Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation.

Four distinct isoforms of the mammalian Na+/H+ exchanger (NHE) have been identified by molecular cloning. Three of these (NHE-1, NHE-2, and NHE-3) have been shown to be functionally active by heterologous expression. Their kinetic and pharmacological properties are well documented, yet comparatively little is known about their regulation. In this report, rat NHE-1, NHE-2, and NHE-3 were stably transfected into antiporter-deficient Chinese hamster ovary cells to study their role in cellular proliferation and their regulation by nucleotides and cell volume. Their ability to influence cell proliferation was assessed by measuring the growth of antiporter-deficient cells and of the different transfectants in media of varying pH. While antiporter-deficient cells were unable to grow at acidic pH levels, all three isoforms supported proliferation under these conditions. Therefore, while the epithelia-specific isoforms (NHE-2 and NHE-3) are thought to play primarily a role in transcellular ion transport, they can also contribute to intracellular pH (pHi) homeostasis and have a permissive role in cell growth. The activity of the three isoforms was markedly inhibited by depletion of cellular ATP. In the pHi 6.0-7.2 range, decreases in the affinity for internal H+ and/or the maximal rate of transport accounted for the inhibitory effect, depending on the isoform. The osmotic responsiveness of the three isoforms was also compared. As reported earlier, NHE-1 was stimulated by hypertonicity. Under similar conditions, NHE-2 was also stimulated to a comparable extent. Conversely, both isoforms were inhibited in hypotonic media. In contrast, NHE-3 was markedly inhibited by hypertonic cell shrinking but was unaffected by hypotonicity. Osmotic inhibition of NHE-3 was rapid, reversible, and apparent throughout the pH range studied. Osmotic inhibition of NHE-3 may play a role in the physiology and pathophysiology of epithelia.