Characterization of Na⁺ uptake in the endangered desert pupfish, Cyprinodon macularius (Baird and Girard)

This study provided an initial characterization of Na⁺ uptake in saline freshwater by the endangered pupfish, Cyprinodon macularius. This species occurs only in several saline water systems in the southwestern USA and northern Mexico, where salinity is largely controlled by water-management practices. Consequently, understanding the osmoregulatory capacity of this species is important for their conservation. The lower acclimation limit of C. macularius in freshwater was found to be 2 mM Na⁺. Fish acclimated to 2 or 7 mM Na⁺ displayed similar Na⁺ uptake kinetics, with K_m values of 4321 and 3672 μM and V_max values of 4771 and 3602 nmol g^-1 h^-1, respectively. A series of experiments using pharmacological inhibitors indicated that Na⁺ uptake in C. macularius was not sensitive to bumetanide, metolazone, or phenamil. These results indicate the Na⁺- K⁺-2Cl^- cotransporter, Na⁺-Cl^- cotransporter, and the Na⁺ channel-H⁺-ATPase system are likely not to be involved in Na⁺ uptake at the apical membrane of fish gill ionocytes in fish acclimated to 2 or 7 mM Na⁺. However, Na⁺ uptake was sensitive to 1 × 10^-3 M amiloride (not 1 × 10^-4 or 1 × 10^-5 M), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and ethoxzolamide. These data suggest that C. macularius relies on a low-affinity Na⁺-H⁺ exchanger for apical Na⁺ uptake and that H⁺ ions generated via carbonic anhydrase-mediated CO₂ hydration are important for the function of this protein.