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Acidocalcisomes and the Contractile Vacuole Complex Are Involved in Osmoregulation in Trypanosoma cruzi

2004; Elsevier BV; Volume: 279; Issue: 50 Linguagem: Inglês

10.1074/jbc.m410372200

1083-351X

Peter Rohloff, Andrea Montalvetti, Roberto Docampo,

Lysosomal Storage Disorders Research

Trypanosoma cruzi, the etiologic agent of Chagas disease, resists extreme fluctuations in osmolarity during its life cycle. T. cruzi possesses a robust regulatory volume decrease mechanism that completely reverses cell swelling when submitted to hypo-osmotic stress. The efflux of amino acids and K+ release could account for only part for this volume reversal. In this work we demonstrate that swelling of acidocalcisomes mediated by an aquaporin and microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of this aquaporin and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Contractile vacuole bladders were isolated by subcellular fractionation in iodixanol gradients, showed a high concentration of basic amino acids and inorganic phosphate, and were able to transport protons in the presence of ATP or pyrophosphate. Taken together, these results strongly support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation. Trypanosoma cruzi, the etiologic agent of Chagas disease, resists extreme fluctuations in osmolarity during its life cycle. T. cruzi possesses a robust regulatory volume decrease mechanism that completely reverses cell swelling when submitted to hypo-osmotic stress. The efflux of amino acids and K+ release could account for only part for this volume reversal. In this work we demonstrate that swelling of acidocalcisomes mediated by an aquaporin and microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of this aquaporin and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Contractile vacuole bladders were isolated by subcellular fractionation in iodixanol gradients, showed a high concentration of basic amino acids and inorganic phosphate, and were able to transport protons in the presence of ATP or pyrophosphate. Taken together, these results strongly support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation. The obligate intracellular parasite Trypanosoma cruzi is the causative agent of Chagas disease, which is the leading cause of cardiac death in endemic areas throughout Latin America. More than 11 million people are infected with the parasite, and some 40 million more are at risk (1Urbina J.A. Docampo R. Trends Parasitol. 2003; 19: 495-501Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). As T. cruzi passes through its digenetic life cycle, it encounters many fluctuations in environmental conditions to which it must adapt in order to survive. Extreme fluctuations in osmolarity occur within the gut of the vector (2Kollien A.H. Grospietsch T. Kleffmann T. Zerbst-Boroffka I. Schaub G.A. J. Insect Physiol. 2001; 47: 739-747Crossref PubMed Scopus (60) Google Scholar, 3Kollien A.H. Schaub G.A. Parasitol. Today. 2000; 16: 381-387Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), and also when the infective form of the parasite passes out of the vector in the highly concentrated excreta and rapidly encounters the interstitial fluid of the mammalian host with a much lower osmolarity. Physiological adaptations to hypo-osmotic stress have been studied extensively in a wide range of mammalian cell types as well as in unicellular eukaryotes. Upon exposure to a reduction in external osmolarity, cells initially swell but soon regain nearly normal cell volume by a process that has been termed the regulatory volume decrease (RVD 1The abbreviations used are: RVD, regulatory volume decrease; AMDP, aminomethylenediphosphonate; CVC, contractile vacuole complex; V-H+-PPase, vacuolar proton pyrophosphatase; Pi, inorganic phosphate; PPi, pyrophosphate; polyP, polyphosphate; DCCD, N,N′-dicyclohexylcarbodiimide; AM, tetraacetoxymethyl ester; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; GFP, green fluorescent protein; IBMX, 3-isobutyl-1-methylxanthine; PBS, phosphate-buffered saline; TcAQP, T. cruzi aquaporin.; reviewed in Ref. 4Lang F. Busch G.L. Ritter M. Volkl H. Waldegger S. Gulbins E. Haussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar), which is accomplished by the efflux of various inorganic ions (such as Na+ and K+) and organic osmolytes to the extracellular environment. Whereas in vertebrate cells inorganic ion efflux is much more important than organic osmolyte efflux, the most functionally significant efflux in unicellular eukaryotes, in terms of total contribution to RVD, seems to involve amino acids. Previous studies (5Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Crossref PubMed Scopus (108) Google Scholar) on the response of T. cruzi to hypoosmotic stress have shown that both insect and vertebrate stages possess a robust RVD mechanism that completely reverses cell swelling following a 50% reduction in extracellular osmolarity. Approximately 50% of this compensatory volume reversal could be accounted for by the efflux of cytosolic amino acids (5Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Crossref PubMed Scopus (108) Google Scholar). Subsequent studies of other major osmolytes, such as sodium, potassium, chloride, inositol, methylamines, Pi, and pyrophosphate (PPi), failed to reveal another efflux mechanism that could account for the remaining volume reversal, except for K+ release that could account for only about 7% of the RVD (6, Rohloff, P. (2003) Osmoregulation in Trypanosoma cruzi: The Role of the Acidocalcisome. Ph.D. thesis, University of Illinois at Urbana-ChampaignGoogle Scholar). In many unicellular eukaryotes the adaptation to hypo-osmotic stress involves, in addition to the release of ions and osmolytes as occurs in mammalian cells (7Steck T.L. Chiaraviglio L. Meredith S. J. Eukaryotic Microbiol. 1997; 44: 503-510Crossref PubMed Scopus (16) Google Scholar), the release of water by a contractile vacuole complex (CVC). Recent work (8Allen R.D. Naitoh Y. Int. Rev. Cytol. 2002; 215: 351-394Crossref PubMed Scopus (86) Google Scholar) has shown that most, if not all, CVCs are composed of a two-compartment system enclosed by two differentiated membranes. One membrane (spongiome), which is often divided into numerous vesicles and tubules, contains a proton-translocating V-H+-ATPase that provides an electrochemical gradient of protons for water transport and that can fuse only with the membrane of the second compartment. The membrane of the second compartment (bladder) expands into a reservoir for water storage and is capable of fusing with the plasma membrane. It is this second compartment that periodically undergoes contraction, with the expulsion of water (8Allen R.D. Naitoh Y. Int. Rev. Cytol. 2002; 215: 351-394Crossref PubMed Scopus (86) Google Scholar). In addition, other vacuoles besides the contractile vacuole bladder have been observed to take up water when protozoa are placed in hypo-osmotic media (9Cronkite D.L. Neuman J. Walker F. Pierce S.K. J. Protozool. 1991; 38: 565-573Crossref PubMed Scopus (9) Google Scholar, 10Temesvari L.A. Rodriguez-Paris J.M. Bush J.M. Zhang L. Cardelli J.A. J. Cell Sci. 1996; 109: 1479-1495Crossref PubMed Google Scholar), and they have been suggested also to play a role in volume homeostasis (11Van Rossum G.D.V. Russo M.A. Schisselbauer J.C. Curr. Top. Membr. Transp. 1987; 30: 45-74Crossref Scopus (29) Google Scholar). We have recently found that an aquaporin or a water channel is present in the contractile vacuole complex as well as in the acidocalcisomes of T. cruzi and suggested a role for these organelles in osmoregulation (12Montalvetti A. Rohloff P. Docampo R. J. Biol. Chem. 2004; 279: 38673-38682Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Acidocalcisomes are acidic calcium-containing organelles present in a number of unicellular eukaryotes (13Docampo R. Moreno S.N. Mol. Biochem. Parasitol. 2001; 114: 151-159Crossref PubMed Scopus (135) Google Scholar, 14Ruiz F.A. Marchesini N. Seufferheld M. Govindjee Docampo R. J. Biol. Chem. 2001; 276: 46196-46203Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15Marchesini N. Ruiz F.A. Vieira M. Docampo R. J. Biol. Chem. 2002; 277: 8146-8153Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), as well as in bacteria (16Seufferheld M. Vieira M.C. Ruiz F.A. Rodrigues C.O. Moreno S.N.J. Docampo R. J. Biol. Chem. 2003; 278: 29971-29978Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), that have been postulated to be involved in osmoregulation because they change their polyphosphate (polyP) (17Ruiz F.A. Rodrigues C.O. Docampo R. J. Biol. Chem. 2001; 276: 26114-26121Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and ionic (18LeFurgey A. Ingram P. Blum J.J. Comp. Biochem. Physiol. A. 2001; 128: 385-394Crossref PubMed Scopus (38) Google Scholar) content when submitted to osmotic changes. It has been postulated that acidocalcisomes could act as a subcellular, osmotically active reservoir linked to the contractile vacuole function in both Chlamydomonas reinhardtii (14Ruiz F.A. Marchesini N. Seufferheld M. Govindjee Docampo R. J. Biol. Chem. 2001; 276: 46196-46203Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and Dictyostelium discoideum (15Marchesini N. Ruiz F.A. Vieira M. Docampo R. J. Biol. Chem. 2002; 277: 8146-8153Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In addition, acidocalcisomes could be the "vesicles" or "vacuoles" that have long ago been identified in free-living protozoa as transferring either water or osmolytes and ions to the spongiome linked to the CV (8Allen R.D. Naitoh Y. Int. Rev. Cytol. 2002; 215: 351-394Crossref PubMed Scopus (86) Google Scholar), because, as acidocalcisomes (13Docampo R. Moreno S.N. Mol. Biochem. Parasitol. 2001; 114: 151-159Crossref PubMed Scopus (135) Google Scholar), they usually appear as indistinctive empty vesicles when examined under conventional transmission electron microscopy (8Allen R.D. Naitoh Y. Int. Rev. Cytol. 2002; 215: 351-394Crossref PubMed Scopus (86) Google Scholar). In this study we have examined the role of the acidocalcisomes and the CVC in osmoregulation in T. cruzi. Hypo-osmotic stress resulted in a significant increase in cyclic AMP, swelling of the acidocalcisomes, and displacement of green fluorescent protein (GFP)-T. cruzi aquaporin (TcAQP) immunofluorescence labeling from the acidocalcisomes to the CVC in a microtubule- and cyclic AMP-dependent fashion. In addition, we isolated the contractile vacuole bladders, showed that they are enriched in alkaline phosphatase, V-H+-ATPase, and calmodulin, and found that they have large amounts of basic amino acids and Pi and that they are able to transport protons in the presence of ATP or PPi. RVD was inhibited by aquaporin inhibitors, by an intracellular Ca2+ chelator, and by a vacuolar H+-ATPase proteolipid subunit inhibitor, suggesting fusion of acidocalcisomes with the CVC. These fusion events with the CVC were also recorded. These results support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation. Cell Culture—T. cruzi epimastigotes (Y strain) were grown at 28 °C in liver infusion tryptose medium (19Bone G. Steinert M. Nature. 1969; 178: 308-309Crossref Scopus (133) Google Scholar) supplemented with 5% heat-inactivated newborn calf serum. GFP-TcAQP- or GFP-expressing epimastigotes (12Montalvetti A. Rohloff P. Docampo R. J. Biol. Chem. 2004; 279: 38673-38682Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) were maintained in liver infusion tryptose medium supplemented with 10% heat-inactivated fetal bovine serum and 1 mg/ml geneticin. Chemicals—Mammalian protease inhibitor mixture, β-glycerophosphate, dipyridamole, 3-isobutyl-1-methylxanthine (IBMX), 2-deoxy-3-AMP, dibutyryl cyclic AMP, bovine serum albumin, Dulbecco's phosphate-buffered saline (PBS), nigericin, EGTA, Dulbecco's modified Eagle medium, and pancreatin were from Sigma. Bafilomycin A1 was from Kamiya Biochemicals (Seattle, WA). Geneticin was from Invitrogen. Newborn calf serum was from BioWhittaker (Walkersville, MD). Fetal bovine serum was from HyClone (Logan, UT). Silicon carbide (400 mesh), chloralin, and fluorescamine were from Aldrich. Trifuralin was from Lilly. Iodixanol (60% solution, Optiprep, from Nycomed) was from Greiner Bio-One (Longwood, FL). Acridine orange was from Molecular Probes (Eugene, OR). Chemiluminescence (ECL) kit and kits for the determination of cyclic AMP and cyclic GMP were from Amersham Biosciences. All other reagents were analytical grade. Mouse polyclonal antiserum was raised against the T. cruzi vacuolar H+-pyrophosphatase (20Luo S. Vieira M.A. Zhong L. Graves J. Moreno S.N.J. EMBO J. 2001; 20: 55-64Crossref PubMed Scopus (79) Google Scholar). Rabbit polyclonal antiserum was raised against a 17-amino acid C-terminal peptide from T. cruzi aquaporin (12Montalvetti A. Rohloff P. Docampo R. J. Biol. Chem. 2004; 279: 38673-38682Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Mouse monoclonal antibody (8B1) raised against the yeast 69-kDa V-H+-ATPase subunit was purchased from Molecular Probes (Eugene, OR). Goat polyclonal antibody raised against an N-terminal 19-amino acid peptide from human calmodulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse Alexa 546 nm conjugate and rabbit anti-goat Alexa 546 nm conjugate were from Molecular Probes. Goat anti-rabbit horseradish peroxidase conjugate was from Sigma. Rabbit anti-goat 10-nm gold conjugate was from Ted Pella (Redding, CA). Cell Treatments—For experiments involving the induction of hypoosmotic stress, cells were washed extensively and resuspended in buffer A (116 mm NaCl, 5. 4 mm KCl, 0.8 mm MgSO4, 5.5 mm glucose, 50 mm Hepes, pH 7.4). The osmolarity of the buffer was adjusted to 300 ± 5 mOsm as verified by an Advanced Instruments 3D3 Osmometer (Norwood, MA). A 50% hypo-osmotic stress was induced by 1:1 dilution of cell suspensions in buffer A with deionized water. Cell Volume Measurements—Relative changes in cell volume after induction of hypo-osmotic stress were followed by using a light scattering technique as described previously (5Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Crossref PubMed Scopus (108) Google Scholar, 6, Rohloff, P. (2003) Osmoregulation in Trypanosoma cruzi: The Role of the Acidocalcisome. Ph.D. thesis, University of Illinois at Urbana-ChampaignGoogle Scholar). Cyclic AMP and Cyclic GMP Determinations—Cells were suspended at a concentration of 2 × 108 per ml and preincubated with 1 mm IBMX for 10 min in Dulbecco's PBS supplemented with 5 mm glucose. Then they were diluted with either water or buffer to 1 × 108 cells per ml. Aliquots of 0.5 ml were taken at the times indicated, centrifuged, and resuspended in 100 μl of PBS and 400 μl of 50 mm sodium acetate, pH 5.5, preheated to 95 °C. These samples were heated at 95 °C for 5 min, centrifuged, and the supernatants collected and stored at –20 °C. Cyclic AMP or cyclic GMP measurements were done using the Amersham Bioscience kits with the acetylation protocol. Electron Microscopy—For conventional electron microscopy, cells or subcellular fractions were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde, washed in Dulbecco's PBS, and then embedded in epoxide resin, sectioned, and stained using standard methods. Grids were observed using an Hitachi 600 transmission electron microscopy operating at 75 kV. For morphometric analysis of acidocalcisomes, the diameter of acidocalcisomes in randomly selected cells was measured at ×40,000 magnification. At least 40 acidocalcisomes were measured from each treatment group (hypo-osmotic, isosmotic) in each of four independent experiments. Fluorescence Microscopy and Immunofluorescence—For immunofluorescence, cells were fixed with 4% paraformaldehyde, adhered to poly-l-lysine coverslips, permeabilized for 5 min with Dulbecco's phosphate-buffered saline (PBS), 0.3% Triton X-100, blocked for 1 h with PBS, 3% bovine serum albumin, 1% fish gelatin, 5% goat serum, 50 mm NH4Cl, and incubated with primary antibody for 1 h and then secondary antibody for 45 min. For visualization of the vacuolar H+-ATPase, the 8B1 monoclonal antibody (Molecular Probes) was used at 1:10, followed by goat anti-mouse Alexa 546 nm conjugate at 1:1000. For detection of calmodulin, goat polyclonal anti-calmodulin antibody (Santa Cruz Biotechnology) was used at 1:50, followed by rabbit anti-goat Alexa 546 conjugate at 1:1000. Specimens were observed with a Leica TCS SP2 laser-scanning confocal microscope. For assessment of GFP-TcAQP translocation, cells were exposed to hypo-osmotic stress for various times, fixed with 4% paraformaldehyde, adhered to poly-l-lysine coverslips, and visualized by epifluorescence microscopy. For each treatment or time point, at least 50 cells were randomly selected and examined. A "nontranslocation" phenotype was recorded when several scattered vesicular localizations of the GFP-TcAQP could be observed; a "translocation phenotype" was recorded when all the GFP-TcAQP fluorescence was concentrated in a single region near the flagellar pocket. Results were expressed as % translocated cells out of the total number of cells examined. Trypanosome Immobilization and Acidocalcisome Tracking—Epimastigotes overexpressing GFP-TcAQP were immobilized onto glass surfaces coated with coral tree lectin (Erythrina cristagalli), which interacts with glycoproteins located on the parasite surface membrane containing oligosaccharides with galactosyl (β-1,4)-N-acetylglucosamine. Cells were immobilized on coverslips in 4- or 8-well plates. The lectin was dissolved in Dulbecco's PBS at a concentration of 0.5 mg/ml. Glass surfaces coated with poly-l-lysine were treated with the lectin solution for 30 min. The solution was then removed, and the lectin-coated surface allowed to air-dry under a laminar flow hood. Epimastigotes in logarithmic phase of growth were collected by centrifugation, washed three times with PBS, and resuspended in buffer A without glucose (116 mm NaCl, 5. 4 mm KCl, 0.8 mm MgSO4, 50 mm Hepes, pH 7.4). The osmolarity of the buffer was adjusted to 300 ± 5 mOsm. Cells were allowed to attach for 30 min at room temperature. Unattached cells were removed, and the wells were rinsed three times with buffer A. To induce translocation of GFP-TcAQP toward the contractile vacuole, buffer A was replaced by the same buffer diluted 1:1 with deionized water or with 500 μm dibutyryl cAMP under isosmotic conditions. Specimens were observed with a Leica TCS SP2 laser-scanning confocal microscope or a two-photon microscope as described before (21Levi V. Ruan Q. Kis-Petikova K. Gratton E. Biochem. Soc. Trans. 2003; 31: 997-1000Crossref PubMed Scopus (0) Google Scholar). Video Microscopy—For microscopic analysis of contractile vacuole filling and vesicle fusion, epimastigotes were immobilized on glass slides with coral tree lectin as described above, bathed in 150 mOsm hypo-osmotic buffer, and viewed with a Zeiss Axiovert 100 inverted microscope. Time lapse photographic data were collected at 1-s intervals using a Roper Scientific Coolsnap camera and MCID software. Video sequences were reconstructed using QuickTime and Final Cut Pro software. Cytochemistry—For cytochemical detection of alkaline phosphatase, a modification of a method described previously was used (22Quiviger B. de Chastellier C. Ryter A. J. Ultrastruct. Res. 1978; 62: 228-236Crossref PubMed Scopus (26) Google Scholar). Cells were fixed in 2.5% glutaraldehyde, 50 mm cacodylate, pH 6.8, 200 mm sucrose, washed twice for 30 min in the same buffer, and then resuspended in 0.5 ml of reaction medium containing 50 mm Tris-HCl, pH 9.0, 200 mm sucrose, 20 mm β-glycerophosphate, and 2.6 mm lead citrate. Lead citrate was prepared as described previously (23Reynolds D. J. Cell Biol. 1963; 17: 208-212Crossref PubMed Scopus (17782) Google Scholar). The cell suspension was frozen in liquid nitrogen for 3 min to permeabilize the cells and then thawed by the addition of 4.5 ml of reaction medium. The suspension was incubated at room temperature for 30 min, washed three times with 50 mm cacodylate, pH 6.8, 200 mm sucrose, and embedded in epoxide resin for visualization by transmission electron microscopy. Western Blotting—Proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membranes using standard methods. All the following steps were performed in Dulbecco's PBS containing 0.1% Tween 20. Membranes were blocked overnight in 5% nonfat dry milk and washed three times, incubated with polyclonal anti-aquaporin antibody at 1:1000 for 1 h and washed three times, incubated with goat anti-rabbit horseradish peroxidase at 1:10,000 for 1 h and washed three times, and then the bands were detected by chemiluminescence. Contractile Vacuole Isolation and Characterization—Contractile vacuoles and acidocalcisomes were isolated simultaneously on discontinuous iodixanol density gradients by using a modification of a method described previously (24Scott D.A. Docampo R. J. Biol. Chem. 2000; 275: 24215-24221Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A cell pellet collected from 100 ml of late-log epimastigote culture was disrupted by grinding with silicon carbide; the lysate was clarified by three low speed centrifugations (two times at 36 × g and one time at 144 × g), and the resulting supernatant was centrifuged at 100,000 × g for 60 min. The resulting pellet was loaded into the 24% layer of an iodixanol gradient containing 4-ml steps of 15, 20, 24, 28, 34, 37, and 40%. The gradient was centrifuged at 50,000 × g for 60 min, and 1-ml fractions (27 in all) were collected from the top. Aliquots of each collected fraction were assayed for enzymatic markers of lysosomes (α-mannosidase), acidocalcisomes (vacuolar-type (AMDP-sensitive) H+-pyrophosphatase), mitochondria (alanine amino-transferase), glycosomes (hexokinase), and contractile vacuole (alkaline phosphatase) as described previously (14Ruiz F.A. Marchesini N. Seufferheld M. Govindjee Docampo R. J. Biol. Chem. 2001; 276: 46196-46203Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15Marchesini N. Ruiz F.A. Vieira M. Docampo R. J. Biol. Chem. 2002; 277: 8146-8153Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 25Scott D.A. Docampo R. Dvorak J.A. Shi S. Leapman R.D. J. Biol. Chem. 1997; 272: 28020-28029Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Vacuolar-type (bafilomycin A1-sensitive) H+-ATPase was assayed as described previously (14Ruiz F.A. Marchesini N. Seufferheld M. Govindjee Docampo R. J. Biol. Chem. 2001; 276: 46196-46203Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Aliquots were also assayed for amino acid, Pi, and polyP content as described previously (5Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Crossref PubMed Scopus (108) Google Scholar, 17Ruiz F.A. Rodrigues C.O. Docampo R. J. Biol. Chem. 2001; 276: 26114-26121Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Acidification of isolated contractile vacuole fractions was assayed by measuring changes in the absorbance of acridine orange (A493–A530 nm) as described previously (26Scott D.A. de Souza W. Benchimol M. Zhong L. Lu H.G. Moreno S.N. Docampo R. J. Biol. Chem. 1998; 273: 22151-22158Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) in an SLM-Aminco DW2000 dual wave-length spectrophotometer. Aliquots of contractile vacuole fractions were incubated at 30 °C in 2.5 ml of 65 mm KCl, 125 mm sucrose, 2 mm MgSO4, 10 mm K+-Hepes, 50 μm EGTA, pH 7.2, containing 3 μm acridine orange. Other compounds (PPi, ATP, NH4Cl, AMDP, and nigericin) were added at the times and concentrations indicated in the appropriate figure legend. Membrane Integrity and Amino Acid Release—Membrane integrity after treatment with AgNO3 and HgCl2 was determined by ethidium bromide exclusion as described before (5Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Crossref PubMed Scopus (108) Google Scholar). Total amino acid content and amino acid analysis of the supernatants of cells exposed to hypotonic or isotonic buffer were determined as described before (5Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Crossref PubMed Scopus (108) Google Scholar). HgCl2 and AgNO3 Inhibit the RVD in Epimastigotes—In a previous study (12Montalvetti A. Rohloff P. Docampo R. J. Biol. Chem. 2004; 279: 38673-38682Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) we identified an aquaporin (TcAQP) that is located in acidocalcisomes and the contractile vacuole complex of T. cruzi epimastigotes. In contrast to other aquaporins that are inhibited by high micromolar concentrations of either HgCl2 or AgNO3 (27Hansen M. Kun J.F. Schultz J.E. Beitz E. J. Biol. Chem. 2002; 277: 4874-4882Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 28Niemietz C.M. Tyerman S.D. FEBS Lett. 2002; 531: 443-447Crossref PubMed Scopus (249) Google Scholar), TcAQP was shown to be exquisitely sensitive to inhibition by very low concentrations of these compounds (12Montalvetti A. Rohloff P. Docampo R. J. Biol. Chem. 2004; 279: 38673-38682Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We therefore treated cells with 1 μm of either HgCl2 or AgNO3, a concentration shown previously to inhibit osmotic swelling in Xenopus laevis oocytes transfected with TcAQP (12Montalvetti A. Rohloff P. Docampo R. J. Biol. Chem. 2004; 279: 38673-38682Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and we then monitored the rate of volume recovery during hypo-osmotic stress. Relative volume was followed over time using the light-scattering technique as described previously (5Rohloff P. Rodrigues C.O. Docampo R. Mol. Biochem. Parasitol. 2003; 126: 219-230Crossref PubMed Scopus (108) Google Scholar), in which changes in absorbance of a cell suspension are negatively correlated with changes in cell volume. Both inhibitors significantly reduced the rate of volume recovery (Fig. 1, A and B). Furthermore, this inhibitory effect was not because of interference with hypo-osmotically induced amino acid efflux (Fig. 1C) and was not because of a general toxic effect of the compounds, because the magnitude of amino acid release was unaffected by pretreatment with either HgCl2 or AgNO3 (Fig. 1C). Epimastigotes under isosmotic conditions treated with either 1 μm HgCl2 or 1 μm AgNO3 for 5 min showed neither morphological nor motility alterations and did not increase their permeability to ethidium bromide (Fig. 1D), indicating the absence of toxic side effects by these low salt concentrations. Taken together, these results suggest a central role for TcAQP in the response of the parasites to hypo-osmotic stress. GFP-TcAQP Translocation during Hypo-osmotic Stress— Based on analogy to vasopressin-stimulated translocation of AQP-2-containing vesicles in the mammalian kidney collecting ducts (29Nielsen S. Chou C.L. Marples D. Christensen E.I. Kishore B.K. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1013-1017Crossref PubMed Scopus (888) Google Scholar, 30Gustafson C.E. Levine S. Katsura T. McLaughlin M. Aleixo M.D. Tamarappoo B.K. Verkman A.S. Brown D. Histochem. Cell Biol. 1998; 110: 377-386Crossref PubMed Scopus (39) Google Scholar), the possibility of a hypo-osmotically induced TcAQP translocation event was studied in GFP-TcAQP expressing epimastigotes. As reported before (12Montalvetti A. Rohloff P. Docampo R. J. Biol. Chem. 2004; 279: 38673-38682Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), GFP-TcAQP localized to acidocalcisomes (arrowheads) and to the contractile vacuole (Fig. 2B, arrow). When cells were stressed and observed microscopically, the GFP-TcAQP-containing acidocalcisomes that were closer to the contractile vacuole could be observed migrating toward it (see below). In many cells (Fig. 2D) only the fluorescent spot corresponding to the contractile vacuole could be observed because it became brighter after hypo-osmotic stress, and less fading of this signal than that of the acidocalcisomes occurred. We therefore investigated whether it was possible to image the movement of acidocalcisomes in live cells submitted to hypo-osmotic stress or in the presence of dibutyryl cyclic AMP (500 μm, see below) either by video fluorescence microscopy or by a new method for three-dimensional particle tracking termed scanning fluorescence correlation spectroscopy (21Levi V. Ruan Q. Kis-Petikova K. Gratton E. Biochem. Soc. Trans. 2003; 31: 997-1000Crossref PubMed Scopus (0) Google Scholar). Because epimastigotes are very motile cells, they needed to be immobilized to follow these changes. This was successfully achieved by attaching them to coverslips covered with coral tree lectin (see "Experimental Procedures"). Periodic filling of the contractile vacuole could be followed by phase microscopy when epimastigotes were incubated in the buffer at 150 mOsm (Fig. 3A; see also movie 1 under Supplemental Material). However, attempts to follow the movement of acidocalcisomes using video fluorescence microscopy, or scanning fluorescence correlation spectroscopy using a two-photon microscope, were unsuccessful, due in part to the small size of the acidocalcisomes and also the fa