Introduction
We have previously provided evidence for the existence of a sodium-proton antiport mechanism (NHE) in the plasma membrane of rat mast cells (Friis & Johansen, 1996) and reported an activation of NHE by hypertonic solutions (Johansen et al., 1988). There are conflicting reports in the literature concerning the role of phosphorylation in relation to NHE activity induced by hypertonic stress (Grinstein et al., 1985; Kapus et al., 1994). The aim of this investigation was to characterize the NHE response to hypertonic stress and to explore if this response would be influenced by inhibition of cellular ATP-synthesis.
Methods
A pH-sensitive probe (2’,7’-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester) was used to measure intracellular pH (pHi) in suspensions of pure populations of mast cells by a cuvette based fluorescence technique using nigericin for calibration. Addition of N-methyl-D-glucamine or NaCl was used to activate NHE. The cells were acid-loaded by incubation with propionic acid.
Results
Addition of increasing concentrations of Na+ (hypertonic stress) caused a proportional rate (ED50 220mM Na+) and extend (ED50 230mM Na+) of pHi-increase, which were inhibited by amiloride analogues, indicating the involvement of a Na+/H+ antiport (NHE). Estimations of the rate of pHi-recovery from an acid load at various intracellular pH demonstrated an increased sensitivity to pHi after cell stimulation by hypertonic stress. The pHi of stimulated cells returned to the initial value by resuspension in bicarbonate buffer. Inhibition of the rate of cellular ATP-synthesis by metabolic inhibitors (2-deoxyglucose and antimycin A) apparently decreased the sensitivity of NHE for the intracellular proton concentration.
Conclusions
The response of mast cells to hypertonic stress is accumulation of Na+ by increased activity of NHE, that is likely to be followed by accumulation of Cl- through Cl--HCO3- exchange. The increased sensitivity of NHE for pHi is attenuated but not blocked by metabolic inhibitors. This may be explained by inhibition of the phosphorylation activity.
References
Friis UG & Johansen T. 1996. Br J Pharmacol 118, 1327-1334
Grinstein S et al. 1985. Proc Natl Acad Sci 82, 1429-1433
Johansen T et al. 1988, Naunyn-Schmiedeberg’s Arch Pharmacol, 358, suppl 2, P 57.13
Kapus, A et al. 1994. J Biol Chem 269, 23544-23552