Pharmacological evidence that DAPI inhibits NHE2 in Fundulus heteroclitus acclimated to freshwater

Ionoregulation in the euryhaline killifish Fundulus heteroclitus has been intensively studied over the last two decades using a variety of techniques. However, there has been limited use of pharmacological inhibitors to identify proteins involved in ion transport for this species. In this study, we used a range of pharmacological inhibitors (EIPA, DAPI, ethoxzolamide, bumetanide, bafilomycin, phenamil, hydrochlorothiazide) to investigate the proteins involved in Na⁺ transport in freshwater (1 mM Na⁺) acclimated F. heteroclitus. Our results indicate that Na⁺ uptake under these conditions is sensitive to both EIPA (NHE-specific inhibitor) and DAPI (putative ASIC-specific inhibitor), but not to any of the other inhibitors. Results for EIPA are consistent with previous studies indicating F. heteroclitus relies solely on NHE2 for Na⁺ transport across the apical membrane of ionocytes. In contrast, results for DAPI are surprising given previous studies that have indicated the H⁺-ATPase is basolaterally located in F. heteroclitus and so cannot contribute to Na⁺ uptake via ASIC. The lack of bafilomycin sensitivity in the current study is consistent with a basolaterally located H⁺-ATPase. This suggests that DAPI is not an ASIC-specific inhibitor as has been previously assumed, and that it may also inhibit NHE2. Finally, we did not observe Na⁺ uptake to be sensitive to ethoxzolamide, suggesting that carbonic anhydrase may not be involved in generating the H⁺ needed to maintain NHE activity in freshwater as has been previously proposed.