THE REGULATION OF AMMONIA UPTAKE IN CHARA-AUSTRALIS

The regulation of ammonia uptake was investigated in internodal cells of the freshwater alga Chara australis. Ammonia uptake was estimated by monitoring (i) its depletion from the bathing solution, (ii) the uptake of radiolabelled methylamine, an analogue of ammonia, and (iii) depletion of ammonia in the unstirred layer with the microelectrode ion-flux estimation technique (MIFE). Distribution of methylamine ((CH3NH3+)-C-14) between the vacuole and cytoplasm was estimated with efflux analysis. When cells were bathed continuously in solutions containing ammonia or methylamine, the uptake rates of both amines decreased over 12 to 48 h despite the continuing existence of a large electrochemical gradient favouring influx of the NH4+ and CH3NH3+ cations. Treatment with 1.0 to 10.0 mM MSX, an inhibitor of glutamine synthetase, caused the internal ammonia concentration to rise and reduced the subsequent uptake of ammonia and methylamine by up to 70% within 2 h. These results suggest that the permease facilitating NH3+/CH3NH3+ influx is under feedback or kinetic regulation from either internal ammonia or an intermediate of nitrogen assimilation. Treatment with metabolic inhibitors (CCCP, azide and DCMU) and some weak acids (DMO and butyric acid) for 30 to 60 min inhibited methylamine uptake, but the changes in the electrical potential difference across the plasma membrane could not account for the magnitude of inhibition. The rate of cytoplasmic streaming, which is an indicator of the cellular ATP concentration in Chara, was inhibited by many of these treatments. However, under certain conditions of external pH and concentration, butyric acid could reversibly inhibit ammonia and methylamine uptake without affecting cytoplasmic streaming, demonstrating that a decrease in cytoplasmic ATP concentration was not responsible for the inhibition. The effect of butyric acid was rapid, causing a 60% inhibition of uptake in 15 min. We conclude that weak acids can inhibit the NH4+/CH3NH3+ permease by acidifying the cytoplasm and suggest that this may also explain the effects of the metabolic inhibitors on ammonia and methylamine uptake.