title: Quantification of presumptive Na(+)/H (+) antiporters of the erythrocytes of trout and eel.
abstract: The presumptive Na(+)/H(+) exchange sites of trout and eel erythrocytes were quantified using amiloride-displaceable 5-(N-methyl-N-[(3)H]isobutyl)-amiloride ((3)H-MIA) equilibrium binding to further evaluate the mechanisms of i) hypoxia-mediated modifications in the trout erythrocyte β-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 μmol l(-1), N = 6) activity (as evaluated by measuring plasma pH changes after addition of catecholamine in vitro) and specific (3)H-MIA binding (IC50 = 257 ± 8.2 nmol l(-1), N = 3) than amiloride, which possessed a proton extrusion IC50 of 26.1 ± 1.6 μmol 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 (3)H-MIA binding. Trout erythrocytes suspended in Na(+)-free saline and maintained under normoxic conditions possessed 37,675 ± 6,678 (N = 6) amiloride-displaceable (3)H-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 β-adrenergic signal transduction system can be explained, in part, by differences in the numbers of Na(+)/H(+) exchange sites.
authors.affiliation: Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada, K1N 6N5.
authors.forename: S D
authors.initials: SD
authors.lastname: Reid
authors.forename: S F
authors.initials: SF
authors.lastname: Perry