Na+-dependent pH Regulation by the Amitochondriate Protozoan Parasite Giardia intestinalis

Giancarlo A Biagini,Leigh A Knodler,Kevin J Saliba,Kiaran Kirk,Michael R Edwards

doi:10.1074/jbc.m102728200

Giancarlo A Biagini, Leigh A Knodler + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m102728200

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Abstract

Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques ((31)P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show that G. intestinalis trophozoites exposed to an extracellular pH range of 6.0--7.5 maintain their cytosolic pH (pH(i)) within the range 6.7--7.1. Maintenance of the resting pH(i) was Na(+)-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH(i) from an intracellular acidosis was also Na(+)-dependent, with the rate of recovery varying with the extracellular Na(+) concentration in a saturable manner (K(m) = 18 mm; V(max) = 10 mm H(+) min(-1)). The recovery of pH(i) from an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na(+)/H(+) exchanger(s) in the regulation of pH(i) in G. intestinalis is discussed.

Highlights

Giardia intestinalis is a parasitic protozoan that colonizes the mucosa of the gastrointestinal tract of humans and animals [1]
In the case of G. intestinalis trophozoites, they must withstand exposure to a wide range of pH ranges as found in the gastrointestinal tract
Similar values for pHi have been reported for other parasitic protozoa; e.g. Trypanosoma brucei epimastigotes, Trypanosoma cruzi epimastigotes, Leishmania major promastigotes, and P. falciparum trophozoites

Summary

Introduction

Giardia intestinalis is a parasitic protozoan that colonizes the mucosa of the gastrointestinal tract of humans and animals [1]. Eukaryote cells use a range of membrane transport mechanisms to import/export Hϩ and HCO3Ϫ from the cytosol. These include the electroneutral Naϩ/Hϩ exchangers (NHEs) and the HCO3Ϫ-dependent exchangers that mediate HCO3Ϫ/ClϪ exchange or NaϩHCO3Ϫ/ClϪ exchange. Hϩ efflux can be mediated by ATP-dependent proton pumps These include P-type Hϩ-ATPases (as found in yeast, e.g. Ref. 7), P-type Hϩ,Kϩ-ATPases (as found in the apical membrane of the gastric mucosa, e.g. Ref. 8), and V-type Hϩ-ATPases (found in the intracellular vacuolar membranes of cells of higher eukaryotes (e.g. Ref. 9) as well as in the plasma membrane of some cell types, including a number of parasitic protozoa (10 –12)). The low inherent sensitivity of the 31P NMR method necessitates the use of high cell densities (which can lead to the metabolic viability of the cells being compromised) as well as limiting the time resolution of the technique

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