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Fish exposed to hypoxia develop decreased heart rate, or bradycardia, the physiological significance of which remains unknown. The general muscarinic receptor antagonist atropine abolishes the development of this hypoxic bradycardia, suggesting the involvement of muscarinic receptors. In this study, we tested the hypothesis that the hypoxic bradycardia is mediated specifically by stimulation of the M2 muscarinic receptor, the most abundant subtype in the vertebrate heart. Zebrafish (Danio rerio) were reared at two levels of hypoxia (30 and 40 Torr P[O.sub.2]) from the point of fertilization. In hypoxic fish, the heart rate was significantly lower than in normoxic controls from 2 to 10 days posffertilization (dpf). At the more severe level of hypoxia (30 Torr P[O.sub.2]), there were significant increases in the relative mRNA expression of M2 and the cardiac type [beta]-adrenergic receptors ([beta]1AR, [beta]2aAR, and [beta]2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at 40 Tort P[O.sub.2]) or significantly attenuated (at 30 Torr P[O.sub.2]) in larvae experiencing M2 receptor knockdown (using morpholino antisense oligonucleotides). Sham-injected larvae exhibited typical hypoxic bradycardia in both hypoxic regimens. The expression of [beta]1AR, [beta]2aAR, [beta]2bAR, and [M.sub.2] mRNA was altered at various stages between 1 and 4 dpf in larvae experiencing [M.sub.2] receptor knockdown. Interestingly, [M.sub.2] receptor knockdown revealed a cardioinhibitory role for the [beta].sub.2]-adrenergic receptor. This is the first study to demonstrate a specific role of the [M.sub.2] muscarinic receptor in the initiation of hypoxic bradycardia in fish. heart rate; cardiovascular function; morpholino; knockdown

Goldfish (Carassius auratus) acclimated to 7[degrees]C and exposed to hypoxia (~10 mmHg) for 7 days exhibited a pronounced remodeling of the gill consisting of the removal of an interlamellar cell mass (ILCM). Subsequent experiments were designed to assess the impact of gill remodeling and the associated increase in functional lamellar surface area on the distribution of branchial ionocytes and [Cl.sup.-] flux across the gill. Despite the increased functional lamellar surface area during hypoxia, there was no corresponding increase in [Cl.sup.-] loss or efflux of the extracellular marker polyethylene glycol (PEG 4000). However, when hypoxic fish were returned to normoxic water for 12 h, rates of [Cl.sup.-] and PEG efflux were markedly stimulated in keeping with an increased surface area for solute movement. Similarly, the rate of branchial [Cl.sup.-1] uptake was reduced (105 [+ or -] 22 vs. 45 [+ or -] 8 [micro]mol x [kg.sup.-1] x [h.sup.-1]) in normoxic and hypoxic fish, respectively, but then stimulated (345 [micro]mol x [kg.sup.-1] x [h.sup.-1] ) upon reestablishment of normoxic conditions. Hypoxia (7 days) was accompanied by a significant decrease in the total cross-sectional area of branchial ionocytes owing to a decrease in their numbers and individual sizes. Thus, despite experiencing an increase in functional lamellar surface area, hypoxic goldfish limit branchial [Cl.sup.-] loss likely by a hypoxia-mediated decrease in paracellular permeability. In normoxic fish, the ionocytes were largely confined to the outer edges of the ILCM. During hypoxia, preexisting ionocytes migrated with the shrinking ILCM, while a smaller proportion of newly differentiated cells appeared below the surface of the ILCM. The capacity to maintain a population of ionocytes in contact with the water is an appropriate strategy to retain ionoregulatory capabilities regardless of whether the lamellae are uncovered or covered. hypoxia; ion regulation; C1 balance; mitochondrion-rich cell; ionocyte; [Na.sup.+ /[K.sup.+]-ATPase; interlamellar cell mass

Hypoxia and increased temperature alter venous blood pressures in teleosts through active changes in venous tone. Elasmobranchs possess a capacious venous system but have limited adrenergic vascular innervation and subambient central venous pressure ([P.sub.cv]). In this study, we explored venous hemodynamic responses to acute temperature increase and moderate (6.9 kPa) and severe (2.5 kPa) hypoxia in the dogfish (Squalus acanthias). Normoxic dogfish at 10[degrees]C had a [P.sub.cv] between -0.08 and -0.04 kPa and a mean circulatory filling pressure ([P.sub.mcf]) of ~0.12 kPa. At 16[degrees]C, heart rate ([f.sub.H]), cardiac output (Q), and [P.sub.mcf] increased but [P.sub.cv] and plasma epinephrine and norepinephrine levels were unchanged. In contrast, moderate and severe hypoxia increased [P.sub.cv] and decreased Q and stroke volume ([V.sub.s]). [f.sub.H] decreased in severe hypoxia, whereas [P.sub.mcf] was unaffected despite elevated catecholamine levels. Atropine abolished hypoxic reductions in Q, [V.sub.s], and [f.sub.H], but [P.sub.cv] still increased. In contrast to the response in teleosts, this study on dogfish suggests that venous capacitance changes associated with warming and hypoxia are minimal and likely not mediated by circulating catecholamines. Thus hemodynamic status of the capacious elasmobranch venous circulation is potentially regulated by blood volume shifts from passive flow-mediated events and possibly through myogenic mechanisms. cardiac filling; cardiac performance; elasmobranch; mean circulatory filling pressure

We tested the hypothesis that endogenously produced hydrogen sulfide ([H.sub.2]S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecfiolamine secretion from chromaffin cells. During acute hypoxia (water [Po.sub.2] = 35 mmHg), plasma [H.sub.2]S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced [H.sub.2]S in vitro when incubated with L-cysteine. In both tissues, the production of [H.sub.2]S was eliminated by adding the cystathionine [beta]-synthase inhibitor, aminooxyacetate. Cystathionine [beta]-synthase and cystathionine [gamma]-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of [H.sub.2]S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x [10.sup.-6] M) or depolarizing levels of KC1 (1 x [l0.sup.-2] M) caused secretion of catecholamines without altering [H.sub.2]S output, suggesting that neuronal excitation is required for [H.sub.2]S release. Addition of [H.sub.2]S (at concentrations exceeding 5 x [10.sup.-7] M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when [Ca.sup.2+] -free perfusate was used. These data, together with the finding that [H.sub.2]S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that [H.sub.2]S is able to directly elicit catecholamine secretion via membrane depolarization followed by [Ca.sup.2+] -mediated exocytosis. adrenaline; noradrenaline; hypoxia; chromaffin cell; stress; cystathionine [gamma]-lyase; cystathionine [beta]-synthase

Members of the [Na.sup.2+]/[Ca.sup.2+] exchanger (NCX) family are important regulators of cytosolic [Ca.sup.2+] in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of [Na.sup.+]-dependent inactivation and requires much higher levels of regulatory [Ca.sup.2+] to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from [Na.sup.+]-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the [Na.sup.+]-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 [micro]M exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously. calcium homeostatis; [Na.sup.+]/[Ca.sup.2+] exchanger; development

Experiments were performed to assess the possible involvement of the [Na.sup.+]/[H.sup.+] 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.sup.+]-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.sup.+] secretion and HC[O.sup.-.sub.3] reabsorption. sodium/hydrogen exchanger; SLC9; proximal tubule; kidney; cortisol; acidosis; sodium/hydrogen exchange; bicarbonate reabsorption

[O.sub.2] chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on [O.sub.2]-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide ([H.sub.2]S) metabolism is involved in [O.sub.2] sensing in vascular smooth muscle. Here, we examined the possibility that [H.sub.2]S is an [O.sub.2] sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic [O.sub.2] sensing and the homolog of the mammalian carotid body. Intrabuccal injection of [H.sub.2]S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited [H.sub.2]S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors, mRNA for [H.sub.2]S-synthesizing enzymes, cystathionine [beta]-synthase and cystathionine [gamma]-lyase, was present in branchial tissue. Homogenized gills produced [H.sub.2]S enzymatically, and [H.sub.2]S production was inhibited by [O.sub.2], whereas mitochondrial [H.sub.2]S consumption was [O.sub.2] dependent. Ambient hypoxia did not affect plasma [H.sub.2]S in unanesthetized trout, but produced a [Po.sub.2]-dependent increase in a sulfide moiety suggestive of increased [H.sub.2]S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and [H.sub.2]S, produced a similar ~10-mV depolarization. These studies are consistent with [H.sub.2]S involvement in [O.sub.2] sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby [H.sub.2]S concentration ([[H.sub.2]S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [[H.sub.2]S] to [Po.sub.2] and may provide an exquisitely sensitive, yet simple, [O.sub.2] sensor in a variety of tissues. hydrogen sulfide concentration

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 [+ or -]1.2 to 32.6 [+ or -] 1.4 mmHg) and an 18% increase in ventral aortic pressure (from 33.0 [+ or -] 1.5 to 38.9 [+ or -] 1.3 mmHg). The hypertension presumably reflected the increase in cardiac output (from 31.0 [+ or -] 0.8 to 36.4 [+ or -] 2.2 ml.[min.sup.-1]. [kg.sup.-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 [alpha]-adrenergic responsiveness of the systemic vasculature is consistent with desensitization or loss of functional [alpha]-adrenoceptors ([alpha]-ARs). In support of this idea, the salt-fed fish exhibited significantly decreased levels of [[alpha].sub.1D]-AR mRNA in the dorsal aorta and the afferent (ABA) and efferent branchial arteries (EBA). In contrast, however, the results obtained from norepinephrine doseresponse curves for EBA and ABA vascular rings in vitro did not provide evidence for loss of function of branchial artery [[alpha].sub.1]-ARs in the salt-fed fish. Indeed, the [EC.sub.50] for the EBA norepinephrine doseresponse curve was significantly reduced (from 3.75 x [10.sup.-7] to 2.12 x [10.sup.-7] M) in the salt-fed fish, indicating an increase in the binding affinity of the [[alpha].sub.1]-ARs. norepinephrine; epinephrine; blood pressure; systemic resistance; salt feeding; hypercapnia; efferent branchial artery; afferent branchial artery

We indirectly tested the idea that the epithelial [Ca.sup.2+] channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of [Ca.sup.2+] uptake. This was accomplished by assessing the levels of gill ECaC mRNA and protein in fish exposed to treatments known to increase or decrease [Ca.sup.2+] uptake capacity. Exposure of trout to soft water ([[Ca.sup.2+]] = 20-30 nmol/1) for 5 days (a treatment known to increase [Ca.sup.2+] 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 Ca[Cl.sub.2] (a treatment known to reduce [Ca.sup.2+] 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 [Ca.sup.2+] 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 [Ca.sup.2+] uptake rates are, in part, regulated by the numbers of apical membrane [Ca.sup.2+] channels that, in turn, modulate the inward flux of [Ca.sup.2+] into gill epithelial cells. transient receptor potential vanilloid 6; hypercapnia; cortisol; hypercalcemia; soft water

[beta]-Adrenoceptors ([beta]-ARs) are seven-transmembrane domain, G proteincoupled receptors that transduce the cellular effects of epinephrine and norepinephrine and play a pivotal role in the vertebrate stress response. This study reports the cloning and characterization of two previously unreported [beta]-ARs from the rainbow trout (Oncorhynchus mykiss). Phylogenetic analysis of amino acid sequences indicates that both [beta]-ARs are homologs of the mammalian [[beta].sub.3]-AR. Analysis of tissue expression patterns indicates that one of these trout [[beta].sub.3]-adrenoceptors ([[beta].sub.3a]-AR) is highly expressed in gill and heart, whereas the second ([[beta].sub.3b]-AR) is highly expressed by red blood cells (RBC). Expression of the [[beta].sub.3b]-AR in the RBC coupled with the finding of a single category of [beta]-AR binding sites on RBC membranes provides strong evidence for the control of the trout RBC [beta]-AR [Na.sup.+]/[H.sup.+] exchanger ([beta]-NHE) activity by signaling through this [[beta].sub.3b]-subtype and not through a [[beta].sub.1]-subtype as previously proposed. The RBC-specific trout [[beta].sub.3b]-AR exhibits binding characteristics that distinguish this receptor from each of the three pharmacologically defined categories of mammalian [beta]-ARs ([[beta].sub.1]-, [[beta].sub.2]-, and [[beta].sub.3]-AR). This study is the first to report the presence of a [[beta].sub.3]-AR subtype in a fish species, and the proposal that the [[beta].sub.3b]-AR controls RBC [beta]-NHE activity represents a novel role for the [[beta].sub.3]-AR subtype in vertebrates. molecular sequence; tissue expression; evolutionary analysis; binding kinetics; [beta]-adrenoceptors

This study addressed the hypothesis that the secretion of catecholamines from trout (Oncorhynchus mykiss) chromaffin cells, during hypoxia, is triggered by stimulation of [O.sub.2] chemoreceptors located within the gills. Sodium cyanide was administered into the inspired water (external cyanide) or injected into the gill circulation (internal cyanide) to pharmacologically stimulate external (water sensing) or internal (blood sensing) [O.sub.2] chemoreceptors, respectively. Both of these treatments caused an elevation of circulating catecholamine levels. The response to external, but not internal, cyanide was abolished by removal of the first gill arch. Hypoxia produced an increase in circulating catecholamine levels that was unaffected by removal of the first gill arch or by denervation of the pseudobranch. Cyanide and hypoxia both caused the well-documented cardiorespiratory reflexes normally observed in this species. This study demonstrates, for the first time, that gill [O.sub.2] chemoreceptors can initiate the reflex that leads to catecholamine release from the chromaffin cells and that stimulation of internally oriented [O.sub.2] receptors on all gill arches appears to be the physiologically important mechanism for initiating release. epinephrine; cardiorespiratory reflexes; gills; hypoxia; Oncorhynchus mykiss

Sensitivity of C[O.sub.2] excretion to blood flow changes in trout is determined by carbonic anhydrase availability. Am J Physiol Regulatory Integrative Comp Physiol 282: R501-R508, 2002.--The blood transit time through the gills of rainbow trout (Oncorhynchus mykiss) was modified by manipulation of cardiac output (Vb). The experiments tested the hypothesis that efficiency of C[O.sub.2] excretion is sensitive to changes in blood flow owing to chemical equilibrium limitations. An extracorporeal blood shunt was used to continuously monitor blood gases in fish in which Vb was elevated (by 13.3 [+ or -] 2.4 ml * [min.sup.-1] * [kg.sup.-1]) by intravascular saline injection or reduced (by 10.8 [+ or -] 1.8 ml * [min.sup.-1] * [kg.sup.-1]) by removal of plasma. The arterial partial pressure of C[O.sub.2] [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]; an index of C[O.sub.2] excretion efficiency) was increased with elevated Vb and was decreased with reduced Vb such that the changes in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exhibited a significant positive sigmoidal relationship with the changes in Vb ([r.sup.2]=0.75; P < 0.05). In contrast, there was no significant relationship between changes in the arterial partial pressure of [O.sub.2] [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]; an index of [O.sub.2] uptake efficiency) and changes in Vb [r.sup.2] = 0.07; P > 0.05). The intravenous administration of carbonic anhydrase (CA; 10 mg/kg) before vascular volume loading eliminated the increase in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] with increased Vb that was observed in control fish. diffusion; perfusion; oxygen uptake; transit time; cardiac output; gill

Researchers have sought to produce direct evidence that angiotensins (ANGs) boost catecholamine secretion from chromaffin cells in the teleost rainbow trout. This involved using both immunohistochemical and pharmacological techniques. It was found that ANGs can prompt the release of catecholamines from the chromaffin tissue of rainbow trout through specific ANG II binding sites. There may be local or systemic sources of ANG II in the head kidney, and the pathways of ANG II synthesis may be sensitive to perfusion pressure.

Kumai Y, Kwong RW, Perry SF. The role of cAMP-mediated intracellular signaling in regulating Na+ uptake in zebrafish larvae. Am J Physiol Regul Integr Comp Physiol 306: R51-R60, 2014. First published November 20, 2013; doi:10.1152/ajpregu.00317.2013.--In the current study, the role of cAMP in stimulating Na+ uptake in larval zebrafish was investigated. Treating larvae at 4 days postfertilization (dpf) with 10 μM forskolin or 1 μM 8-bromo cAMP significantly increased Na+ uptake by three-fold and twofold, respectively. The cAMP-dependent stimulation of Na+ uptake was probably unrelated to protein trafficking via microtubules because pretreatment with 200 μM colchicine or 30 μM nocodazole did not attenuate the magnitude of the response. Na+ uptake was stimulated markedly following acute (2 h) exposure to acidic water. The acid-induced increase in Na+ uptake was accompanied by a twofold elevation in whole body cAMP levels and attenuated by inhibiting PKA with 10 μM H-89. Knockdown of Na+-H+ exchanger 3b (NHE3b) attenuated, but did not abolish, the stimulation of Na+ uptake during forskolin treatment. In glial cell missing 2 morphants, in which the role of NHE3b in Na+ uptake is diminished and the Na+-Cl- cotransporter (NCC) becomes the predominant route of Na+ entry, forskolin treatment continued to increase Na+ uptake. These data suggest that at least NHE3b and NCC are targeted by cAMP in zebrafish larvae. Staining of larvae with fluorescent forskolin and propranolol revealed the presence of transmembrane adenylyl cyclase within multiple subtypes of ionocytes expressing β-adrenergic receptors. Taken together, results of the present study demonstrate that cAMP-mediated intracellular signaling may regulate multiple Na+ transporters and plays an important role in regulating Na+ uptake in zebrafish larvae during acute exposure to an acidic environment. [ABSTRACT FROM AUTHOR]

The functional role of the tight junction protein claudin-b in larval zebrafish (Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac. Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate- dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression. Additionally, Na+ efflux was substantially increased in the claudin-b morphants, which contributed to a significant reduction in whole-body Na+ levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na+. Nevertheless, the elevated loss of Na+ in the morphants was compensated by an increase in Na+ uptake. Notably, we observed that the increased Na+ uptake in the morphants was attenuated in the presence of the selective Na+/Cl--cotransporter (NCC) inhibitor metolazone, or during exposure to Cl--free water. These results suggested that the increased Na+ uptake in the morphants was, at least in part, mediated by NCC. Furthermore, treatment with an H+-ATPase inhibitor bafilomycin A1 was found to reduce Na+ uptake in the morphants, suggesting that H+-ATPase activity was essential to provide a driving force for Na+ uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na+ handling in zebrafish. [ABSTRACT FROM AUTHOR]

The potential role of adrenergic systems in regulating Na+ uptake in zebrafish (Danio rerio) larvae was investigated. Treatment with isoproterenol (a generic β-adrenergic receptor agonist) stimulated Na+ uptake, whereas treatment with phenylephrine (an α1-adrenergic receptor agonist) as well as clonidine (an α2-adrenergic receptor agonist) significantly reduced Na+ uptake, suggesting opposing roles of α- and β-adrenergic receptors in Na+ uptake regulation. The increase in Na+ uptake associated with exposure to acidic water (pH = 4.0) was attenuated in the presence of the nonselective β-receptor antagonist propranolol or the β1-receptor blocker atenolol; the β2-receptor antagonist ICI-118551 was without effect. The stimulation of Na+ uptake associated with ion-poor water (32-fold dilution of Ottawa tapwater) was unaffected by β-receptor blockade. Translational gene knockdown of β-receptors using antisense oligonucleotide morpholinos was used as a second method to assess the role of adrenergic systems in the regulation of Na+ uptake. Whereas β1- or β2B-receptor knockdown led to significant decreases in Na+ uptake during exposure to acidic water, only β2A-receptor morphants failed to increase Na+ uptake in response to ion-poor water. In support of the pharmacology and knockdown experiments that demonstrated an involvement of β-adrenergic systems in the control of Na+ uptake, we showed that the H+-ATPase-rich (HR) cell, a subtype of ionocyte known to be a site of Na+ uptake, is innervated and appears to express β-adrenergic receptors (propranolol binding sites) at 4 days postfertilization. These data indicate an important role of adrenergic systems in regulating Na+ uptake in developing zebrafish. [ABSTRACT FROM AUTHOR]

The involvement of a Na+/H+ exchanger (NHE) in mediating Na+ uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na+ uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na+ uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na+ uptake in fish reared in acidic water (pH ~ 4.0). A role for NHE in Na+ uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na+/H+ exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na+ uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na+ uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na+ uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H+-ATPase) significantly reduced Na+ uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na+ uptake in larval zebrafish in acidic water: one in which Na+ uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H+-ATPase. [ABSTRACT FROM AUTHOR]

Fish exposed to hypoxia develop decreased heart rate, or bradycardia, the physiological significance of which remains unknown. The general muscarinic receptor antagonist atropine abolishes the development of this hypoxic bradycardia, suggesting the involvement of muscarinic receptors. In this study, we tested the hypothesis that the hypoxic bradycardia is mediated specifically by stimulation of the M2 muscarinic receptor, the most abundant subtype in the vertebrate heart. Zebrafish (Danio rerio) were reared at two levels of hypoxia (30 and 40 Torr Po2) from the point of fertilization. in hypoxic fish, the heart rate was significantly lower than in normoxic controls from 2 to 10 days post-fertilization (dpf). At the more severe level of hypoxia (30 Ton Po2), there were significant increases in the relative mRNA expression of M2 and the cardiac type β-adrenergic receptors (β1AR, β2aAR, and β2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at 40 Ton Po2) or significantly attenuated (at 30 Torr Po2) in larvae experiencing M2 receptor knockdown (using morpholino antisense oligonucleotides). Sham-injected larvae exhibited typical hypoxic bradycardia in both hypoxic regimens. The expression of β1AR, β2aAR, β2bAR, and M2 mRNA was altered at various stages between 1 and 4 dpf in larvae experiencing M2 receptor knockdown. Interestingly, M2 receptor knockdown revealed a cardioinhibitory role for the β2-adrenergic receptor. This is the first study to demonstrate a specific role of the M2 muscarinic receptor in the initiation of hypoxic bradycardia in fish. [ABSTRACT FROM AUTHOR]

Presently, three α1-adrenoceptor (AR) types are recognized in vertebrates: α1A-, α1B-, and α1D-ARs. These α1-subtypes have distinct pharmacology and molecular profiles, play crucial roles in metabolic and vascular control, and are the targets for numerous pharmaceuticals, especially those affecting blood pressure and vascular resistance. To better understand the functional divergence within the α1-AR gene family, we sequenced these α1-AR paralogs in the rainbow trout and performed an extensive phylogenetic analysis. We show that these AR genes evolved by duplication events just before the origin of the jawed vertebrates. Our computational analyses suggest that the differences between the three α1-AR subtypes may affect their tissue specificity, ligand specificity, and possibly signal transduction processes and desensitization. We also show that, within each subtype, differences exist between fish and mammalian receptors, both at the transcriptional and at the physiological level. These differences, however, suggest that the role of α1-ARs in fish is more complex than previously thought. Our integrated analysis of the α1-AR gene family suggests that these receptors evolved these distinct features very early within vertebrates. [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]

This study addressed the hypothesis that the secretion of catecholamines from trout (Oncorhynchus mykiss) chromaffin cells, during hypoxia, is triggered by stimulation of O[sub 2] chemoreceptors located within the gills. Sodium cyanide was administered into the inspired water (external cyanide) or injected into the gill circulation (internal cyanide) to pharmacologically stimulate external (water sensing) or internal (blood sensing) O[sub 2] chemoreceptors, respectively. Both of these treatments caused an elevation of circulating catecholamine levels. The response to external, but not internal, cyanide was abolished by removal of the first gill arch. Hypoxia produced an increase in circulating catecholamine levels that was unaffected by removal of the first gill arch or by denervation of the pseudobranch. Cyanide and hypoxia both caused the well-documented cardiorespiratory reflexes normally observed in this species. This study demonstrates, for the first time, that gill O[sub 2] chemoreceptors can initiate the reflex that leads to catecholamine release from the chromaffin cells and that stimulation of internally oriented O[sub 2] receptors on all gill arches appears to be the physiologically important mechanism for initiating release. [ABSTRACT FROM AUTHOR]

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish ( Danio rerio ) is expressed abundantly in Na + -absorbing/H + -secreting H + -ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a -/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a -/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na + uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na + content remained constant. While Cl - uptake was significantly reduced in ca17a -/- mutants, Cl - content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl - uptake, implicating Ca17a in the mechanism of Cl - uptake by larval zebrafish. H + secretion, O 2 consumption, CO 2 excretion, and ammonia excretion were generally unaltered in ca17a -/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl - uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

Convective water flow across respiratory epithelia in water-breathing organisms maintains transcutaneous oxygen (O 2 ) partial pressure (Po 2 ) gradients that drive O 2 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 O 2 uptake in larvae. We hypothesized that the pectoral fins, which can generate water flow across the skin in larvae, promote transcutaneous O 2 transfer and thus aid in O 2 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 O 2 uptake, and was absent by 21 dpf. In rainbow trout ( Oncorhynchus mykiss ), Po 2 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 Po 2 boundary layers. Lack of pectoral fins compromised whole animal O 2 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.

Purely diffusive O 2 transport typically is insufficient to sustain aerobic metabolism in most multicellular organisms. In animals that are small enough, however, a high surface-to-volume ratio may allow passive diffusion alone to supply sufficient O 2 transfer. The purpose of this study was to explore the impacts of internal convection on respiratory gas transfer in a small complex organism, the larval zebrafish ( Danio rerio). Specifically, we tested the hypothesis that internal convection is required for the normal transfer of the respiratory gases O 2 and CO 2 and maintenance of resting aerobic metabolic rate in larvae at 4 days postfertilization (dpf). Morpholino knockdown of the vascular endothelial growth factor (VEGF) or cardiac troponin T (TNNT2) proteins allowed an examination of gas transfer in two independent models lacking internal convection. With the use of a scanning micro-optrode technique to measure regional epithelial O 2 fluxes ( Jo 2 ), it was demonstrated that larvae lacking convection exhibited reduced Jo 2 in regions spanning the head to the trunk. Moreover, the acute loss of internal convection caused by heart stoppage resulted in reduced rates of cutaneous Jo 2 , an effect that was reversed upon the restoration of internal convection. With the use of whole body respirometry, it was shown that loss of internal convection was associated with reduced resting rates of O 2 consumption and CO 2 excretion in larvae at 4 dpf. The results of these experiments clearly demonstrate that internal convection is required to maintain resting rates of respiratory gas transfer in larval zebrafish.

Carbon monoxide (CO) is a gaseous signaling molecule and is produced in vivo from the intracellular breakdown of heme via the heme oxygenase (HO) family of enzymes. In this study we investigated the role of the HO-1/CO system in the control of ventilation in zebrafish, Danio rerio Immunohistochemistry revealed the presence of HO-1 in the chemoreceptive neuroepithelial cells (NECs) of larvae (4 days postfertilization) and adults, indicating the potential for endogenous CO production in the NECs. Hypoxia (20 min, water Po 2 of 30 mmHg) caused a significant increase in HO-1 activity in whole larvae and in the gills of adult fish. Zebrafish with reduced HO-1 activity (via HO-1 knockdown in larvae or zinc protoporphyrin IX treatment in adults) exhibited increased ventilation frequency (V f ) under normoxic but not hypoxic conditions. The addition of exogenous CO restored resting V f in fish with diminished CO production, and in some cases (e.g., hypoxic sham larvae) CO modestly reduced V f below resting levels. Larval fish were treated with phenylhydrazine (PHZ) to eliminate the potential confounding effects of CO-hemoglobin interactions that might influence ventilation. PHZ treatment did not cause changes in V f of normoxic larvae, and the addition of CO to PHZ-exposed larvae resulted in a significant decrease in sham and HO-1-deficient fish under normoxic conditions. This study demonstrates for the first time that CO plays an inhibitory role in the control of breathing in larval and adult zebrafish., (Copyright © 2016 the American Physiological Society.)

The effects of acute exposure to acidic water on Na + and Cl - homeostasis, and the mechanisms underlying their compensatory regulation, were investigated in the larval zebrafish Danio rerio Exposure to acidic water (pH 4.0; control pH 7.6) for 2 h significantly reduced Na + uptake and whole body Na + content. Nevertheless, the capacity for Na + uptake was substantially increased in fish preexposed to acidic water but measured in control water. Based on the accumulation of the Na + -selective dye, Sodium Green, two ionocyte subtypes exhibited intracellular Na + enrichment after preexposure to acidic water: H + -ATPase rich (HR) cells, which coexpress the Na + /H + exchanger isoform 3b (NHE3b), and a non-HR cell population. In fish experiencing Na + -Cl - cotransporter (NCC) knockdown, we observed no Sodium Green accumulation in the latter cell type, suggesting the non-HR cells were NCC cells. Elimination of NHE3b-expressing HR cells did not prevent the increased Na + uptake following acid exposure. On the other hand, the increased Na + uptake was abolished when the acidic water was enriched with Na + and Cl - , but not with Na + only, indicating that the elevated Na + uptake after acid exposure was associated with the compensatory regulation of Cl - Further examinations demonstrated that acute acid exposure also reduced whole body Cl - levels and increased the capacity for Cl - uptake. Moreover, knockdown of NCC prevented the increased uptake of both Na + and Cl - after exposure to acidic water. Together, the results of the present study revealed a novel role of NCC in the compensatory regulation of Na + and Cl - uptake following acute acidosis., (Copyright © 2016 the American Physiological Society.)

Hydrogen sulfide (H2S) can act as a signaling molecule for various ion channels and/or transporters; however, little is known about its potential involvement in Ca(2+) balance. Using developing zebrafish (Danio rerio) as an in vivo model system, the present study demonstrated that acute exposure to H2S donors increased Ca(2+) influx at 4 days postfertilization, while chronic (3-day) exposure caused a rise in whole body Ca(2+) levels. The mRNA expression of Ca(2+)-transport-related genes was unaffected by H2S exposure, suggesting that posttranscriptional modifications were responsible for the altered rates of Ca(2+) uptake. Indeed, treatment of fish with the protein kinase A inhibitor H-89 abolished the H2S-mediated stimulation of Ca(2+) influx, suggesting that H2S increased Ca(2+) influx by activating cAMP-protein kinase A pathways. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are two key enzymes in the endogenous synthesis of H2S. Using an antisense morpholino knockdown approach, we demonstrated that Ca(2+) influx was reduced in CBS isoform b (CBSb)- but not in CSE-deficient fish. Interestingly, the reduction in Ca(2+) influx in CBSb-deficient fish was observed only in fish that were acclimated to low-Ca(2+) water (i.e., 25 μM Ca(2+); control: 250 μM Ca(2+)). Similarly, mRNA expression of cbsb but not cse was increased in fish acclimated to low-Ca(2+) water. Results from whole-mount immunohistochemistry further revealed that CBSb was expressed in Na(+)-K(+)-ATPase-rich cells, which are implicated in Ca(2+) uptake in zebrafish larvae. Collectively, the present study suggests a novel role for H2S in promoting Ca(2+) influx, particularly in a low-Ca(2+) environment., (Copyright © 2015 the American Physiological Society.)

The involvement of the calcium-sensing receptor (CaSR) in Ca(2+) homeostasis was investigated in larval zebrafish, Danio rerio. The expression of CaSR mRNA was first observed at 3 h posfertilization (hpf) and increased with development until plateauing at ∼48 hpf. At 4 dpf, CaSR mRNA was increased in fish acclimated to low Ca(2+) water (25 μM vs. 250 μM in normal water). Using immunohistochemistry and confocal microscopy, we demonstrated that the CaSR is expressed in the olfactory epithelium, neuromasts, ionocytes on the yolk sac epithelium, and corpuscles of Stannius. Results of double immunohistochemistry and/or in situ hybridization indicated that the CaSR is localized to a subset of mitochondrion-rich ionocytes enriched with Na(+)/K(+)-ATPase and epithelial Ca(2+) channel (ecac). Translational knockdown of the CaSR prevented 4 dpf larvae from regulating whole body Ca(2+) levels when exposed to a low Ca(2+) environment. Further, the increases in ecac mRNA expression and Ca(2+) influx, normally associated with exposure to low-Ca(2+) water, were prevented by CaSR knockdown. These findings demonstrate that larval zebrafish lacking the CaSR lose their ability to regulate Ca(2+) when confronted with a low-Ca(2+) environment. Results from real-time PCR suggested that the mRNA expression of the hypocalcemic hormone stanniocalcin (stc-1) remained elevated in the CaSR morphants following acclimation to low-Ca(2+) water. Overall, the results suggest that the CaSR is critical for Ca(2+) homeostasis in larval zebrafish exposed to low environmental Ca(2+) levels, possibly owing to its modulation of stanniocalcin mRNA expression.