The Na/K-ATPase in the gills of Antarctic and New Zealand nototheniids: The Physiologic and Molecular Effects of Warm-acclimation.

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Guynn, Sierra Renee
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Antarctic fish, living in -2°C waters, have a serum osmolality of « 600mOsm/kg nearly double that of temperate teleosts. The increase in serum osmolality is due to proportional increases in the Na" and CT concentrations as compared to temperate teleosts, including members of the same family. The sodium and potassium adensosine triphosphatase (Na/K-ATPase) in the chloride cells of the gill is responsible for maintenance of serum ion concentrations. The effects of thermal acclimation in two Nototheniid species, the stenothermal Antarctic Trematomous bernacchii and the eurythermal New Zealand Notothenia angustata. were investigated by measuring serum osmolality, gill Na/K-ATPase activity, sodium pump density and ouabain affinity. Both fishes were acclimated at their upper and lower viable thermal temperatures. Warm acclimation (+4°C) of the 7. bernacchii significantly decreased their serum osmolality from 550 mOsm/kg to 450 mOsm/kg compared to cold-acclimation (-1.5°C) and this was accompanied by a two-fold increase in gill Na/K-ATPase activity. Warm-acclimation(+14°C) of the N. angustata did not significantly change their serum osmolality from 330 mOsm/kg or gill Na/K-ATPase activity compared to the cold-acclimated (+4°C) N. angustata. Using [3H]-ouabain binding techniques, the Bmax and K(| values of gill Na/K-ATPase enzymes were determined. No difference in the Bmax or Kd of the warm-acclimated T. bernacchii accounted for the increase in Na/K-ATPase activity. The change in gill Na/K-ATPase activity in the warm-acclimated T. bernacchii is not mediated by an increase in the number of enzyme sites and is not reflected by a change in ouabain affinity for Na/K-ATPase. The Na/K-ATPase is composed of two subunits; a catalytic a-subunit with 4 isoforms and a |3-subunit with 3 isoforms. In previous teleost studies, several isoform mRNAs and al and a3 proteins have been identified in gills. As a first step towards quantifying the Na/K-ATPase a-subunit, we determined the mRNA and protein isoform composition in the gills of the Antarctic T. bernacchii. In other teleost studies, especially with salinity acclimation experiments, changes in Na/K-ATPase a isoform expression accompanied changes in Na/K-ATPase activity. As the first step in determining the molecular effects of warm acclimation, the gill Na/K-ATPase a isoform expression was determined. Partial cDNA sequences of T. bernacchii al, a2, and a3 isoforms were derived from RT-PCR using total RNA from brain, muscle, kidney and gill and subsequent sequencing of sub-cloned products. There is 72-74% nucleotide identity between the three T. bernacchii isoform sequences and 71%, 76% and 78% compared to the rat isoforms for the al, a2, and a3, respectively. The T. bernacchii al, a2, and a3 amino acid (AA) sequences, within the 13 AA region that defines the isoforms, share 65%. 65% and 94% AA identity with the rat al, a2, and a3 sequences, espectively. There are two unique and radical substitutions in the T. bernacchii sequences; in the al a glutamine for a histidine at position 503(rat) and in the a2 an alanine for an arginine at position 493(rat). Using isoform specific primers, the mRNA for all three isoforms was found in T. bernacchii brain, gill, heart, trunk kidney and muscle. Using isoform specific antibodies, the Na/K-ATPase al, a2 and a3 subunit isoforms were found in the tissues of both T. bernacchii and N. angustata, a temperate nototheniid. This is the first report detecting all three isoforms at the nucleotide and protein level in teleosts. Additional experiments determined the T. bernacchii gill Na/K-ATPase a isoform mRNA and protein expression during warm acclimation. The Na/K-ATPase al, a2 and a3 isoforms have different kinetic and physiologic properties. It was hypothesized that the increase in activity is due to a change in the gill Na/K-ATPase a isoform expression. Using western blotting and isoform specific antibodies, the band density of the Na/K-ATPase al, a2 and a3-subunit isoform proteins was measured in cold and warm-acclimated T. bernacchii. Na/K-ATPase a3-subunit isoform protein decreased 56±0.1% (p<0.05) during warm acclimation. There were no significant changes in al or a2-subunit isoform proteins. The NazK-ATPase a3-subunit isoform has a lower intracellular Na ’ affinity, increases intracellular Na‘ concentrations([Na ]ic) when transfected into HeLa cells, and inhibits the activity of the al isoform. Our results suggest a relationship between the decreased serum osmolality of warm acclimated T. bernacchiii and the expression of the a3 isoform, possibly due to the expected decrease in [Na4]ic with decreaseing serum osmolality.
Creighton University
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