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Convective water flow across respiratory epithelia in water-breathing organisms maintains transcutaneous oxygen (O2) partial pressure (PO2) gradients that drive O2 uptake. Following hatch, larval fishes lack a developed gill and the skin is the dominant site of gas transfer, yet few studies have addressed the contribution of convective water flow to cutaneous O2 uptake in larvae. We hypothesized that the pectoral fins, which can generate water flow across the skin in larvae, promote transcutaneous O2 transfer and thus aid in O2 uptake. In zebrafish (Danio rerio), the frequency of pectoral fin movements increased in response to hypoxia at 4 days postfertilization (dpf), but the response was blunted by 15 dpf, when the gills become the dominant site of O2 uptake, and was absent by 21 dpf. In rainbow trout (Oncorhynchus mykiss), PO2 measured at the skin surface of ventilating larvae was lower when the pectoral fins had been surgically removed, directly demonstrating that fins contribute to convective flow that dissipates cutaneous PO2 boundary layers. Lack of pectoral fins compromised whole animal O2 consumption in trout during hypoxia, but this effect was absent in zebrafish. Overall, our findings support a respiratory role of the pectoral fins in rainbow trout, but their involvement in zebrafish remains equivocal. [ABSTRACT FROM AUTHOR]

The structural features of the fish gill necessary for oxygen uptake also favor undesirable, passive movements of ions and water. Reversible gill remodeling is one solution to this conflict. Cell masses that limit functional surface area are lost when oxygen availability decreases in hypoxia or oxygen demand increases with exercise or high temperature. However, much remains to be learned about how widespread reversible gill remodeling is among fish species, and how and why it occurs. [ABSTRACT FROM AUTHOR]

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

We tested the hypothesis that endogenously produced hydrogen sulfide (H2S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water P02 = 35 mmHg), plasma H2S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H2S in vitro when incubated with L-cysteine. In both tissues, the production of H2S was eliminated by adding the cystathionine β-synthase inhibitor, aminooxyacetate. Cystathionine β-synthase and cystathionine γ-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H2S and catecholamines in the outfiowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 X 10-6 M) or depolarizing levels of KCI (1 X 10-2 M) caused secretion of catecholamines without altering H2S output, suggesting that neuronal excitation is required for H2S release. Addition of H2S (at concentrations exceeding 5 X 10-7 M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca2+-free perfusate was used. These data, together with the finding that H2S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H2S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca2+-mediated exocytosis. [ABSTRACT FROM AUTHOR]

Experiments were performed to assess the possible involvement of the Na+/H+ exchanger isoform 3 (NHE3; SLC9A3) in renal acid-base regulation in adult rainbow trout (Oncorhynchus mykiss). NHE3 mRNA was expressed at high levels in the kidney relative to its paralog, NHE2. The results of in situ hybridization demonstrated an abundance of NHE3 mRNA in renal tubules. The combination of immunocytochemistry and histological staining revealed that NHE3 was confined to the apical membrane of proximal tubules, where it was colocalized with the vacuolar-type H+-ATPase. Levels of NHE3 protein (assessed by Western blotting) were increased during hypercapnia, likely as a result of increased transcription, as indicated by increasing levels of NHE3 mRNA (as determined by real-time PCR). Plasma cortisol concentration was increased during hypercapnia, and administration of exogenous cortisol caused a marked increase in NHE3 mRNA and protein. Thus we speculate that the elevation of plasma cortisol during hypercapnia contributes to transcriptional activation of NHE3 that ultimately promotes acid-base regulation by stimulating H+ secretion and HCO3- reabsorption. [ABSTRACT FROM AUTHOR]

We have taken advantage of the unique properties of nucleated rainbow trout (Oncorhynchus mykiss) red blood cells (rbcs) to demonstrate that b-adrenergic stimulation with the agonist, isoproterenol, significantly enhanced the heat-induced induction of heat-shock proteins (Hsps) in trout rbcs without affecting hsp expression on its own. Furthermore, this β-adrenergic potentiation of hsp expression occurred only at physiologically relevant concentrations of adrenergic stimulation. In further experiments, we found that adrenaline increased 100-fold and noradrenaline increased 50-fold in trout after a 1-h heat shock at 25°C, ~2°C above acclimation temperature. This is the first time the adrenergic heat-shock response has been described for a temperate fish species. We conclude that β-adrenergic stimulation enhances hsp expression in trout rbcs following heat stress, indicating physiological regulation of the cellular stress response in fish. [ABSTRACT FROM AUTHOR]

Experiments were conducted on rainbow trout to determine the impact of dietary salt on arterial blood pressure. After 4-6 wk, fish fed a salt-enriched diet exhibited a 37% elevation of dorsal aortic pressure (from 23.8 ± 1.2 to 32.6 ± 1.4 mmHg) and an 18% increase in ventral aortic pressure (from 33.0 ± 1.5 to 38.9 ± 1.3 mmHg). The hypertension presumably reflected the increase in cardiac output (from 31.0 ± 0.8 to 36.4 ± 2.2 ml·min-1·kg-1) because systemic and branchial resistances were statistically unaltered by salt feeding. The chronic hypertension was associated with a decrease in the pressor responses of the systemic vasculature to catecholamines and hypercapnia in the salt-fed fish. The reduction in α-adrenergic responsiveness of the systemic vasculature is consistent with desensitization or loss of functional α-adrenoceptors (α-ARs). In support of this idea, the salt-fed fish exhibited significantly decreased levels of α1D-AR mRNA in the dorsal aorta and the afferent (ABA) and efferent branchial arteries (EBA). In contrast, however, the results obtained from norepinephrine dose-response curves for EBA and ABA vascular rings in vitro did not provide evidence for loss of function of branchial artery α1-ARs in the salt-fed fish. Indeed, the EC50 for the EBA norepinephrine dose-response curve was significantly reduced (from 3.75 × 10-7 to 2.12 × 10-7 M) in the salt-fed fish, indicating an increase in the binding affinity of the α1-ARs. [ABSTRACT FROM AUTHOR]

Numerous fish species, including rainbow trout (Oncorhynchus mykiss), are able to inhabit both freshwater and seawater and routinely migrate between the two environments. One of the most critical adjustments allowing such successful migrations is a remodelling of the gill in which a suite of morphological and molecular changes ensure optimal function in the face of reversing requirements for salt and water balance. The remodelling leads to specific freshwater and seawater gill phenotypes that are readily identified by the orientation and/or quantities of specific ion transporters and the presence or absence of specific cell types. The proximate cues promoting gill phenotypic plasticity are unknown. Here, by assessing the consequences of a salt-enriched diet (in the absence of any changes in external salinity) in the freshwater rainbow trout, we demonstrate that internal salt loading alone, is able to induce various elements of the seawater gill phenotype. Specifically, we show upregulation of three ion transport genes, cystic fibrosis transmembrane conductance regulator (CFTR), Na+/K+/2Cl- co-transporter (NKCC1) and Na+/K+-ATPase, which are essential for ionic regulation in seawater, and the appearance of chloride cell-accessory cell complexes, which are normally restricted to fish inhabiting seawater. These data provide compelling evidence that gill remodelling during migration from freshwater to seawater may involve sensing of elevated levels of internal salt. [ABSTRACT FROM AUTHOR]

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC rnRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+] = 20-30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (~7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an ~100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells. [ABSTRACT FROM AUTHOR]

Experiments were performed to test the hypothesis that exposure of rainbow trout to repetitive hypoxia would result in a decreased capacity of chromaffin cells to secrete catecholamines owing to increased production of nitric oxide (NO), a potent inhibitor of catecholamine secretion. A partial sequence of trout neuronal nitric oxide synthase (nNOS) was cloned and its mRNA was found to be present in the posterior cardinal vein (PCV), the predominant site of chromaffin cells in trout. Using heterologous antibodies, nNOS and endothelial NOS (eNOS) were localized in close proximity to the chromaffin cells of the PCV. Exposure of trout to acute hypoxia (5.33 kPa for 30 mm) in vivo resulted in significant increases in plasma catecholamine and NO levels. However, after 4 days of twice-daily exposures to hypoxia, the elevation of plasma catecholamine levels during hypoxia was markedly reduced. Associated with the reduction in plasma catecholamine levels during acute hypoxia was a marked increase in basal and hypoxia-evoked circulating levels of NO that became apparent after 2-4 days of repetitive hypoxia. The capacity of the chromaffin cells of the hypoxia-exposed fish to secrete catecholamine was assessed by electrical stimulation of an in situ saline-perfused PCV preparation. Compared with control (normoxic) fish, the PCV preparations derived from fish exposed to repeated hypoxia displayed a significant reduction in electrically evoked catecholamine secretion that was concomitant with a marked increased in NO production. This additional rise in NO secretion in preparations derived from hypoxic fish was prevented after adding NOS inhibitors to the perfusate; concomitantly, the reduction in catecholamine secretion was prevented. The increased production of NO during hypoxia in vivo and during electrical stimulation in situ was consistent with significant elevations of nNOS mRNA and protein; eNOS protein was unaffected. These results suggest that the reduced capacity of trout chromaffin cells to secrete catecholamines after repeated hypoxia reflects an increase in the expression of nNOS and a subsequent increase in NO production during chromaffin-cell activation. [ABSTRACT FROM AUTHOR]

The goal of the present study was to assess the catecholamine secretory capabilities of rainbow trout Oncorhynchus mykiss chromaffin cells experiencing desensitization of the nicotinic receptor. It was hypothesized that the potential to secrete catecholamines could be maintained under conditions of nicotinic receptor desensitization owing to activation of non-cholinergic release pathways. An in situ model for chromaffin cell nicotinic receptor desensitization was developed by perfusing a posterior cardinal vein preparation with saline containing 10[sup -5] mol l[sup -1] nicotine. Under such conditions of desensitization, the chromaffin cells were largely unresponsive to high-frequency (20 Hz) electrical stimulation; the minimal remaining secretory response was abolished by addition of the nicotinic receptor antagonist hexamethonium (10[sup -3] mol l[sup -1]). In marked contrast, however, the capacity to secrete catecholamines in response to low-frequency (1 Hz) electrical stimulation was unaffected by nicotinic receptor desensitization or by cholinergic receptor blockade (hexamethonium plus atropine). In preparations experiencing nicotinic receptor desensitization, the stimulatory effect of low-frequency (1 Hz) stimulation on catecholamine secretion was reduced by 43% in the presence of the VPAC receptor antagonist, VIP[sub 6-28]. The stimulatory effect of high-frequency (20 Hz) stimulation was unaffected by VIP[sub 6-28]. Catecholamine secretion evoked by cod VIP (10[sup -11] mol kg[sup -1]) and homologous angiotensin II ([Asn¹, Val[sup 5]] Ang II; 5×10[sup -7] mol kg[sup -1]) was markedly enhanced (107 and 97%, respectively) in desensitized preparations. However, the secretory response to the muscarinic receptor agonist methylcholine (1×10[sup -3] mol kg[sup -1]) was unchanged by desensitization. The results of this study demonstrate that exploitation of non-cholinergic mechanisms, including peptidergic pathways activated during low-frequency neuronal stimulation, is a potential strategy whereby catecholamine secretion from trout chromaffin cells can be maintained during periods of nicotinic receptor desensitization. [ABSTRACT FROM AUTHOR]

The current model for the neuronal control of catecholamine release from piscine chromaffin cells advocates that the neurotransmitters vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are co-released with acetylcholine from preganglionic fibres upon nerve stimulation. Both VIP and PACAP elicit the secretion of exclusively adrenaline from rainbow trout chromaffin cells, which presumably arises from the activation of VPAC type receptors. Thus, the goals of the present study were (1) to localize VPAC receptors in the chromaffin cell fraction of the posterior cardinal vein (PCV) of trout and (2) to test the hypothesis that the selective secretion of adrenaline elicited by VIP could be explained by the absence of the VPAC receptors from the noradrenaline-containing cells. Fluorescent labelling of chromaffin cells using aldehyde-induced fluorescence of catecholamines and antisera raised against dopamine β-hydroxylase (DβH) revealed a distinct layer of chromaffin cells lining the walls of the PCV. Furthermore, specific VIP-binding sites were demonstrated on chromaffin cells using a biotinylated VIP that was previously established as being bioactive. Although multiple labelling experiments revealed that a number of DβH-positive cells were immunonegative for phenylethanolamine N-methyl transferase (PNMT; noradrenaline-containing cells versus adrenaline-containing cells, respectively), labelling of VIP- binding sites was similar to that of DβH labelling, suggesting that all chromaffin cells possess VIP-binding sites. Pharmacological assessment of the VIP-binding sites indicated that they exhibited characteristics of VPAC receptors. Specifically, the labelling of VIP-binding sites was prevented after pre-treatment of PCV tissue sections with unlabelled VIP, PACAP or the specific VPAC receptor antagonist VIP 6-28. By contrast, sections pretreated with the PAC[sub 1] receptor blocker PACAP 6-27... [ABSTRACT FROM AUTHOR]

Experiments were performed to test the hypothesis that the marked ventilatory and cardiovascular responses to hypercarbia in rainbow trout Oncorhynchus mykiss arise from specific stimulation of chemoreceptors localised to the first gill arch. This was accomplished by measuring cardiorespiratory variables during acute hypercarbia (20 min at P[sub CO[sub 2]]=8mmHg; 1mmHg=0.133kPa) in fish subjected to selective bilateral extirpation of the first gill arch. The cardiovascular responses to hypercarbia in the intact fish included a significant bradycardia (from 75.0±1.6 to 69.0±2.0 beats min[sup -1]; means ± S.E.M.; N=16), an increase in dorsal aortic blood pressure (from 30.8±1.5 to 41.9±2.5 mmHg; N=16) and a rise in systemic vascular resistance (from 1.1±0.1 to 1.4±0.1 mmHg ml[sup -1] kg[sup -1] min[sup -1]; N=16). Removal of the first gill arch or pre-treatment with the muscarinic receptor antagonist atropine prevented the hypercarbic bradycardia without affecting the pressure or resistance responses. Correlation analysis, however, revealed shallow but significant inverse relationships between water P[sub CO[sub 2]] and cardiac frequency in both atropinised (r²=0.75) and gill-extirpated (r²=0.90) fish, suggesting a direct mild effect of CO[sub 2] on cardiac function. The ventilatory response to hypercarbia in the intact fish consisted of an increase in ventilation amplitude (from 0.62±0.06 to 1.0±0.13cm; N=16) with no change in breathing frequency. Removal of the first gill arch lowered resting breathing frequency and prevented the statistically significant elevation of breathing amplitude. Gill extirpation, however, did not totally abolish the positive correlation between water P[sub CO[sub 2]] and ventilation amplitude (r²=0.84), suggesting the presence of additional (although less important) chemoreceptive sites that are not confined to the first gill arch. Plasma catecholamine... [ABSTRACT FROM AUTHOR]

Assesses the role of renin-angiotensin system in catecholamine release during hypoxia periods in rainbow trout. Increase of plasma catecholamine levels upon nicotinic receptor desensitization; Activation of the renin-angiotensin system during hypoxia; Role of angiotensin II in facilitating catecholamine release.

The buffering capacity (β) of rainbow trout (Oncorhynchus mykiss) plasma was manipulated prior to intravascular injection of bovine carbonic anhydrase to test the idea that proton (H+) availability limits the catalysed dehydration of HCO3– within the extracellular compartment. An extracorporeal blood shunt was employed to continuously monitor blood gases in vivo in fish exhibiting normal plasma β (–3.9±0.3 mmol l–1 pH unit–1), and in fish with experimentally (using N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) elevated plasma β (–12.1±1.1 mmol l–1 pH unit–1). An injection of 5 mg kg–1 carbonic anhydrase equally reduced (after 90 min) the arterial partial pressure of CO2 in trout with regular (–0.23±0.05 Torr) or high (–0.20±0.05 Torr) plasma β; saline injection was without effect. Because ventilation and venous blood gases were unaffected by carbonic anhydrase, the effect of extracellular carbonic anhydrase in lowering arterial partial pressure of CO2 was likely caused solely by a specific enhancement of CO2 excretion owing to acceleration of HCO3– dehydration within the plasma. The lowering of arterial partial pressure of CO2 in trout after injection of exogenous carbonic anhydrase provides the first in vivo evidence that the accessibility of plasma HCO3– to red blood cell carbonic anhydrase constrains CO2 excretion under resting conditions. Because the velocity of red blood cell Cl–/HCO3– exchange governs HCO3– accessibility to red blood cell carbonic anhydrase, the present study also provides evidence that CO2 excretion at rest is limited by the relatively slow rate of Cl–/HCO3– exchange. The effect of carbonic anhydrase in lowering arterial partial pressure of CO2 was unrelated to plasma buffering capacity. While these data could suggest that H+ availability does not limit extracellular HCO3– dehydration in vivo at resting rates of CO2 excretion, it is more likely that the degree to which plasma β was elevated in the present study was insufficient to drive a substantially increased component of HCO3– dehydration through the plasma. [ABSTRACT FROM AUTHOR]

Focuses on a study which assessed the potential direct and modulating effects of acute hypoxia on catecholamine secretion in rainbow trout, Oncorhynchus mykiss using a perfused cardinal vein preparation and chronically cannulated fish. Materials and methods; Effects of hypoxic perfusate on in situ basal catecholamine secretion rates; Influence of acute moderate hypoxia on nicotine-evoked catecholamine release in vivo.

Discusses a study which assessed the relative involvement of nicotinic and muscarinic cholinergic receptors in the neuronal control of catecholamine secretion from the chromaffin tissue of rainbow trout. Background information on the mechanism of catecholamine secretion in teleosts; Materials and methods; Results; Discussion.

Determines the contribution of angiotensin II (Ang-II)-elicited increases in humoral catecholamines levels to rainbow trout cardiovascular control. Effect of homologous rainbow trout Ang-II in plasma catecholamine levels; Cardiovascular effects of Ang-II with and without alpha-adrenoreceptor blockade; Relationship between plasma concentrations and their cardiovascular effects.

Investigates the relationships between angiotensins and catecholamine release from the chromaffin tissue of rainbow trout. Evidence that angiotensins can elicit catecholamine release; Method in which angiotensins effects this release.

Reports that repeated physical stress significantly enhances the adrenergic responsiveness of the rainbow tout red blood cell. Experiments; Results; Discussion; Interrelationships between chronic and acute stress responses in fish.

Characterizes the acid-base disequilibrium in the arterial blood of rainbow trout Oncorhynchus mykiss during environmental hypoxia, hyperoxia or hypercapnia. Extracorporeal circulation; Measurement of pH, partial pressure of oxygen and carbon dioxide; Ventilation.

Examines the response of cannulated rainbow trout to acute hypoxia in fish acclimated to two temperatures (5 and 15 degrees Celsius). Effects of acclimation on catecholamine release; Effects of acclimation temperature on hemoglobin oxygen-affinity; Other potential causes/correlates of catecholamine release.

Evaluates several potential factors limiting carbon dioxide excretion in the rainbow trout. Hematocrit level; Lysis of red blood cells (RBC); Carbonic anhydrase; RBC intracellular acidity.