Differential expression and regulation of Na(+)/H(+) exchanger isoforms in rabbit parietal and mucous cells.

Several Na(+)/H(+) exchanger (NHE) isoforms are expressed in the stomach, and NHE1 and NHE2 knockout mice display gastric mucosal atrophy. This study investigated the cellular distribution of the NHE isoforms NHE1, NHE2, NHE3, and NHE4 in rabbit gastric epithelial cells and their regulation by intracellular pH (pH(i)), hyperosmolarity, and an increase in cAMP. Semiquantitative RT-PCR and Northern blot experiments showed high NHE1 and NHE2 mRNA levels in mucous cells and high NHE4 mRNA levels in parietal and chief cells. Fluorescence optical measurements in cultured rabbit parietal and mucous cells using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and NHE isoform-specific inhibitors demonstrated that in both cell types, intracellular acidification activates NHE1 and NHE2, whereas hyperosmolarity activates NHE1 and NHE4. The relative contribution of the different isoforms to pH(i)- and hyperosmolarity-activated Na(+)/H(+) exchange in the different cell types paralleled their relative expression levels. cAMP elevation also stimulated NHE4, whereas an increase in osmolarity above a certain threshold further increased NHE1 and not NHE4 activity. We conclude that in rabbit gastric epithelium, NHE1 and NHE4 regulate cell volume and NHE1 and NHE2 regulate pH(i). The high NHE1 and NHE2 expression levels in mucous cells may reflect their special need for pH(i) regulation during high gastric acidity. NHE4 is likely involved in volume regulation during acid secretion.

Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells.

Concomitant Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activation occurs during stimulation of acid secretion in cultured rabbit parietal cells, possibly related to a necessity for volume regulation during the secretory process. We investigated whether cytoplasmic volume changes occur during secretagogue stimulation of cultured rabbit parietal cells. Cells were loaded with the fluorescent dye calcein, and the calcein concentration within a defined cytoplasmic volume was recorded by confocal microscopy. Forskolin at 10(-5) M, carbachol at 10(-4) M, and hyperosmolarity (400 mosmol) resulted in a rapid increase in the cytoplasmic dye concentration by 21 +/- 6, 9 +/- 4, and 23 +/- 5%, respectively, indicative of cell shrinkage, followed by recovery to baseline within several minutes, indicative of regulatory volume increase (RVI). Depolarization by 5 mM barium resulted in a decrease of the cytoplasmic dye concentration by 10 +/- 2%, indicative of cell swelling, with recovery within 15 min, and completely prevented forskolin- or carbachol-induced cytoplasmic shrinkage. Na(+)/H(+) exchange inhibitors slightly reduced the initial cell shrinkage and significantly slowed the RVI, whereas 100 microM bumetanide had no significant effect on either parameter. We conclude that acid secretagoguges induce a rapid loss of parietal cell cytoplasmic volume, followed by RVI, which is predominantly mediated by Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange.

Different acid secretagogues activate different Na+/H+ exchanger isoforms in rabbit parietal cells.

Rabbit parietal cells express three Na+/H+ exchanger isoforms (NHE1, NHE2, and NHE4). We investigated the effects of carbachol, histamine, and forskolin on Na+/H+ exchange activity and acid formation in cultured rabbit parietal cells and tested the effect of NHE isoform-specific inhibition on agonist-induced Na+/H+ exchange. Carbachol (10(-4) M) was the weakest acid secretagogue but caused the strongest Na+/H+ exchange activation, which was completely blocked by 1 microM HOE-642 (selective for NHE1); histamine (10(-4) M) and forskolin (10(-5) M) were stronger stimulants of [14C]aminopyrine accumulation but weaker stimulants of Na+/H+ exchange activity. HOE-642 (1 microM) reduced forskolin-stimulated Na+/H+ exchange activity by 35%, and 25 microM HOE-642 (inhibits NHE1 and -2) inhibited an additional 13%, but 500 microM dimethyl amiloride (inhibits NHE1, -2, and -4) caused complete inhibition. The presence of 5% CO2-HCO-3 markedly reduced agonist-stimulated H+ efflux rates, suggesting that the anion exchanger is also activated. Hyperosmolarity also activated Na+/H+ exchange. Our data suggest that, in rabbit parietal cells, Ca2+-dependent stimulation causes a selective activation of NHE1, whereas cAMP-dependent stimulation activates NHE1, NHE2, and more strongly NHE4. Because intracellular pH (pHi) did not change in the presence of CO2-HCO-3 and concomitant activation of Na+/H+ and anion exchange is one of the volume regulatory mechanisms, we speculate that the physiological significance of secretagogue-induced Na+/H+ exchange activation may not be related to pHi but to volume regulation during acid secretion.