EXPERIMENTAL PARASITOLOGY ARTICLE NO. PR974197
87, 133–141 (1997)
Giardia lamblia: Evidence for Carrier-Mediated Uptake and Release of Conjugated Bile Acids Siddhartha Das,*,†,1 Claudio D. Schteingart,‡,2 Alan F. Hofmann,‡ David S. Reiner,† Stephen B. Aley,*,† and Frances D. Gillin†,§ *Department of Biological Sciences, University of Texas, El Paso, Texas 79968, U.S.A.; †Department of Pathology, §Center for Molecular Genetics, and ‡Division of Gastroenterology, University of California, San Diego, California 92103, U.S.A. DAS, S., SCHTEINGART, C. D., HOFMANN, A. F., REINER, D. S., ALEY, S. B., AND GILLIN, F. D. 1997. Giardia lamblia: Evidence for carrier-mediated uptake and release of conjugated bile acids. Experimental Parasitology 87, 133–141. Giardia lamblia trophozoites colonize the human small intestine, where they are exposed to high concentrations of conjugated bile acids. Previous work has shown that bile acids enhance trophozoite survival, multiplication, and differentiation into the cyst stage. Therefore, experiments were performed to test whether carrier-mediated uptake of conjugated bile acids is present in this primitive parasite. Uptake of both cholyltaurine (C-tau) and cholylglycine (C-gly) was increased manyfold after culturing trophozoites in medium lacking bile acids. Absence of uptake at 4°C and inhibition by other conjugated bile acids provided additional evidence for carrier-mediated uptake. Uptake of C-tau was greater than that of C-gly under all experimental conditions and appeared to be mediated by a different carrier. The major evidence for different carriers is that C-tau uptake was Na+-dependent, while C-gly uptake was not. In addition, C-tau uptake was more strongly inhibited by DTNB and several organic anions than C-gly uptake. Radiolabeled C-tau and C-gly were each released rapidly from trophozoites at 37°C but not at 4°C, suggesting that release of conjugated bile acids was also carrier-mediated. These findings are consistent with the notion that multiple transporters for conjugated bile acids are present in a lower eukaryote. We speculate that intracellular bile acids may facilitate lipid trafficking and membrane biosynthesis. © 1997 Academic Press INDEX DESCRIPTORS AND ABBREVIATIONS: Giardia lamblia; bile acid; transport; efflux; c-tau, cholyltaurine or taurocholate; c-gly, cholylglycine or glycocholate; PC, phosphatidylcholine; DTNB, 5, 59-dithio-bis(2-nitrobenzoic acid); DTT, dithiothreitol; 12OHC-tau, 12-hydroxycholanoyltaurine; DC-tau, deoxycholyltaurine, CDC-tau, chenodeoxycholyltaurine, CDC-gly, chenodeoxycholylglycine.
INTRODUCTION Giardia lamblia is a major cause of waterborne enteric disease worldwide. The flagellated trophozoites colonize the small intestine of infected humans below the entrance of the common bile duct and are therefore exposed to high concentrations of conjugated bile acids (Adam 1991). Bile acids, which are end products of cholesterol metabolism, are synthesized in the liver, conjugated in N-acyl linkage with glycine or taurine, and secreted into the bile. Efficient absorption in the terminal ileum leads 1 To whom correspondence should be addressed. Fax: 915-747-5808. E-mail:
[email protected]. 2 Present address: Ferring Research Institute Inc., San Diego, CA 92121.
to the accumulation of a large recycling pool of bile acids which is stored in the gallbladder between meals and discharged into the small intestine during digestion, a process termed the enterohepatic circulation (Hofmann 1989). Conjugated bile acids are planar amphipathic molecules (MW ∼500 Da) that are important for the solubilization of various dietary lipids (Hofmann 1989; Hofmann and Mysels 1988). Because they are strong acids, conjugated bile acids are fully ionized at the pH prevailing in the small intestine. Because of their charge and size, bile acid molecules do not passively cross either cell membranes or the paracellular tight junctions of the small intestinal mucosa in the adult mammal. The lack of passive absorption of bile acids contributes to the high intraluminal
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concentration of conjugated bile acids in the proximal small intestinal lumen (5–30 mM) (Hofmann 1989). The detergent property of these bile acids plays a role in inhibiting bacterial growth in the normal human small intestine (Simon and Gorbach 1981). As early as 1937, bile or bile acids were proposed as a determinant of Giardia growth in the intestine of rodents (Hegner and Eskridge 1937). More recently, cultured G. lamblia trophozoites were reported to have an extremely limited capacity for de novo lipid biosynthesis, and to obtain their lipids from serum in culture (Jarroll et al. 1981). Farthing et al. (1985) proposed that in vivo, trophozoites obtain much of their membrane phospholipid from the PC present in bile. Bile from a number of mammals stimulated in vitro growth of trophozoites (Farthing et al. 1983, 1985; Keister 1983), and bile acids increased uptake of phospholipids (Halliday et al. 1995) and cholesterol (Lujan et al. 1996). We reported that a defined mixture of biliary lipids containing bile acids, PC, and cholesterol supported growth of G. lamblia in a serum-free medium (Gillin et al. 1986). Moreover, we showed that trophozoites were able to tolerate concentrations of conjugated bile acids in vitro (Gillin 1987) that are cytotoxic for cultured mammalian cells (Quist et al. 1991). In addition, we found that conjugated bile acids, when present above their critical micellar concentrations, protected trophozoites from lysis by unconjugated fatty acids which are formed in the small intestinal lumen during digestion of dietary triglycerides (Das et al. 1988). Finally, in studies aimed at inducing the complete life cycle of G. lamblia in vitro, we found that exposure to bile is an absolute requirement for differentiation of the trophozoite into the cyst form (Gillin et al. 1989). Bile or bile acids stimulated expression of cyst antigens, as well as formation of encystation-specific secretory vesicles that transport cyst antigens to the nascent cyst wall (Reiner et al. 1989; Faubert et al. 1991). Thus, under different conditions, biliary constituents not only stimulate trophozoite growth but are also required for encystation. Here we report that uptake of conjugated bile
acids by G. lamblia trophozoites is carriermediated and that at least two distinct transport mechanisms are present. MATERIALS AND METHODS Materials. Unless otherwise specified, all chemicals were purchased from Sigma Chemical Co., St. Louis, MO, and were of the highest purity available. C-gly was obtained from Fluka Chemicals (St. Louis, MO). Sodium-free C-tau (Ca2+ salt) was prepared by an ion exchange procedure. 12-Hydroxycholanoyltaurine (12OHC-tau) was synthesized from 12a-hydroxycholan-24-oic acid and taurine as described (del Vecchio et al. 1995). DIDS (4,49diisothiocyanatostilbene-2,29-disulfonic acid, disodium salt), SITS (4-acetoamido-49-isothiocyanotostilbene-2-29disulfonic acid, disodium salt), and Lucifer Yellow (lithium salt) were purchased from Molecular Probes (Eugene, OR). Rose Bengal was purchased from Aldrich Chemicals (Milwaukee, WI). [3H(G)]Cholyltaurine ([3H]C-tau) and [glycine-1-14C]cholylglycine ([14C]C-gly) were purchased from New England Nuclear (Boston, MA). Organisms. G. lamblia strain WB (ATCC No. 30957, clone C6) belongs to the most common group of human isolates (Weiss et al. 1992). Trophozoites were grown at 37°C with subculture twice weekly in filter-sterilized Diamond’s TYI-S-33 medium with 10% adult bovine serum and bovine bile (500 mg/ml) (Diamond et al. 1978; Keister et al. 1983), but without added vitamins, iron, or antibiotics. Uptake of radiolabeled bile acids. Trophozoites were grown to late log phase in tissue culture flasks in TYI-S-33 growth medium containing bovine bile as above. In early experiments (not shown), we found that depriving trophozoites of bile led to greatly increased uptake of radiolabeled bile acids. Therefore, monolayers were deprived of bile for 14 to 18 hr by refeeding with TYI-S-33 medium, pH 7.1, without bovine bile (not shown). Bile-replete control monolayers were refed with TYI-S-33 medium containing bovine bile (500 mg/ml). Unless otherwise indicated, the following protocol was utilized for uptake of bile acids. After incubation for 14 to 18 hr without bile, the media and unattached trophozoites were discarded and the attached parasites were harvested by chilling and centrifugation in labeling buffer consisting of 25 mM Hepes buffer, pH 7.1, containing 150 mM NaCl with 5 mM cysteine and 1.7 mM ascorbic acid, a medium which promotes survival (Gillin and Reiner 1982). The cells were then washed twice and resuspended in the same buffer. Uptake was initiated by addition of [3H]C-tau or [14C]C-gly at a final concentration of 20 mM (∼1–2 × 105 cpm) to 1 × 107 cells in a final volume of 0.5 ml. The cells were incubated at 37°C with occasional gentle shaking for 90 min, then chilled for 15 min to release any attached cells, and collected by centrifugation (10 min, 10,000g) in the cold. The cells were resuspended in 0.5 ml of the same cold buffer and washed three times. Finally, cell pellets were lysed in 0.2 ml water, and the radioactivity in 50 ml of cell
BILE ACID TRANSPORT BY extract was measured in duplicate by liquid scintillation spectrometry using Aqua-Mix scintillation fluid (Reiner et al. 1993). The sodium dependence of uptake was determined by substituting 88 mM choline chloride or 150 mM KCl for the NaCl in the washing and labeling buffer. Initial experiments showed that this was an optimal choline chloride concentration (trophozoites are rather osmotolerant) and that the absence of Na+ did not decrease viability and attachment of trophozoites (not shown). [3H]C-tau and [14C]C-gly release. To measure bile acid release from trophozoites, [3H]C-tau or [14C]C-gly uptake was first carried out as described above for 120 min at 37°C. Radiolabeled conjugated bile acid-containing cells were then centrifuged and washed in the cold, resuspended in fresh labeling buffer without C-tau, and incubated at 37°C for various times between 0 and 60 min with shaking. The cells were collected by centrifugation and the radioactivity in buffer and cell pellets was counted as above. To determine whether bile acids were metabolized by G. lamblia, bile acids released into the buffer were analyzed by thin-layer chromatography (Hofmann 1962) and highpressure liquid chromatography (Rossi et al. 1987). Competition and inhibition experiments. Uptake of [3H]C-tau or [14C]C-gly (20 mM, unless otherwise specified) was carried out as above in the presence of the indicated natural conjugated bile acids or the potent ileal bile acid uptake inhibitor 12OHC-tau at the concentrations indicated. The data from the inhibition experiments of Fig. 4 were analyzed by a nonlinear curve fit. Effects of DTNB and DTT on [3H]C-tau and [14C]C-gly uptake were tested at 1 mM. Effects of various organic anions were examined using a concentration of 50 mM. These concentrations were chosen from prior dose response curves and were not toxic to G. lamblia under the conditions of the assay. Each experiment shown was performed in duplicate and is typical of two to six independent determinations. Statistical analyses are described in each section.
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but the maximal uptake rate was less than that of C-tau and tended to occur earlier (not shown). Uptake of C-tau by G. lamblia trophozoites was largely Na+-dependent (Fig. 1). Uptake continued at a fairly constant velocity and reached a plateau at ∼60 min. In contrast, uptake of C-gly by trophozoites was identical in the presence and the absence of Na+ ions (with either choline chloride or KCl, not shown), supporting a second carrier for C-gly. Bile acid uptake was not detectable at 4°C, consistent with a transporter-mediated process (data not shown). Release of Conjugated Bile Acids To establish the fate of C-tau and C-gly after uptake, trophozoites which had been prelabeled for 120 min with C-tau or C-gly were washed extensively at 4°C and then incubated at 37°C, and the kinetics of efflux determined (Fig. 2).
RESULTS +
Na -Dependent and -Independent Bile Acid Transport In initial studies, we reported (Reiner et al. 1993) that the rate of [3H]C-tau uptake was increased by bile starvation. In additional experiments, transport of each conjugated bile acid was greatly stimulated by preincubating trophozoite monolayers in growth medium lacking its normal supplement of bovine bile. Transport of C-tau gradually increased from
