Kinetic properties of Na⁺/H⁺ exchange in cultured bovine pigmented ciliary epithelial cells

Abstract

Uptake studies with²²Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na⁺ transport. A large part of Na⁺ uptake was sensitive to amiloride, quinidine and harmaline. Na⁺ uptake was stimulated by intracellular acidification (using the NH ⁺₄ prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentK _M for Na⁺ from 38 to 86 mM without considerable changes inV _max. In the presence of 5 mM Na⁺ half maximal inhibition of amiloride sensitive Na⁺ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na⁺ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H⁺ seemed to be competitive with aK _i of 20–40 nM. 10 μM amiloride increased the apparentK _M for Na⁺ from 33 mM to 107 mM, whileV _max remained nearly unchanged. IC₅₀ for amiloride was 6 μM at 5 mM Na⁺ and 36 μM in the presence of 150 mM Na⁺. Thus, amiloride behaves as a competitive inhibitor with aK _i of about 5 μM. The affinities of Na⁺ to the transport site (K _M≈16 mM), to the inhibitory site for protons (K _M≈21 mM), and to the inhibitory site for amiloride (K _M≈26 mM) were in the same order of magnitude.

In summary, we have presented evidence for the presence of a Na⁺/H⁺ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na⁺, H⁺ and amiloride.