QUANTIFICATION OF PRESUMPTIVE NA+ H+ ANTIPORTERS OF THE ERYTHROCYTES OF TROUT AND EEL/

The presumptive Na+/H+ exchange sites of trout and eel erythrocytes we re quantified using amiloride-displaceable 5-(N-methyl-N-[H-3]isobutyl )-amiloride (H-3-MIA) equilibrium binding to further evaluate the mech anisms of i) hypoxia-mediated modifications in the trout erythrocyte b eta-adrenergic signal transduction system and ii) the marked differenc es in the catecholamine responsiveness of this system between the trou t and eel. MIA was a more potent inhibitor of both trout apparent eryt hrocyte proton extrusion (IC50 = 20.1 +/- 1.1 mumol l-1, N = 6) activi ty (as evaluated by measuring plasma pH changes after addition of cate cholamine in vitro) and specific H-3-MIA binding (IC50 = 257 +/- 8.2 n mol l-1, N = 3) than amiloride, which possessed a proton extrusion IC5 0 of 26.1 +/- 1.6 mumol l-1 (N = 6) and a binding IC50 of 891 +/- 113 nmol l-1 (N = 3). The specific Na+ channel blocker phenamil was withou t effect on adrenergic proton extrusion activity or specific H-3-MIA b inding. Trout erythrocytes suspended in Na+-free saline and maintained under normoxic conditions possessed 37,675 +/- 6,678 (N = 6) amilorid e-displaceable H-3-MIA binding sites per cell (B(max) presumptive Na+/ H+ antiporters) with an apparent dissociation constant (K(D)) of 244 /- 29 nmol l-1 (N = 6). Acute hypoxia (PO2 = 1.2 kPa; 30 min) did not affect the K(D), yet resulted in a 65 % increase in the number of pres umptive Na+/H+ antiporters. Normoxic eel erythrocytes, similarly suspe nded in Na+-free saline, possessed only 17,133 +/- 3,716 presumptive N a+/H+ antiporters (N = 6), 45 % of that of trout erythrocytes, with a similar K(D) (246 +/- 41 nmol l-1, N=6). These findings suggest that i nter- and intra-specific differences in the responsiveness of the tele ost erythrocyte beta-adrenergic signal transduction system can be expl ained, in part, by differences in the numbers of Na+/H+ exchange sites .