemic pHa=7.73±0.04vs. control pHa=7.99±0.04). However, after 15–20 days of anaemia (hct=6.6±0.8%) induced by blood withdrawal, the arterial CO2 tension was significantly lower than the control value, suggesting that physiological adjustments occurred within this time period to compensate for the lowered haematocrit. Compensation probably did not involve alterations in ventilation, which was unaffected by 5 days of anaemia (anaemic ;w=786±187 ml min-1 kg-1vs. control ;w=945±175 min-1 kg-1), based on indirect Fick principle measurements.

Rainbow trout, Oncorhynchus mykiss, were exposed to 48h of environmental hypoxia (water partial pressure of oxygen = 8.0 kPa) at either 5 or 15°C. Blood was sampled during hypoxia via a dorsal aorta cannula to measure arterial blood partial pressure of oxygen and plasma catecholamine concentrations. After 48h, the number (Bmax) and dissociation constant (Kd) of red blood cell surface ß-adrenoceptors were determined using a radioligand-displacement binding assay. At 5°C, plasma catecholamine levels were elevated at 24h whereas at 15°C, levels were elevated at 48h. At either temperature, following 48h of hypoxia, there was no change in Bmax or Kd of red blood cell surface ß-adrenoceptors, compared to normoxic control fish. This study demonstrates that chronic exposure to moderate hypoxia does not affect the number or affinity of cell surface ß-adrenoceptors on trout red blood cells.

The effects of serotonin (5-hydroxytryptamine; 5-HT) on continuously recorded dorsal and ventral aortic blood pressures, (PDA, PVA), arterial oxygen tension arterial carbon dioxide tension extracellular pH (pHa), buccal pressure (Pbuccal) and plasma catecholamine levels were investigated in rainbow trout, Oncorhynchus mykiss. Intra-arterial injections of serotonin (50–250 nmol kg−1) caused a rapid decrease in PDA and an increase in PVA, suggesting vasoconstriction of the branchial vasculature. The blood pressure changes were accompanied by a reduction in (approximately 3–8 kPa depending on the dose injected), an increase in (approximately 0.03–0.07 kPa) and a decrease in pHa (approximately 0.02–0.12 pH units). These changes, indicative of impaired gas transfer, occurred despite obvious hyperventilation based on measurements of buccal pressure. After pre-treatment with the serotonergic receptor antagonist methysergide, injections of 100nmolkg−1 serotonin caused an increase in a reduction in and an increase in pHa. Methysergide treatment did not affect the usual serotonin-induced hyperventilation or the reduction in PDA but did abolish the rise in PVA; indeed, PVA was lowered significantly by serotonin after methysergide treatment. This reduction in PVA was eliminated by pre-treatment of fish with the combination of methysergide and sotalol (β-adrenoceptor antagonist), suggesting an adrenergic component to the overall blood pressure response. Analysis of plasma catecholamines after injection of serotonin revealed that high doses (50, 100, 250 nmol kg−1) caused significant increases in concentrations of both noradrenaline and adrenaline. We conclude that the cardio-respiratory effects of exogenous serotonin injections are complex and arise from several integrated responses, including the direct action of serotonin on receptors within the branchial and systemic vasculatures, indirect action on ganglionic receptors, and the stimulation of catecholamine release from adrenergic nerves and/or chromaffin cells.

Experiments were performed invitro specifically to elucidate the underlying mechanism(s) of the attenuated adrenergic responses of eel (Anguillarostrata) erythrocytes. This was achieved by comparing β-adrenoceptor numbers and affinities in addition to (i) Na+/H+ exchange activity, (ii) cell swelling and (iii) cyclic AMP formation mediated by catecholamines in eel and trout (Oncorhynchusmykiss) erythrocytes under normoxic and hypoxic conditions. Under normoxic conditions, eel erythrocytes displayed a total absence of Na+/H+ exchange activity (as determined from measurements of extracellular pH) after addition of noradrenaline (50–1000 nmol l− 1) in contrast to a pronounced dose-dependent response in trout. Incubation of the blood under hypoxic conditions, to achieve approximately 50% haemoglobin O2-saturation, further increased the extent of Na+/H+ exchange activation in trout and elicited a statistically significant, although physiologically small (10% of the response in trout), activation of H+ extrusion activity in eel. Catecholamine-mediated cell swelling, although obvious in trout, was absent in eel when estimated under hypoxic conditions. Eel erythrocytes possessed approximately 50% fewer surface β-adrenoceptors than did trout erythrocytes, although the dissociation constants (KD) of these receptors did not differ between eel and trout. The numbers and affinities of the erythrocyte β-adrenoceptors were not significantly affected by the hypoxic incubation. Both eel and trout erythrocytes displayed a dose-dependent elevation of cyclic AMP concentration in response to noradrenaline that was further increased by hypoxia. Surprisingly, eel erythrocytes produced larger quantities of cyclic AMP despite the lower numbers of surface ^adrenoceptors. Thus, the absence of adrenergic swelling and the attenuated H+ extrusion response in eel erythrocytes cannot be attributed to insufficient numbers of β-adrenoceptors or to functional uncoupling of these receptors from adenylate cyclase. Instead, the differences between trout and eel may reflect differing numbers of Na+/H+ exchangers or fundamental differences in the manner by which these exchangers are activated by cyclic AMP.

A spontaneously ventilating, blood-perfused trout preparation is described and its suitability for the study of gas exchange in fish assessed. Cardiovascular dynamics closely approximated those found in vivo; perfusion flow rate .100 g−1, ventral aortic pressure (VAP) = 58·8 cm H2O, dorsal aortic pressure (DAP) = 34·8 cm H2O. Gas exchange characteristics in the branchial and systemic circulations also were similar to those described for resting, intact rainbow trout. All preparations showed consistent oxygen uptake . Min−1.100 g−1) and carbon dioxide excretion rates . Min−1.100 g−1) acro the gills. Across the systemic circulation, oxygen was extracted .min−1.100 g−1) and carbon dioxide produced by the metabolizing tissue (.min−1, 100 g−1). The respiratory quotient (REJ for gas transfer across the gills was 1·85. This high value was a reflection of the fact that much more oxygen than carbon dioxide was added to venous blood in the tonometer. The respiratory quotient for the tissues (RQs) was 0·83, a more reasonable value. Breathing rate (fg) was maintained at 69·4 ventilations. Min−1. The mean vascular resistance of blood-perfused gills (Rg) was 14·2 cm H2O.ml−1.min. 100 g−1, a value higher than that usually measured in vivo. Mean systemic vascular resistance (Rs) was 19·2 cm H2O. ml−1. min. 100 g−1 which is similar to that measured in intact fish. Cardiovascular responses to hypoxia and adrenergic responses in the branchial and systemic circulations of these preparations also closely approximated those found in vivo. This preparation is deemed suitable for studies of the cardiovascular system as well as gas transfer. The results from these experiments are representative of the in vivo condition in fish.

A spontaneously ventilating, blood-perfused trout preparation was used to examine gas exchange across the gills. The blood flow rate and input oxygen content to the branchial circulation were manipulated to assess the contributions of perfusion and diffusion limitations to oxygen transfer. Increases in the flow rate or the haematocrit (Hct) were positively correlated with increases in the oxygen uptake across the gills Manipulation of pulse pressure or frequency of the pump, with no changes to had no effect on Addition of adrenaline (1 × 10−6 M) to the blood also did not effect Calculations of cardiac output from the Fick principle always yielded values which were overestimates of the actual cardiac output set by the mechanical pump. The difference between the measured oxygen uptake by the fish from the water and the amount of oxygen transferred to the blood across the gills was a reflection of gill tissue metabolism. It is concluded that trout gills, like mammalian lungs, are primarily perfusion limited for oxygen uptake under resting normoxic conditions, but decreases in diffusion limitations come into play under stress conditions, such as environmental hypoxia or exercise.

A spontaneously ventilating blood-perfused trout preparation and saline perfused gill preparations were utilized to investigate the role of the erythrocyte and branchial epithelium in CO2 excretion and acid-base regulation. CO2 excretion blood-perfused preparations was positively correlated with haematocrit (Het), and was abolished completely during plasma-perfusion. Elevating HCO3− concentration of input blood from 10 to 25 mm significantly increased , fourfold in blood-perfused preparations as a result of increased entry of HCO3− into the red blood cell and not into the gill epithelium. Increased HCO3− concentration was without effect in totally saline-perfused coho salmon (Onchorynchus kisutch). The addition of 4-acetamido-4′-iro-thiocyanatostilbene-2,2′ disulfonic acid (SITS; 10−4 M) to input blood significantly reduced and oxygen uptake in blood-perfused fish due to inhibition of erythrocytic HCO3Ȓ/Cl −exchange. Unlike blood-perfused preparations, no saline-perfused preparation (isolated holobranchs or totally perfused rainbow trout or coho salmon) displayed measureable CO2 excretion at physiological and pH. Increased input in both blood-perfused and saline-perfused preparations significantly increased due to enhanced branchial diffusion of molecular CO2. It is concluded that the entry of HCO3− into the erythrocyte is the rate-limiting step in CO2 excretion and that movement of HCO3− from plasma to gill epithelium cells in no way contributes to overall CO2 elimination.

Fishes living in fresh water counter the passive loss of salts by actively absorbing ions through specialized cells termed ionocytes. Ionocytes contain ATP-dependent transporters and are enriched with mitochondria; therefore ionic regulation is an energy-consuming process. The purpose of this study was to assess the aerobic costs of ion transport in larval zebrafish (Danio rerio). We hypothesized that changes in rates of Na+uptake evoked by acidic or low Na+rearing conditions would result in corresponding changes in whole-body oxygen consumption (ṀO2) and/or cutaneous oxygen flux (JO2), measured at the ionocyte-expressing yolk sac epithelium using the scanning micro-optrode technique (SMOT). Larvae at 4 days post-fertilization (dpf) that were reared under low pH (pH 4) conditions exhibited a higher rate of Na+uptake compared with fish reared under control conditions (pH 7.6), yet they displayed a lower ṀO2 and no difference in cutaneous JO2. Despite a higher Na+uptake capacity in larvae reared under low Na+conditions, there were no differences in ṀO2 and JO2 at 4 dpf. Furthermore, although Na+uptake was nearly abolished in 2 dpf larvae lacking ionocytes after morpholino knockdown of the ionocyte proliferation regulating transcription factor foxi3a, ṀO2 and JO2 were unaffected. Finally, laser ablation of ionocytes did not affect cutaneous JO2. Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na+, are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence that ion regulation in zebrafish larvae incurs a low aerobic cost.

Freshwater fishes absorb Na+from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na+uptake is coordinated by (1) Na+/H+exchanger 3b (Nhe3b) and (2) H+-ATPase-powered electrogenic uptake in H+-ATPase-rich (HR) cells and by (3) Na+-Cl−-cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na+uptake by larval zebrafish under ‘normal’ (800 µmol l−1) and ‘low’ (10 µmol l−1) Na+conditions. We hypothesized that Na+uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2(encoding Nhe3b), particularly in low Na+where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na+uptake was sustained in nhe3b KO larvae under both Na+conditions, which led to the exploration of whether compensatory regulation of H+-ATPase or Ncc was responsible for maintaining Na+uptake in nhe3b KO larvae. mRNA expression of the genes encoding H+-ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H+-ATPase, which significantly reduced H+flux by HR cells, did not reduce Na+uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na+-Cl−-cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H+-ATPase morpholino and chloride-free conditions did not reduce Na+uptake under normal or low Na+. These findings highlight the flexibility of the Na+uptake system and demonstrate that Nhe3b is expendable to Na+uptake in zebrafish and that our understanding of Na+uptake mechanisms in this species is incomplete.