SUMMARYWe identified five Na+/K+-ATPase α-isoforms in rainbow trout and characterized their expression pattern in gills following seawater transfer. Three of these isoforms were closely related to other vertebrate α1 isoforms (designated α1a, α1b and α1c),one isoform was closely related to α2 isoforms (designated α2) and the fifth was closely related to α3 isoforms (designated α3). Na+/K+-ATPase α1c- and α3-isoforms were present in all tissues examined, while all others had tissue specific distributions. Four Na+/K+-ATPase α-isoforms were expressed in trout gills (α1a, α1b, α1c and α3). Na+/K+-ATPase α1c- and α3-isoforms were expressed at low levels in freshwater trout gills and their expression pattern did not change following transfer to 40% or 80% seawater. Na+/K+-ATPase α1a and α1b were differentially expressed following seawater transfer. Transfer from freshwater to 40% and 80% seawater decreased gill Na+/K+-ATPaseα1a mRNA, while transfer from freshwater to 80% seawater caused a transient increase in Na+/K+-ATPase α1b mRNA. These changes in isoform distribution were accompanied by an increase in gill Na+/K+-ATPase enzyme activity by 10 days after transfer to 80% seawater, though no significant change occurred following transfer to 40% seawater. Isoform switching in trout gills following salinity transfer suggests that the Na+/K+-ATPase α1a- andα1b-isoforms play different roles in freshwater and seawater acclimation, and that assays of Na+/K+-ATPase enzyme activity may not provide a complete picture of the role of this protein in seawater transfer.

Maintenance of ion balance requires that ionoregulatory epithelia modulate ion flux in response to internal or environmental osmotic challenges. We have explored the basis of this functional plasticity in the gills of the euryhaline killifish Fundulus heteroclitus. The expression patterns of several genes encoding ion transport proteins were quantified after transfer from near-isosmotic brackish water [10 parts/thousand (ppt)] to either freshwater (FW) or seawater (SW). Many changes in response to SW transfer were transient. Increased mRNA expression occurred 1 day after transfer for Na(+)-K(+)-ATPase-alpha(1a) (3-fold), Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) (3-fold), and glucocorticoid receptor (1.3-fold) and was paralleled by elevated Na(+)-K(+)-ATPase activity (2-fold). The transient increase in NKCC1 mRNA expression was followed by a later 2-fold rise in NKCC protein abundance. In contrast to the other genes studied in the present work, mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel generally remained elevated (2-fold) in SW. No change in protein abundance was detected, however, suggesting posttranscriptional regulation. The responses to FW transfer were quite different from those to SW transfer. In particular, FW transfer increased Na(+)-K(+)-ATPase-alpha(1a) mRNA expression and Na(+)-K(+)-ATPase activity to a greater extent than did SW transfer but had no effect on V-type H(+)-ATPase expression, supporting the current suggestion that killifish gills transport Na(+) via Na(+)/H(+) exchange. These findings demonstrate unique patterns of ion transporter expression in killifish gills after salinity transfer and illustrate important mechanisms of functional plasticity in ion-transporting epithelia.

The common killifish, Fundulus heteroclitus, inhabits brackish water estuaries and salt marshes along the eastern coast of North America. The species distribution is latitudinal, from Newfoundland to Florida, and thus spans a cline of environmental temperatures. Correspondingly, many previous studies have investigated thermal adaptations in populations across the range. Differences between populations include latitudinal differences in glycolytic enzyme expression and activity (Powers et al., 1986;Pierce and Crawford, 1996), endocrinology (DeKoning et al., 2004;Picard and Schulte, 2004), metabolism (Podrabsky et al., 2000), morphology and behaviour (Powers et al., 1993), and, as a result, these fish are sometimes divided into two subspecies, F.h. macrolepidotus (northern) and F.h. heteroclitus (southern). By contrast, few studies have assessed whether intraspecific physiological differences exist between populations of F. heteroclitus in response to other environmental factors (e.g. tidal cycle; DiMichele and Westerman, 1997).Species within the genus Fundulus are suggested to have arisen from brackish water ancestors, and there is substantial variation in both the salinity of their native habitats (ranging from freshwater to seawater) and their salinity tolerance (Griffith, 1974). Intraspecific differences in salinity tolerance and distribution also appear to exist within some Fundulus species. For example, northern populations of F. heteroclitus have higher fertilization success and larval survival in hyposmotic salinities than southern populations (Able and Palmer, 1988). Furthermore, the proportion of northern genotypes increases in freshwater habitats, even at latitudes and temperatures that are typical for the southern subspecies (Powers et al., 1993). It is therefore likely that molecular or physiological differences exist within F. heteroclitus that form the basis for variation in freshwater tolerance. We examined intraspecific variation in ionoregulatory physiology within euryhaline killifish, Fundulus heteroclitus, to understand possible mechanisms of freshwater adaptation in fish. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations after transfer from brackish water (10·g·l -1 ) to freshwater. Differences in Na + regulation between each population might partially account for this difference in tolerance, because plasma Na + was decreased for a longer period in southern survivors than in northerns. Furthermore, northern fish increased Na + /K + -ATPase mRNA expression and activity in their gills to a greater extent 1-14·days after transfer than did southerns, which preceded higher whole-body net flux and unidirectional influx of Na + at 14·days. All observed differences in Na + regulation were small, however, and probably cannot account for the large differences in mortality. Differences in Cl -regulation also existed between populations. Plasma Cl -was maintained in northern fish, but in southerns, plasma Cl -decrease...

. Lipid oxidation fuels recovery from exhaustive exercise in white muscle of rainbow trout. Am J Physiol Regulatory Integrative Comp Physiol 282: R89-R99, 2002; 10.1152/ajpregu.00238.2001.-The oxidative utilization of lipid and carbohydrate was examined in white muscle of rainbow trout (Oncorhynchus mykiss) at rest, immediately after exhaustive exercise, and for 32-h recovery. In addition to creatine phosphate and glycolysis fueling exhaustive exercise, near maximal activation of pyruvate dehydrogenase (PDH) at the end of exercise points to oxidative phosphorylation of carbohydrate as an additional source of ATP during exercise. Within 15 min postexercise, PDH activation returned to resting values, thus sparing accumulated lactate from oxidation. Glycogen synthase activity matched the rate of glycogen resynthesis and represented near maximal activation. Decreases in white muscle free carnitine, increases in long-chain fatty acyl carnitine, and sustained elevations of acetyl-CoA and acetyl carnitine indicate a rapid utilization of lipid to supply ATP for recovery. Increases in malonyl-CoA during recovery suggest that malonyl-CoA may not regulate carnitine palmitoyltransferase-1 in trout muscle during recovery, but instead it may act to elongate short-chain fatty acids for mitochondrial oxidation. In addition, decreases in intramuscular triacylglycerol and in plasma nonesterified fatty acids indicate that both endogenous and exogenous lipid fuels may be oxidized during recovery. pyruvate dehydrogenase; glycogen synthase; carbohydrate; lactate; metabolism; malonyl-coenzyme A; nonesterified fatty acids OVER THE PAST several decades many studies have examined the metabolic responses of fish white muscle to high-intensity, exhaustive exercise together with the pattern of metabolite recovery (16). These studies have led to the development of a model of fuel selection during exhaustive exercise based on hydrolysis of highenergy phosphates [i.e., creatine phosphate (CrP) and ATP] and "anaerobic" glycolysis leading to lactate accumulation. Furthermore, it has been demonstrated that there is a temporal shift in fuel selection during exhaustive exercise from an initial hydrolysis of CrP (7,8) to an activation of glycogenolysis and glycolysis (25). As a result, exhaustion in rainbow trout is characterized by a 40 to 60% decrease in white muscle ATP and CrP concentrations and up to a 90% decrease in muscle glycogen concentrations with reciprocal and stoichiometric increases in inosine monophosphate (IMP), free creatine (Cr), and lactate, respectively (e.g., Ref. 39).During recovery, pathways must be activated to resynthesize ATP, CrP, and glycogen in preparation for another possible bout of exercise. To this end, trout experience an excess postexercise O 2 consumption (EPOC) (34), in part, representing a stimulation of oxidative phosphorylation for ATP production during recovery. The tricarboxcylic acid (TCA) cycle supplies reducing equivalents for mitochondrial oxidative phosphorylation through the utilization of acetyl-Co...

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