Halotolerant Alga Dunaliella safina'

Na+/H+ exchange activity in whole cells of the halotolerant alga DunaIfeila saUna can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Nat were followed. Following a mild intracellular acidification, intracellular Nat content increased dramatically and then decreased. We interpret the phase of Nat influx as due to the activation of the plasma membrane Na+/Hf antiporter and the phase of Nat efflux as due to an active Nat extrusion process. The following observations are in agreement with this interpretation: (a) the Na+ influx phase was sensitive to Li+, which is an inhibitor of the Na /H+ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na concentration is due to a combination of Na uptake via the Na /H antiporter and an active, ATPase-dependent, Na+ extrusion. The Na+/Hf antiporter seems, therefore, to play a principal role in internal pH regulation in DunalietIa. Estimations of intracellular pH in Dunaliella indicate that the cytoplasmic pH is maintained at a constant value, around 7.4, in a wide range of extracellular pH (5.0-9.0), suggesting the existence of an efficient pH control mechanism (8, 9). Na+/H' antiporters are ubiquitously distributed in bacterial, plant, and animal plasma membranes and have been suggested to be involved both in the regulation ofintracellular pH and Nae concentration (14, 19). Plasma membrane vesicles isolated from the halotolerant unicellular alga Dunaliella salina were found to contain an active Na+/H' antiporter (12). The antiporter was highly specific to Na' and competitively inhibited by Li' or amiloride. It was solubilized and reconstituted into active proteoliposomes (13). Dunaliella can grow in media containing a very wide range of salt concentrations (0.1-5.5 M), but the intracellular Na' concentration under all these conditions is low (10-100 mM) (2, 5, 7, 11). It was therefore suggested that the antiporter in Dunaliella may play a major role in the intracellular regulation of Na' concentration. In the present paper, we report on an investigation exam'In memory of Professor Jacob B. Biale. ining the role of the Na+/H' antiporter in intracellular pH regulation in vivo. The intracellular pH was artificially decreased by the introduction of a permeant weak acid at an appropriate external pH (1, 8, 10, 16), and the resultant Na+ and proton flows were followed. MATERIALS AND METHODS Culture of the Algae Dunaliella salina were grown in media containing 1 M NaCl and 25 mm NaHCO3 as described previously (4). Cultures were grown under continuous illumination at 26TC with slow continuous shaking and under pH stat control. The pH was maintained at 7.0 by addition of gaseous CO2 Lowering Intracellular pH and Measurements Algae in the log phase were centrifuged and resuspended at a cell density of 3 to 5 x 108 cells/ml in a reaction medium composed of the growth medium, buffered with 50 mM Mes and 20 mm Mops at pH 5.5 (or as indicated). The suspension was preincubated for 30 min in the light, and then the desired weak acid, adjusted to pH 5.5 (or as indicated), was added. At the times indicated, 0.1 mL of the suspension was applied to a 5-mL column of Dowex-Tris for estimation of intracellular Na+ as described previously (20). Elution from the Dowex columns was with 4 mL of 1.6 M glycerol, 5 mM MopsTris (pH 5.5) at 00C. Na', K+, and Li' content were determined in a flame photometer. Cell number and volume were measured in a model ZM Coulter counter. 02 evolution was measured with a Rank Brothers electrode.