Quantification of presumptive Na+/H+ antiporters of the erythrocytes of trout and eel |
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| Authors: | Scott D Reid Steve F Perry |
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| Institution: | (1) Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada, K1N 6N5;(2) Present address: Department of Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario;(3) Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1, Canada |
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| Abstract: | The presumptive Na+/H+ exchange sites of trout and eel erythrocytes were quantified using amiloride-displaceable 5-(N-methyl-N-3H]isobutyl)-amiloride (3H-MIA) equilibrium binding to further evaluate the mechanisms of i) hypoxia-mediated modifications in the trout erythrocyte  -adrenergic signal transduction system and ii) the marked differences in the catecholamine responsiveness of this system between the trout and eel. MIA was a more potent inhibitor of both trout apparent erythrocyte proton extrusion (IC50 = 20.1 ± 1.1  mol l–1, N = 6) activity (as evaluated by measuring plasma pH changes after addition of catecholamine in vitro) and specific 3H-MIA binding (IC50 = 257 ± 8.2 nmol l–1, N = 3) than amiloride, which possessed a proton extrusion IC50 of 26.1 ± 1.6 mol l–1 (N = 6) and a binding IC50 of 891 ± 113 nmol l–1 (N = 3). The specific Na+ channel blocker phenamil was without effect on adrenergic proton extrusion activity or specific 3H-MIA binding. Trout erythrocytes suspended in Na+-free saline and maintained under normoxic conditions possessed 37,675 ± 6,678 (N = 6) amiloride-displaceable 3H-MIA binding sites per cell (Bmax, presumptive Na+/H+ antiporters) with an apparent dissociation constant (KD) of 244 ± 29 nmol l–1 (N = 6). Acute hypoxia (PO2 = 1.2 kPa; 30 min) did not affect the KD, yet resulted in a 65% increase in the number of presumptive Na+/H+ antiporters. Normoxic eel erythrocytes, similarly suspended in Na+-free saline, possessed only 17,133 ± 3,716 presumptive Na+/H+ antiporters (N = 6), 45% of that of trout erythrocytes, with a similar KD (246 ± 41 nmol l–1, N = 6). These findings suggest that inter- and intra-specific differences in the responsiveness of the teleost erythrocyte -adrenergic signal transduction system can be explained, in part, by differences in the numbers of Na+/H+ exchange sites. |
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| Keywords: | erythrocyte trout eel hypoxia Na+/H+ antiporter catecholamines |
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