0031-3998/95/3702-0227$03.00/0 PEDIATRIC RESEARCH Vol. 37, No. 2, 1995 Copyright © 1995 International Pediatric Research Foundation, Inc. Printed in U.S.A. The Relative Roles of External Taurine Concentration and Medium Osmolality in the Regulation of Taurine Transport in LLC-PKI and MDCK Cells DEBORAH P. JONES, LESLIE A. MILLER, AND RUSSELL W. CHESNEY Department of Pediatrics, University of Tennessee, Memphis, Le Bonheur Children 's Medical Center, Center for Pharmacokinetics and Therapeutics, Le Bonheur Children's Medical Center, Memphis, Tennessee 38163 ABSTR CT Taurine is a l3-sulfonic amino acid that serves as a nutrient whereas L-alanine had no effect. The concentration of taurine or important for developing brain and retina and as an osmolyte in structurally similar analogs in the external medium might modify the medullary collecting duct. The activity of the taurine trans­ the response of taurine accumulation after exposure to hypertonic port system is regulated by substrate supply and by the external medium, in that taurine-replete cells behave differently than osmolality; these two stimuli induce changes in taurine transport. taurine-depleted cells. These studies indicate that there are at Increased medium osmolality (500 mosmol) stimulates taurine least two distinct mechanisms involved in the regulation of uptake into MDCK cells but not LLC-PKI cells. The enhanced taurine transport: external taurine concentration and medium taurine uptake that occurs in response to hyperosmolality is osmolality, with taurine concentration seeming to be the predom­ localized primarily to the basolateral surface of MOCK cells, inant stimulus. Thus, the changes in cell taurine transport depend whereas the adaptive response to medium taurine concentration on the physiologic stimulus as well as the cell studied, a phe­ is expressed on both the apical and the basolateral surfaces of nomenon that might be related to the renal tubular site of origin. both cell lines. The response of MOCK cells to medium osmo­ (Pediatr Res 37: 227-232, 1995) lality requires protein synthesis and RNA transcription and is expressed in the presence of microtubular toxins. When cell Abbreviations monolayers were loaded with taurine by incubation in high­ GABA, aminoisobutyric acid taurine medium before increasing medium osmolality, the ex­ act D, actinomycin D pected increase in taurine uptake was blunted. Similarly, in­ cyt D, cytochalasin 0 creased external l3-alanine (500 p,M) also prevented the Coleh, colchicine anticipated increase in taurine accumulation in response to hy­ Cycle, cyclohexamide pertonicity; aminoisobutyric acid and betaine (500 p,M) partially GPC, glycerophosphocholine prevented the increase in taurine transport after hypertonicity, EBSS , Earle's balanced salt solution T aurine, a f3-sulfonic amino acid that is found in millimolar The kidney taurine transp orter serves at least two im po rtant intracellular concentrati ons in the kidney of many mammali an function s: 1) to reabsorb filte red taurine as a nutrient (this sp eci es, is pos tulated to ha ve several ke y functions in the occurs in the proximal tubule and has been shown to be kidney; among these are the fun cti on of a nutritional substrate, fun cti on ally immature in the neon atal rat, to be regulated by as well as that of an osmolyte (1). T aurine is transported by the dietary taurine intake, and to be abnormal in the hypertaurinin­ ren al tubular cell by a sodium- and chlo ride- dependent trans­ ur ic mouse model) and 2) to offe r protection to medullary porter, w hich accepts the f3-amino acid taurine and structural coll ecting du ct cells or to MDCK (M adin-Darby canine kid­ analogs such as f3-alanine (2-5) . ne y) cells in response to increased external os mola lity by serv ing as an osmol yte. In addition to sorbitol, inositol , gl yc­ erophosphorylcholine, and bet aine, taurine is increased in the Received April 26, 1994; accepted September 20, 1994. inner medulla of salt-loaded rats and MDCK cells. In the first Correspondence and reprint requests: Deborah P. Jones, M.D., University of Tennessee, example, taurine transporter activity changes in response to Memphis Department of Pediatrics, 956 Court Ave., Room B31O, Memphis, TN 38163. taurine supply, and in the latter, in response to external tonic­ Supported in part by grants from the National Institutes of Health (DK 37223), The National Kidney Foundation of West Tennessee, LeBonheur Children's Medical Center, ity. W e have been interested in contrasting the nature of the and The American Heart Association (91- 004470, Clinician Scientist Award). stimulus as well as the cell type: the proximal tubular cell, in 227 228 JONES ET AL. which regulation of taurine transport by substrate concentration radioactivity counted in a Packard Tricarb 2000-CA Liquid may be most important, in comparison with the collecting duct Scintillation Analyzer (Packard Instrument Co., Downers or distal cell, in which regulation by external osmolality is of Grove, IL). greater importance to cell survival. Total cell protein was measured by a modification of the Taurine transport is regulat ed by the dietary intake of taurine Lowry method (12). Duplicate lO-J.LL samples of cell homo­ in the rat (6), by the extracellular concentration in kidney cells genate and standards were pipetted into a 96-well microtiter in culture (7), and by medium osmolality in MDCK cells (8). plate. A Oo4-mL volume of solution A, consisting of 2% We have previousl y demonstrated that taurine transport in both Na2C03, 1.0% CuS0 4, and 2.0% KNaC4H406 (100:1:1) was MDCK and LLC-PK1 cells is increased after incubation of cell added to each well, followed by 0.04 mL of 1.0 N Folin­ monolayers in taurin e-free medium and is decreased after Diocalteau phenol reagent. After a 30-min incubation, the OD incubation of cells in high levels of extracellular taurine (8). was measured using an automated microtiter plate spectropho­ The adaptive response to medium taurine concentration was tometer at a wavelength of 680. The protein concentration of prevented when protein synthesis and protein export were the samples were determined by linear regression analysis from inhibited (9). Inhibition of RNA transcription had no effect on standards of BSA. Uptake was then expressed as pmol of the ability of the cells to respond to medium taurine concen­ taurine per mg of cell protein per min. tration (9). Adaptive response to medium substrate concentration. The following studies were performed to examine the inter­ Confluent cell monolayers were exposed for 24 h to hormonally action between medium hypertonicity and medium taurine defined, serum-free medium with 0, 50, or 500 J.LM taurine. concentration in regulation of taurine transport in cultured Uptake studies were performed after two 37°C washes in EBSS. renal epithelial cells. Response of cells to hypertonicity. Cell monolayers were exposed for 24 h to standard, hormon ally defined, serum-free METHODS medium with 50 J.LM taurine or with medium made hypertonic to 500 mosmol by the addition of raffinose. Monolayers were Cell culture methods. LLC-PK1 and MDCK cell lines were then gently washed twice in 37°C EBSS follow ed by measure­ obtained from American Type Culture Collection (Rockville, ment of taurine uptake in the presence and absence of sodium. MD) and maintained in 5% CO2, 95% air. Stand ard medium, MDCK monolayers were incubated in the presence of either consisting of Dulbecco's modified Eagle's medium (1000 act 0 (0.5 J.L glmL) (9, 13), which inhibits RNA transcription; mglL glucose, 584 mglL t -glutarnine, and 110 mglL sodium Cyclo (70 J.LM ) (9, 13), which inhibits protein synthesis; and pyruvate) in a 1:1 mixture with Ham's F12 Nutrient Mixture two inhibitors of microtubul ar depolymerization , cyt 0 (10 (GIBCO/BRL, Life Technologies, Grand Island, NY), plus J.LM ) (14) and Coleh (50 J.LM) (9), for 2 h before and during a 10% FCS and penicillin (100 U/ml) and streptomycin (100 12-h incubation in either standard or hypertonic medium . J.LglmL), was used for routine cell carriage (7). Cells were Materials. Media, penicillin/streptomycin, trypsin, and FCS subcultured by trypsinization and seeded onto Oo4-J.Lm polycar­ were purchased from GIBCO(Grand Island, NY). Radiolabeled bonate filter supports (Costar, Cambridge, MA, Transwell) taurine and inulin were purchased from New England Nuclear (10). For experiments, medium was replaced with a hormon­ Corp. (Boston, MA). Insulin, 'hydrocortisone, thyroxin, prostag­ ally defined, serum-free formulation consisting of Dulbecco's landin E1, EBSS, choline chloride, choline bicarbonate, and other modified Eagle's medium/F 12 with insulin (5 J.LglmL) , trans­ chemicals were from Sigma Chemical Co. (St. Louis, MO) and 5 ferrin (5 J.LglmL) , prostaglandin E1 (2.5 X 10- mglmL) , transferrin was from Calbiochem (La Jolla, CA). hydrocortisone (5 X 10-8 M), and thyroxin (5 X 10- 12 M). Data analysis. Data comparisons were made by t test for Transport studies. Uptake studies were perform ed on con­ independent data and by analysis of variance with assistance fluent monolayers 10-14 d after seeding. Briefly, cells were from the computer program STATVIEW 512+ (Brainpower, washed with EBSS at 3rC. Uptake was initiated by the Inc., Calabasas, CA). addition of EBSS with or without sodium, pH 704 , with 50 J.LM taurine (0.5 J.LCi 3H-taurine) at 37°C (10). Uptake was termi­ RESULTS nated by the removal of uptake solution followed by three rapid washes with cold EBSS. The uptake solution contain ed 14C _ Effect ofhypertonicity and medium taurine concentration inulin (0.1 J.LCi/mL) in addition to 3H-taurine. Luminal uptake on taurine uptake in LLC-PKI and MDCK cells.