The effect of medium hypo-osmolality on cell volume and intracellular amino acid composition was studied in the protozoan parasite Giadia intestinalis. When G. intestinalis was exposed to hypotonic medium, it initially swelled by 40% of its original volume and then decreased its volume, thus demonstrating a regulatory volume decrease process (RVD). These processes were accompanied by a rapid release of intracellular neutral amino acids, especially alanine, but not by amino acids with net charges such as glutamate and ornithine. The net alanine efflux was Na+ and CI-independent, and sensitive to medium osmolality. Alanine efflux was sigmoidal with respect to medium osmolality, with an approximately linear relationship over a range of 250 mOsm kg-l. Alanine efflux was also sensitive to temperature, and an Arrhenius plot gave a Q l o of 36 and an activation energy of 25 kcal mol-l (105 kJ mol-l), suggesting that a carrier-type transport protein, or uniport was involved in the net alanine efflux under hypotonic conditions. This volumeactivated (VA) alanine efflux was not inhibited by ionophores or chloride channel blockers. Of the potential inhibitors tested, only phydroxymercuribenzoate inhibited net alanine efflux. This thiol reagent also inhibited giardial RVD, suggesting that alanine efflux plays a significant role in this process. The VA (alanine) uniport was able to transport 2-aminoisobutyric acid (AIB), a structural analogue of alanine which is frequently used for the characterization of eukaryotic alanine transport but which is not transported by Giardia under isotonic conditions. On the basis of AIB uptake under hypotonic conditions and lack of transactivation of AIB efflux from AIB-loaded cells by external 10 mM alanine or glycine under isotonic conditions, it is evident that the VA (alanine) uniport is different from the previously reported (alanine) antiport.

Giardia intestinalis and Hexamita inflata are microaerophilic protozoa which rely on fermentative metabolism for energy generation. These organisms have developed a number of antioxidant defence strategies to cope with elevated O 2 tensions which are inimical to survival. In this study, the ability of pyruvate, a central component of their energy metabolism, to act as a physiological antioxidant was investigated. The intracellular pools of 2-oxo acids in G. intestinalis were determined by HPLC. With the aid of a dichlorodihydrofluorescein diacetate-based assay, intracellular reactive oxygen species generation by G. intestinalis and H. inflata suspensions was monitored on-line. Addition of physiologically relevant concentrations of pyruvate to G. intestinalis and H. inflata cell suspensions was shown to attenuate the rate of H 2 O 2 -and menadione-induced generation of reactive oxygen species. In addition, pyruvate was also shown to decrease the generation of low-level chemiluminescence arising from the oxygenation of anaerobic suspensions of H. inflata. In contrast, addition of pyruvate to suspensions of respiring Saccharomyces cerevisiae was shown to increase the generation of reactive oxygen species. These data suggest that (i) in G. intestinalis and H. inflata, pyruvate exerts antioxidant activity at physiological levels, and (ii) it is the absence of a respiratory chain in the diplomonads which facilitates the observed antioxidant activity.