Molecular & Biochemical Parasitology 126 (2003) 219–230 Regulatory volume decrease in Trypanosoma cruzi involves amino acid efflux and changes in intracellular calcium Peter Rohloff, Claudia O. Rodrigues, R. Docampo ∗ Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USA Received 3 July 2002; received in revised form 30 October 2002; accepted 30 October 2002 Abstract A regulatory volume decrease (RVD) in response to hyposmotic stress has been characterized in different life-cycle stages of Try- panosoma cruzi. Hyposmotic stress initially caused swelling, but this was rapidly reversed by a compensatory volume reversal that was essentially complete by 5min. Volume recovery was associated with an amino acid efflux that accounted for approximately 50% of the regulatory volume decrease in all three life-cycle stages. The amino acid efflux was selective for neutral and anionic amino acids, but excluded cationic amino acids. Acidocalcisomes contained an amino acid pool over four times more concentrated than whole-cell levels, but about 90% of this was composed of Arg and Lys, so involvement of this pool in amino acid efflux was ruled out. Hy- posmotic stress induced a rise in intracellular calcium that was dependent on influx of calcium across the plasma membrane, since chelation of extracellular calcium abolished the response. Influx of calcium was confirmed by demonstration of manganese-mediated quenching of intracellular fura-2 fluorescence and partial inhibition of the rise in calcium by calcium channel blockers. Manipulation of intra- and extracellular calcium levels had minor effects on the initial rate of amino acid efflux and no effect on the rate of volume recovery. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma cruzi; Regulatory volume decrease; Calcium; Acidocalcisome 1. Introduction The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas’ disease, which is the leading cause of cardiac death in young adults in endemic areas through- out Mexico, Central, and South America. Over 16 mil- lion individuals are infected and another 90 million are Abbreviations: RVD, regulatory volume decrease; EGTA, ethylene glycol-bis(-amino-ethyl ether) N,N,N ′ ,N ′ -tetraacetate; EtBr, ethidium bromide; Fluorescamine, phenylspiro[furan-2(3H),1 ′ - phthalan]-3,3 ′ -dione; AM, tetraacetoxymethyl ester; Calcein, 4 ′ ,5 ′ -bis- [N,N bis(carboxymethyl)aminomethyl]fluorescein; Fura-2, 1-[2-(5- carboxyozol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2 ′ -amino-5 ′ -methylphe- noxy)-ethane, N,N,N ′ N ′ -tetraacetate; BAPTA, 1,2-bis(o-aminophe- noxy)ethane tetraacetate; Half-BAPTA, N-(2-methoxyphenyl)imi- doacetate; iso-Cl, isotonic chloride buffer; Hepes, N-(2-hydroxyethyl)- piperazine-N ′ -(2-ethanesulfonate); AA, amino acid; H 2 DIDS, 4,4 ′ -diiso- thiocyanatodihydrostilbene-2,2 ′ -disulfonic acid; VSOAC, volume- sensitive organic osmolyte anion channel; HAAC, hypotonically-activated amino acid channel ∗ Corresponding author. Tel.: +1-217-333-3845; fax: +1-217-244-7421. E-mail address: rodoc@uiuc.edu (R. Docampo). at risk of infection. However, chemotherapy for this dis- ease remains unsatisfactory [1,2], and consequently there is great interest in the description of novel cell biological pro- cesses which might serve as suitable targets for rational drug design. One novel feature of T. cruzi biology is its ability to sur- vive the wide range of environmental conditions that it en- counters as it progresses through its life cycle, including dramatic fluctuations in external osmolarity. These osmotic fluctuations occur both within the gut of the triatomine vec- tor as the insect’s fed state changes [3] and also as the par- asite moves from the insect gut through the vertebrate host interstitium, acidic phagolysosomes, and cytosol of verte- brate host cells. Physiological responses to hyposmotic stress have been studied extensively in a number of mammalian cell types. Here, exposure to a reduction in external osmolarity leads to initial cell swelling, but cells soon regain nearly normal volume by a process that has been termed the regulatory vol- ume decrease (RVD) and is accomplished by the efflux of various osmolytes to the extracellular environment followed 0166-6851/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0166-6851(02)00277-3