Activation of Na+/H+ exchange is required for regulatory volume decrease after modest "physiological" volume increases in jejunal villus epithelial cells.

Abstract

Epithelial cell volume increases that occur because of the uptake of Na+-cotransported solutes or hypotonic dilution are followed by a regulatory volume decrease (RVD) due to the activation of K+ and Cl- channels. We studied the relationship of Na+/H+ exchange (NHE) to this RVD in suspended guinea pig jejunal villus cells, using electronic sizing to measure cell volume changes and fluorescent spectroscopy of cells loaded with 2', 7'-bis(carboxyethyl)-5()-carboxyfluorescein to monitor intracellular pH (pHi). When the volume increase achieved by these cells during Na+ solute absorption was duplicated by a modest 5-7% hypotonic dilution, their pHi first acidified and then alkalinized. This alkalinization was blocked by 5-(N-methyl-N-isobutyl) amiloride (MIA; 1 microM), an inhibitor of NHE. The RVD subsequent to 5-7% hypotonic dilution was prevented by Na+-free medium and by amiloride and non-amiloride derivatives. The order of potency of these inhibitors was as follows: MIA > 5-(N,N-dimethyl) amiloride > cimetidine > clonidine, in keeping with the pattern attributable to NHE-1 as the isoform of NHE responsible for increase in pHi after modest volume increases. A substantial 30% hypotonic dilution caused acidification, and RVD following this larger volume increase was not affected by MIA. To assess the effect of hypotonicity on the activity of NHE, we measured the rate of MIA-sensitive pHi recovery from an acid load (dpHi/dt) in 5 and 30% hypotonic media. pHi recovery was faster in 5% hypotonic medium compared with isotonic medium and slowest in 30% hypotonic medium, which suggested that the activity of NHE was stimulated in the slightly hypotonic medium, but inhibited in the 30% hypotonic medium. To determine the role of activated NHE in RVD after a modest volume increase, cells were hypotonically diluted 7% in MIA to prevent RVD and then alkalinized by NH4Cl or acidified by propionic acid addition. Only after alkalinization was there complete volume regulation. We conclude that in Na+-absorbing enterocytes, the NHE-1 isoform of Na+/H+ exchange is stimulated by volume increases that duplicate the "physiological" volume increase occurring when these cells absorb Na+-cotransported solutes. The subsequent alkalinization of pHi is a required determinant of the osmolyte loss that underlies this distinct volume regulatory mechanism.