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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