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.

A novel perspective on the evolutionary loss of plasma-accessible carbonic anhydrase at the teleost gill

“…Finally, we found evidence for a translocation of β-NHE protein in adrenergically stimulated rainbow trout RBCs, from the cytoplasm into the membrane ( Fig. 2 ), which is consistent with previous findings in white seabass ( Harter et al, 2021 ) and work describing an increase in radiolabelled β-NHE binding sites in hypoxia-exposed RBCs ( Reid and Perry, 1994 ). The translocation of β-NHE protein in adrenergically stimulated trout RBCs was less prominent than that observed in white seabass RBCs ( Harter et al, 2021 ), perhaps indicating interspecific differences in this novel cellular mechanism that are worthy of investigation.…”

“…In contrast, stimulated RBCs treated with the NHE inhibitor amiloride (ISO+Am) had a significantly lower EC 50 P CO 2 (0.96±0.04 kPa) compared with the ISO treatment, and were not different from DMSO controls. Therefore, the increase in Hb–O 2 saturation after adrenergic stimulation of RBCs can be attributed to β-NHE activity, likely through its well-described effects on RBC pH i ( Nikinmaa, 1992 ); Na + channels that are also affected by amiloride are not involved in the RBC β-NHE response ( Reid and Perry, 1994 ). More importantly, however, the addition of extracellular CA to ISO-treated RBCs (ISO+CA) significantly increased EC 50 P CO 2 (1.55±0.07 kPa) compared with DMSO and ISO+Am controls and was not different from RBCs treated with ISO alone.…”

Effects of Acute Hypoxia and Reoxygenation on Physiological and Immune Responses and Redox Balance of Wuchang Bream (Megalobrama amblycephala Yih, 1955)

“…Our results show that Hb and MetHb are more significantly induced at 6 h for moderate hypoxia treatment, suggesting that Hb and MetHb may be more sensitive to hypoxia time rather than oxygen levels in fish. Na + influx in fish erythrocytes can indicate the activation of Na + /H + exchanger and the increase of the pH value, inducing a decrease of hemoglobin affinity to oxygen in fish erythrocytes, as a result the oxygen is delivered into tissues (Nikinmaa, 1992; Reid and Perry, 1994; Nielsen, 1997). In this study, Na + stayed at high levels with the decrease of DO, but decreased at 6 h for hypoxia, indicating that Na + probably flows into M. amblycephala erythrocytes (Figure 1).…”

The Effects Of Repeated Physical Stress On The β-Adrenergic Response Of The Rainbow Trout Red Blood Cell

“…Instead, the dual effects of hypoxia on increasing the affinity and the maximal activity of Na + /H + exchange are presumably related to regulation at the level of the exchanger itself, as proposed Stress and trout red blood cells initially by Motais et al (1987). This could involve a direct effect of hypoxia on the numbers and/or affinities of the Na + /H + exchanger (Reid and Perry, 1994a) or an indirect effect mediated by the oxygenation state of haemoglobin (Motais et al 1987;Nikinmaa and Jensen, 1992). The results of experiments using 8bromo cyclic AMP revealed that the responsiveness of the Na + /H + exchanger to cyclic AMP was modified by hypoxia since there were significant increases in maximal Na + /H + exchange activity (Figs 6, 7) and reductions in EC50 values (Table 4).…”

“…plasma membrane. But, since ␤-adrenergic cell swelling is also enhanced by hypoxic conditions in vitro independently of intracellular cyclic AMP levels, regulation of the rbc adrenergic response is also likely to occur at the level of the Na + /H + exchanger itself (Reid and Perry, 1994a).…”