Effects of osmotic stresses on isolated rat hepatocytes. II. Modulation of intracellular pH

Abstract

To assess the roles of acid-base transport systems in cell volume regulation in rat hepatocytes, intracellular pH (pHi) was measured in subconfluent monolayers loaded with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) after exposure to hypotonic and relative hypertonic media, interventions that stimulate regulatory volume decrease (RVD) and increase (RVI), respectively. During RVD, pHi decreased from 6.98 +/- 0.11 to 6.85 +/- 0.08 in the absence of HCO3- and from 7.26 +/- 0.10 to 7.19 +/- 0.06 in its presence. Omission of Na+ or addition of 1 mM amiloride prevented the decline in pHi. Acute withdrawal or replacement of Na+ in hypotonic medium resulted in a slower rate of fall or recovery in pHi, respectively, than when the same maneuvers were carried out in isotonic medium. In contrast, during RVI, pHi increased from 6.86 +/- 0.11 to 7.15 +/- 0.15 in the absence of HCO3-, a rise in pHi that was also completely abolished by Na+ removal or by 1 mM amiloride. In the presence of HCO3-, the rise in pHi was less marked than in its absence, although net acid efflux was greater because of a greater intracellular buffering capacity. Cl- removal in the presence of HCO3- had no effect on the change in pHi during either RVD or RVI. Perfusion with 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) during RVD lowered pHi further and accentuated the subsequent pHi rise seen after the return to isotonic medium. These data suggest that Na(+)-H+ exchange in rat hepatocytes is downregulated during RVD and activated during RVI. Cl(-)-HCO3- exchange does not appear to be involved in hepatocyte volume regulation.