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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.