Some Characteristics of Threonine Transport Across the Blood-Brain Barrier of the Rat

Abstract: Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals,
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  Journal zyxwvusrqpo f Neurochemislry zyxwvusrqpon aven Press, Ltd., New zyxwvutsrqp ork zyxwvusrqpo   1988 International Society for Neurochemistry zyxwvutsrqpo Some Characteristics of Threonine Transport Across the Blood-Brain Bamer of the Rat Armando Tovar, Jean K. Tews, Nimbe Torres, and Alfred E. Harper Departments of Nutritional Sciences and Biochemistry University of Wisconsin-Madison Madison Wisconsin U.S.A. Abstract: Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, an- alogues, and transport models) on transport of threonine and phenylalanine across the blood-brain bamer. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2-amino-n- butyrate (AABA) were stronger competitors than norleucine. 2-Aminobicyclo[2.2.l]heptane-2-carboxylate BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2- (methy1amino)isobutyrate MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleu- cine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25 (p zyxw   0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium-dependent ASC system. Key Words: Blood-brain bamer-Large neutral amino acids-Small neutral amino acids-Phenylalanine-Sodium-Threo- nine-Transport. Tovar A. zyx t al. Some characteristics of threonine transport across the blood-brain bamer of the rat. J. Neurochem. 51, 1285-1293 (1988). An adequate supply of nutrients is required to maintain normal function of tissues and organs. The supply of nutrients for the brain is regulated in part by the blood-brain bamer (BBB). Diet-induced distortions of the blood patterns of amino acids can produce se- lective and severe depressions of amino acid concen- trations in brain (Peng et al., 1972; Tews et al., 1980; Gietzen et al., 1986; Tews and Harper, 1986b). Brain amino acid patterns can also be modified, but usually much less, by changes in the protein content of the diet (Peng et al., 1972; Fernstrom and Faller, 1978; Glaeser et al., 1983; Glanville and Anderson, 1985; Peters and Harper, 1985, 1987; Tews et al., 19870). Amino acids are transported across the BBB by car- riers which are specific for different classes of amino acids (Oldendorf and Szabo, 1976). Because ofthe lack of separate carriers for individual amino acids, com- petition for transport occurs among amino acids of a given class (neutral, acidic, or basic). Furthermore, ki- netic measurements have shown that the K, values for amino acid transport into brain are similar to, or even below, usual concentrations in plasma (Pardridge, 1977, 1983; Smith et al., 1987); hence, alterations in plasma amino acid concentrations can alter flux of amino acids into brain. Competition for amino acid transport across the BBB is influenced, therefore, by the relative plasma concentrations of a specific amino acid and its competitor(s), and the affinity of the carrier for each amino acid. Diet-induced alterations in plasma amino acid patterns are seen to be accompanied by concomitantly measured changes in the rate of amino acid entry into brain (Tews et al., 1987a,b, 1988); this can be reflected in selective changes in the concentra- tions of amino acids in brain (Tews et al., 1988). Both large (LNAA) and small neutral amino acids (SNAA) are considered to be transported across the BBB by a single, sodium-independent, L system having high and low affinities, respectively, for these two types Received December 16, 1987; evised manuscript received April 18, 1988; ccepted April 29, 1988. Address correspondence and reprint requests to Dr. J. K. Tews at Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, U.S.A. Abbreviations used: AABA, 2-amino-n-butyrate; AIB, 2-amino- isobutyrate; BBB, blood-brain barrier; BCAA, branchedchain amino acids; BCH, 2-aminobicyclo[2.2.1]heptane-2-carboxylate; UI, brain uptake index; LNAA, large neutral amino acids; MeAIB, 2-(meth- y1amino)isobutyrate; SNAA, small neutral amino acids. 285   286 A. TOVAR ET AL. of amino acids (Pardridge and Oldendorf, 1977). SNAA, such as threonine and the dispensable serine or alanine, have relatively low rates of entry into brain, whereas LNAA, such zyxwvut s phenylalanine or the branched- chain amino acids (BCAA), enter the brain far more rapidly (Oldendorf, 1971; Pratt, 1980; Smith et al., 1987). However, Sershen and Lajtha (1 979) concluded that the ASC system for SNAA described by Christen- sen (1982, 1985) represents a small, but significant, component for SNAA transport in brain. In contrast, Wade and Brady (1981) concluded from studies with cysteine as a model that the ASC system is unimportant in brain. Lefauconnier et al. (1985) found evidence for the presence of more than one transport system for SNAA and LNAA in 16-day-old rats. We recently reported that changes in the rate of threonine transport across the BBB were correlated more closely with plasma concentrations of SNAA than with those of the BCAA, amino acids which are present in high concentrations in plasma of rats fed high protein diets (Tews et al., 19876). In contrast, the rate of valine entry into brain was correlated more strongly with plasma concentrations of leucine, isoleucine, and other LNAA than with those of SNAA (Tews et al., 1987~). We have expanded, therefore, our previous studies on threonine transport across the zyxwvut BB by using the single injection technique of Oldendorf (1 97 1) to ex- amine the competitive effects of individual LNAA and SNAA and of several analogues and model amino ac- ids. Similar experiments camed out with phenylalanine have shown clear differences between the responses of these two amino acids to the presence of the various competitors. The absence of sodium ion also affected differently hreonine and phenylalanine passage across the BBB. M TERI LS ND METHODS Male Sprague-Dawley rats weighing between 250 and 300 g were fed commercial rat chow and were housed in a room lighted from 0700 to 1900 h. Transport across the BBB of radioactive threonine or phenylalanine was measured as de- scribed by Oldendorf( 197 1). The rats were anesthetized with sodium pentobarbital (55 mg/kg rat i.p.), and the right com- mon carotid artery was exposed. The rats were decapitated and the brain removed 15 s after the intracarotid injection of 0.2 ml of a mixture of 3H20 nd I4C-labeled amino acid, with or without competitor amino acid; the ratio of 3H to I4C was about zyxwvuts : 1. The solutions for injection were prepared in Krebs-Ringer bicarbonate buffer containing 4 mMHEPES (pH 7.55). Final concentration of the radioactive amino acid was 0.05 mM, and competitors (L form) were present at 1 mM unless oth- erwise indicated (2 mM for DL-P-hydroxynorvaline). These concentrations were chosen because they are similar to con- centration ranges often found in plasma after various dietary treatments. 2-Aminobicyclo[2.2.l]heptane-2-carboxylic cid (BCH) was a mixture of the endo and exo isomers and was used at twice the concentrations indicated in the figures (to compensate for the presence of inactive isomer). Cysteine was maintained in the reduced state by addition of dithio- threitol [2 or 5 zyxw M (Wade and Brady, 1981)l. [We found that 5 mM dithiothreitol alone had no effect on the brain uptake index (BUI) for threonine: BUI = 11.9 & 0.4, mean & SEM; n = 3.1 Experiments on the effects of sodium ion were camed out in buffers containing choline chloride and potassium bicarbonate as replacements for the usual sodium chloride and bicarbonate. Portions of the injectate and of acid-soluble extracts of the ipsilateral cerebrum were counted in Aquasol scintillation fluid (Dupont NEN, Boston, MA, U.S.A.) in a counter with automatic quench corrections (LKB Model 12 17, Gaithers- burg, MD, U.S.A.). Results (based on dpm) are expressed as the BUI: [(brain 14C/3H)/(injectate 4C/3H)] 100. Residual blood was measured by determining the BUI for ['4C]sucrose, a substance which does not cross the BBB; this value (2.9 k 0.1, n = 5) was subtracted from the BUIs determined for the amino acids. Radioactive amino acids were obtained from Dupont NEN; unlabeled amino acids were purchased from Sigma (St. Louis, MO, U.S.A.). Analysis of variance was performed on the BUI values to determine if differences were statistically significant; if so, differences between treatment groups were tested by Fisher's protected least significant difference test (Snedecor and Cochran, 1980). RESULTS Competition by natural amino acids Several natural amino acids were effective compet- itors for transport of threonine (0.05 mM across the BBB (Fig. 1A). Of these amino acids, leucine reduced the BUI for threonine to the greatest extent (by 67 ). Other LNAA, such as phenylalanine, isoleucine, valine, and tryptophan, were somewhat less effective than leu- cine. The SNAA, alanine and serine, also significantly reduced the BUI for threonine, but to a smaller degree (by about 20 ) han did any of the tested LNAA. Rais- ing the threonine concentration to 1 mM depressed the BUI by 70 , thereby demonstrating the saturability of threonine transport across the BBB. Threonine transport was little affected by the presence of acidic glutamate or cationic lysine (not shown; BUI = 10.6 k 0.8 or 10.7 * 0.9, respectively). In a similar experiment (Fig. lB), the BUI for phe- nylalanine, a typical LNAA, was depressed by the BCAA (by about 55 ), not affected by threonine, and inhibited slightly by serine or alanine (by 13 or 22 , respectively). The greatest depression of transport of labeled phenylalanine (76 ) occurred in the presence of an increased concentration of phenylalanine itself. Threonine uptake into the brain was also measured in the presence of different concentrations of serine and phenylalanine, either alone or in combination (Fig. 2). Low concentrations of phenylalanine, 0.1 and 0.2 mM, reduced the BUI for threonine by about 20 and 40% respectively; the maximum reduction in threo- nine transport occurred with a phenylalanine concen- tration of about 1 mM. In contrast, the effect of serine J. Neurochem., Vol. 51. No. 4, 1988   287 HREONINE TRANSPORT INTO BRAIN 4- zyxwv 2- A Thr Ala Ser Trp Val Ile Phe LeuThr zyxwvutsrq Competitor ab C I B i L he Thr Ser AIa Ile BCAAPhe Competitor FIG. 1. A: BUI for threonine (0.05 mM) in the absence or presence of other amino acids (1 zyxwvutsr M); threonine at 1 mM. B: BUI for phe- nylalanine (0.05 mM) in the absence zyxwvutsr r presence of other amino acids (1 mM); 'phenylalanine at 1 mM. Note difference in scales for A and B. BCAA indicates equimolar concentrations of ism leucine, leucine, and valine (0.33 mM each). Bars show means zyxwvu   EM; n = 3-8 rats per group. Means differ where letters above bars differ (p < 0.05). Note that the results shown in Figs. 1, 3, and 4 were obtained during the entire course of the studies and, therefore, some results are repeated in these figures. zyxwvutsr was minor at the lower concentrations (about 10 re- duction at zyxwvutsrq .2 mM ; however, stronger competitive effects of serine occurred at the higher concentrations, and at 5 mM its effect was similar to that of 1 mM phenylalanine. In the presence of equimolar concen- trations of serine plus phenylalanine, the competitive effects were similar to those for phenylalanine alone (not shown). The trend toward a lower BUI when the combined concentration was 5 mM 2.5 mM each), rather than 5 mM phenylalanine alone, was not sta- tistically significant. zyx i values of 0.14 t 0.03 and 0.87 t .25 mM, respectively, were calculated for phenyl- alanine and serine by weighted, nonlinear regression analysis (Cleland, 1979), with the diffusion component eliminated as described by Pardridge 1 977). Competition by amino acid analogues 2-Amino-n-butyric acid (AABA) and norvaline sig- nificantly depressed the BUI for threonine by about 45 and 55 , respectively (Fig. 3A). P-Hydroxynorvaline, a threonine antagonist in bacteria (Shiio et al., 1970), was less effective as a competitor than norvaline, but at least as effective as several four-carbon amino acids, such as allothreonine or homoserine, which decreased the BUI by less than 30 ; 2-aminoisobutyric acid (AIB) caused only a minor depression of 10 n the BUI for BCH Ser Phe Phe+Ser . he+Cys 04' 1. 8.1. I 0 1 2 3 4 5 Total Concentration mM) a ab bc cd d FIG. 2. BUI for threonine (0.05 mM) as affected by increasing con- centrations of phenylalanine, serine, or BCH (total BCH was twice the indicated concentration n order to compensate for presence of inactive isomer). Bars show means 2 SEM; n = 3. Different letters indicate significant differences between treatments when total competitor concentration was 5 mM (p < 0.05). J. Neurochern.. Vol 51. No. 4, 1988   288 A. TOVAR ET AL. rj A Thr AIB HSer NLeu AlloThrflOHNV AABA NVal Thr Competitor b T zyx   C T d T Phe AABA AlloThr ROHNV NVal NLeu Phe zyxwvutsrqponm Competitor FIG. 3. BUI for threonine (A) or phenylalanine zyxwvuts B) n the presence of amino acid analogues. HSer, homoserine; NLeu, norleucine; BOHNV, DL-p-hydroxynorvaline 2 mnn); AlloThr, allothreonine; NVal, norvaline. Other details are described in Fig. 1. threonine. Norleucine, the six-carbon homologue of norvaline, reduced the BUI less than did norvaline. In contrast to their differing effects on threonine transport, norleucine and norvaline were equally ef- fective competitors for phenylalanine transport, re- ducing its uptake by 50-55 (Fig. 3B). Allothreonine and hydroxynorvaline moderately depressed phenyl- alanine uptake by 25 and 33 , espectively. AABA did not significantly lower the BUI for phenylalanine, whereas it was one of the most effective competitors for threonine transport. Competition by amino acids that are models for transport of SNAA and LNAA Christensen's ongoing classification of amino acid transport systems, including representative model amino acids for identifying these systems, has been summarized recently (Christensen, 1985; Stein, 1986). Although these models have been studied mainly with preparations other than brain, we have measured BUIs for threonine and phenylalanine in the presence of 2- (methy1amino)isobutyrate MeAIB), BCH, or cysteine. Our results show that none of these proposed models were as effective in reducing the BUI for threonine as was an equimolar amount of threonine itself (Fig. 4A). MeAIB, a recognized model for the A system for SNAA transport, when present at either 1 or 10 mM had no inhibitory effects on threonine transport across the BBB (Figs. 4 and 5). BCH, a standard model for the L system for LNAA, was a moderately effective competitor for threonine transport (Figs. 2 and 4A). Reductions in BUI induced by BCH at concentrations at or below the effective concentration of 1 mM were clearly less than that induced by 1 mM phenylalanine; raising the effective BCH concentration to 5 mM reduced the BUI for threonine to a value statistically similar to that ob- served in the presence of 5 mM serine, but significantly higher than that with 5 mM phenylalanine (Fig. 2). Doubling the concentration of BCH (10 mM further lowered threonine transport only slightly to 5.2 0.1 (n = 3), a value similar to that obtained with 1 mM phenylalanine (not shown). A Ki value for BCH of0.60 z   .12 mM was obtained from these data. Although cysteine has been reported to serve as a specific model for the ASC system in hepatocytes (Kil- berg et al., 1979), it seems unsatisfactory for this pur- pose in brain (Wade and Brady, 1981). Cysteine (1 mM reduced the BUI for threonine by 40 , insignif- icantly more than the 30 reduction caused by a com- parable concentration of BCH (Fig. 4A). An equimolar mixture of phenylalanine and cysteine (5 mM total concentration) reduced the mean BUI to a value sig- nificantly below those for BCH, serine, or phenylala- nine alone (Fig. 2). Finally, the combination of BCH and cysteine (1 mM each) reduced the BUI to 6.6 .2 (n = 3), close to the value of 5.5 observed in the pres- ence of phenylalanine and serine at 1 mM each (not shown). When the concentrations were each raised 2.5- fold, the combination of BCH and cysteine again did not reduce the BUI for threonine (5.4 .1; n 3) below that observed when phenylalanine and serine were combined at zyxw .5 mM each (Fig. 2). Phenylalanine uptake was depressed by each of the model amino acids (Fig. 4B), with BCH, which reduced the BUI by about 60 , being most effective. The BUI was lowered in the presence of MeAIB and cysteine by about 20 and 30 , respectively. A combination of BCH and cysteine (1 mM each) did not depress the BUI for phenylalanine below the value observed in the presence J Neurochem., Vol. 51, NO. 4, 988   289 HREONINE TRANSPORT INTO BRAIN a zyxwvutsrqpo 5 zyxwvutsrqponml   T zyx la 10 z zyxwvutsr   m5 A b 0 Thr MeAlB BCH Cys Thr' Competitor zyxwvut 6 b C d e T Phe MeAlB Cys BCH Phe' Competitor FIG. 4. BUI for threonine (A) or phenylalanine (B) in the presence of amino acids suggested as models for LNAA or SNAA transport systems; n = 3-10 rats per group. Other details are described in Figs. 1 and 2. of the same concentration of BCH alone (mean = 18.6; n = 2 . Effects of sodium It is believed that sodium is not required for amino acid transport across the BBB (Pardridge and Olden- dorf, 1977; Momma et al., 1987). In agreement with this view, removal of sodium ion from the injectate had no effect on phenylalanine entry into the brain (BUI = 47.0 zyx   .5 and 47.0 .6 in the absence and presence of sodium, respectively). However, the ab- sence of sodium clearly affected threonine transport. In order to characterize further the mode of threonine transport across the BBB, we have carried out the pro- cedures proposed by Shotwell et al. (198 1) (for Chinese hamster ovary cells) and used by Tayarani et al. (1 987) to identify in isolated brain capillaries contributions of the A, ASC, and L systems to transport of various neu- tral amino acids. MeAIB (10 mM had no detectable competitive effect on threonine transport across the BBB (Fig. 5), implying that significant entry of threo- nine into brain does not occur via the A system for neutral amino acids. Removal of sodium from the me- dium caused a highly significant reduction in the BUI for threonine (p < 0.00 1, Student's zyx   test), thereby sug- gesting that, under our conditions, about 25 of thre- onine uptake may have occurred via the ASC system (both the A and ASC systems are sodium-dependent). Further reduction of the BUI for threonine in the ab- sence of sodium and in the presence of BCH (effective concentration of 10 mM showed that about 40 of threonine uptake presumably occurred via the sodium- independent L system. Under these conditions, resid- ual, noninhibitable (presumably nonsaturable) uptake accounted for about zyx 5 of the total. Other results (Fig. 6) also suggest the presence of both a sodium-dependent and -independent compo- 151 a a Cation Sodium Sodium Choline Choline Competitor MeAlB BCH FIG. 5. BUI for threonine in the presence or absence of sodium in the injectate: competition by models for the A and L systems. MeAlB (A system) and BCH (L system) were used at the effective concentration of 10 mM each; threonine concentration was 0.05 mM. Bars show means k SE; n = 6, 3, 6. and 4 rats, respectively, for groups shown from left to right. Means differ where letters above bars differ (p < 0.05). J. Neurochem., Vol. 51, No. 4, 1988

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May 17, 2018
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