003 1-3998/92/3201-0118$03.00/0 PEDIATRIC RESEARCH Vol. 32, No. 1, 1992 Copyright O 1992 International Pediatric Research Foundation, Inc. Printed in U.S.A. Taurine and Osmoregulation. IV. Cerebral Taurine Transport Is Increased in Rats with Hypernatremic Dehydration1 HOWARD TRACHTMAN, STEPHEN FUTTERWEIT, AND RICHARD DEL PIZZO Department of Pediatrics, Schneider Children's Hospital, Long Island Jewish Medical Center, Long Island Campus for Albert Einstein College of Medicine, New Hyde Park, New York 11042 ABSTRACT. Taurine is an organic osmolyte in brain cells. Regulation of cerebral cell volume in the face of osmolal stress We studied whether cerebral taurine transport is enhanced is an important biological function in all species (1). In terrestrial as part of the cell volume regulatory adaptation to hyper- mammals, the brain is encased in a rigid, bony vault. Therefore, natremia. Hypernatremic dehydration was induced for 48 this organ poorly tolerates sudden changes in cell size induced h. Synaptosomes, metabolically active nerve terminal ves- by changes in serum osmolality (2). Cerebral cells have developed icles, were isolated by homogenization of brain and purifi- the capacity to modulate the intracellular content of osmopro- cation on a discontinuous Ficoll gradient. Taurine transport tective molecules in response to hypo- and hyperosmolal states was evaluated in vitro using a rapid filtration assay. After and to minimize fluctuations in cell size (3). The important 48 h of hypernatremia, there was a 22.4% increment in classes of compatible, organic osmolytes include inorganic ions, Na+-specific taurine transport from 2.99 f 0.16 to 3.66 2 amino acids, polyhydric sugar alcohols, and methylamines (4). 0.13 pmollmg protein130 min (p < 0.001). Dehydration for Taurine (2-aminoethane sulfonic acid) is a constituent of the 48 h without hypertonic saline loading had no effect on amino acid pool of nonperturbing osmoprotective molecules in taurine uptake. Glycine transport was unaltered by hyper- natremia. The adaptation in taurine uptake resulted from invertebrate and vertebrate species (5, 6). It has been thought an enhanced V,,, of the high affinity-low capacity trans- that the primary means of regulating the cytosolic pool of os- port system [265 +: 17, control versus 337 f 19 nmol/min/ molytes during osmolal stress is release or sequestration of these mg protein, experimental (p < 0.03)] without a change in molecules in subcellular organelles (2, 7). However, exogenous the Km (260 pM). Under both control and hypernatremic taurine supplements the cerebral cell content of osmolytes and conditions, Na+ and C1- were required for maximal total confers protection against brain shrinkage in rats with hyperna- Na+-mediated taurine uptake. Oubain (1 mM) decreased tremic dehydration (8). This suggests that altered transmembrane taurine uptake by 25%, whereas addition of B-alanine or flux of osmoprotective molecules is part of the coordinated hypotaurine (500 pM) to the external media reduced tau- cerebral response to osmolal stress. Therefore, we conducted the rine transport by 4565% in both control and experimental following investigations to test the hypothesis that brain taurine conditions (p < 0.01). Synaptosomal taurine uptake in uptake is increased during hypernatremic dehydration. The hypernatremic rats was inhibited by 1520% (p c 0.01) methodologic approach involved the use of synaptosomes, which after addition of 4-acetamido-4'-isothiocyanostilbene-2,2'- are metabolically active nerve terminal vesicles formed by me- disulfonic acid (0.1 mM) or 4,4'-diisothiocyanostilbene- chanical shearing forces during homogenization of the brain (9, 2,2'-disulfonic acid (0.1 mM) to the external medium. We 10). They can be used for in vitro assessment of changes in conclude that hypernatremic dehydration of moderate se- intrinsic cerebral membrane transport function. verity and duration results in stimulation of brain taurine uptake, mediated by increased activity of the B-amino acid carrier. An intact anionic binding site is required for max- imal taurine uptake during hypernatremia. (Pediatr Res MATERIALS AND METHODS 32: 118-124,1992) Animals. Male Sprague-Dawley rats (Taconic Farms, German- town, NY) weighing 200-350 g were used in these experiments. They were kept in an animal facility that was maintained at 25°C Abbreviations with a 12-h lightldark cycle. They were fed rodent chow contain- DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfoc acid ing 22.8% protein (Ralston Purina, St. Louis, MO) and were MOPS, 3-[N-morpholino]propanesulfonic acid provided water to drink ad libitum before the onset of the SITS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disul- experiments. fonic acid All experimental protocols were approved by the Animal Utilization Review Committee of Long Island Jewish Medical Center. Hypernatremic dehydration regimen. Hypernatremic dehydra- Received February 18, 199 I; accepted March 16, 1992. tion was induced for 48 h according to previously described Correspondence: Howard Trachtman, M.D., Schneider Children's Hospital, methods (1 1). After weighing the rats and obtaining a pretreat- Division of Pediatric Nephrology, 271-16 76th Avenue, New Hyde Park, NY 1 1042. ment serum sample for chemical analysis,-. animals were totallv Supported by grants from the American Heart Association, New York affiliate deprived of water for 24 h. The rats were weighed the next da; (Grant No. 88-0236) and the Long Island Jewish Medical Center Research Award and received intraperitoneal injections of 1 M N~C~in a dose Program. I Presented in part at the meeting of the Society for Pediatric Research, Wash- designed raise the serum Naf 180 mmO1/L ington, DC, May 1989. over the next 24 h. The dose was calculated according to the 118 CEREBRAL TAURINE TRANS1PORT IN HYPERNATREMIA 119 following formula: medium after equilibration and centrifugation was used to cal- culate the internal volume of the synaptosomes, expressed as pL/ Volume of 1 M NaCl (mL) = 0.6 x body weight x (180 - 140) mg protein. Assays of synaptosome integrity and purity. The integrity of Rats were provided food but not water during the period of the synaptosomes was determined by assaying the lactate dehy- hypernatremia. Animals with dehydration due to water depri- drogenase enzyme activity in the medium before and after adding vation alone were denied access to drinking water for a total of Triton X-100, 0.5% vol, to the sample. The rate of NADH 48 h, but did not receive hypertonic saline injections. Control formation was measured by monitoring the conversion of lactate rats, which had free access to water and were given sham injec- to pyruvate at 340 nm. The purity of the synaptosome prepara- tions, were studied in parallel with experimental animals. In each tion was evaluated by measuring rotenone-insensitive NADH synaptosome preparation procedure, two to four animals evenly and NADPH cytochrome c reductase activities in 3 mL of a divided between the control and hypernatremic conditions were reaction mixture containing (in mM) KCN 0.3, NAD(P)H 0.1, studied. At the completion of each regimen, animals were killed cytochrome c 0.1, rotenone 1.5, and K2HP0450, pH 7.4. Syn- by decapitation. A free-flowing, nonhemolyzed blood sample aptosomal protein, 25 pL, was added to start the reaction, which was obtained from the stump for determination of serum Na+ was monitored at 550 nm in a temperature-controlled spectro- concentration. The brains were excised and placed in an ice-cold photometer (Perkin Elmer, Nonvalk, CT) at 25°C. The extinction isolation medium containing (in mM) sucrose 320, Tris-HC1 10, coefficient of cytochrome c was taken to be 21.1 (1 3). and K-EDTA 1, pH 7.4, for preparation of synaptosomes. All Transport assay. Taurine uptake was measured using 1 mL of subsequent procedures were performed at 0-4°C. synaptosomal protein containing 4-6 mg/mL. Thawed samples Isolation of synaptosomes. Synaptosomeswere isolated accord- were incubated in 5 mL of the preequilibration medium for 10 ing to the procedure described by Fraser et al. (12). Brains were min at 37°C. The suspension was spun at 20 000 X g for 5 min minced with a scalpel, washed three times to remove blood and and the pellet was resuspended in 250-300 pL of the preequili- extraneous tissue fragments, brought up to 15 mL with isolation bration medium. The pellet was gently aspirated through a 25- medium, and then homogenized with 15 strokes of a glass gauge needle 3 to 5 times to ensure a homogeneous synaptosome Dounce homogenizer (Wheaton, Millville, NJ). Cellular debris suspension. Synaptosomes were maintained at 0-4°C until ali- was removed by centrifuging the homogenates at 1 300 X g for quots of the suspension were added to individual assay tubes. 3 min. The supernatant was respun at 18 000 X g for 10 min to External media contained (in mM) NaCl or KC1 140, MgCl2 5, obtain the crude synaptosome pellet. The pellet was resuspended EGTA 0.2, MOPS 5, and taurine 0.1. Ten pCi of 3H-taurine in 15 mL of isolation medium by homogenization with three were added to 4 mL of external medium. Glycine uptake was strokes. This suspension, obtained from one to two animals, was assayed using Na+ and K+ external media containing 0.1 mM layered onto a discontinuous Ficoll gradient composed of 11 and glycine and 10 pCi of 3H-glycine. In the ionic requirement 7.5% layers, each 12.5 mL in volume. The Ficoll was dialyzed studies, external medium contained an equimolar amount of for 5 h before being used to remove low molecular weight choline or gluconate instead of Na+ or C1-, respectively. In the fragments. The gradients were spun at 100 000 x g for 70 min, inhibitor studies, oubain (1 mM), p-alanine (500 pM), hypotau- and the synaptosomes were isolated from the interface between rine (500 pM), P-amino-isobutyric acid (500 pM), SITS (0.1 and the 7.5 and 11 % Ficoll layers.