The role of alanine in the acute response of Giardia intestinalis to hypo-osmotic shock

Abstract

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.

The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity

“…Thus damage to the plasma membrane, as indicated by the progressive loss of ability to exclude the anionic voltage-sensitive dye DiBAC % (3) (Lloyd & Hayes, 1995), led to swelling of organisms and loss of the regulatory volume control process, described in fully functional organisms (Park et al, 1995). Sites known to be O # -inhibited in G. intestinalis include a (4Fe-4S) iron-sulphur cluster (Ellis et al, 1993) and redox-active thiols involved in volume regulation (Park et al, 1995). The fact that these effects could also be produced almost immediately by 60 µM H # O # suggests that exposure to inhibitory levels of O # results in accumulation of this partial reduction product.…”

Nitrosative stress induced cytotoxicity in Giardia intestinalis

“…Previous work on G. intestinalis has shown that thiol‐reactive agents (e.g. N ‐ethylmaleimide) decrease the capacity of this organism to undergo volume regulation as a response to hypo‐osmotic shock (Park et al. 1995).…”

Osmosensing and osmoregulation in unicellular eukaryotes

“…Protozoans (kinetoplastids, apicomplexans and early eukaryotes) can osmoregulate by changing the concentration of alanine (Burrows and Blum 1991;Cronkite et al 1993;Park et al 1995;Ehud et al 2013). They also employ efflux of ions and water from their contractile vacuoles and intracellular compartments (LeFurgey et al 2001;Grønlien et al 2002;Rohloff and Docampo 2008).…”