Advertisement

Role of Oxoproline in the Regulation of Neutral Amino Acid Transport across the Blood-Brain Barrier*

  • Wha-Joon Lee
    Affiliations
    Department of Physiology and Biophysics, Finch University of Health Science/The Chicago Medical School, North Chicago, Illinois 60064-3095, and
    Search for articles by this author
  • Richard A. Hawkins
    Correspondence
    To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Finch University of Health Science/The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064-3095. Tel.: 847-578-3218; Fax: 847-578-3404.
    Affiliations
    Department of Physiology and Biophysics, Finch University of Health Science/The Chicago Medical School, North Chicago, Illinois 60064-3095, and
    Search for articles by this author
  • Darryl R. Peterson
    Affiliations
    Department of Physiology and Biophysics, Finch University of Health Science/The Chicago Medical School, North Chicago, Illinois 60064-3095, and
    Search for articles by this author
  • Juan R. Viña
    Footnotes
    Affiliations
    Departamento de Bioquímica y Biología, Molecular Facultades de Medicina y Farmacia, Universitat de Valencia, Valencia 46010, Spain
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by Grant NS31017 from the NINDS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    Supported by Dirección General de Investigaciones Ciencia y Técnica Grant PM 91-0198, Spain, and Fondo de Investigaciones Sanitarias Grant FISS 94/1573, Spain.
Open AccessPublished:August 09, 1996DOI:https://doi.org/10.1074/jbc.271.32.19129
      Regulation of neutral amino acid transport was studied using isolated plasma membrane vesicles derived from the bovine blood-brain barrier. Neutral amino acids cross the blood-brain barrier by facilitative transport system L1, which may allow both desirable and undesirable amino acids to enter the brain. The sodium-dependent amino acid systems A and Bo,+ are located exclusively on abluminal membranes, in a position to pump unwanted amino acids out. γ-Glutamyl transpeptidase, the first enzyme of the γ-glutamyl cycle, is an integral protein of the luminal membrane of the blood-brain barrier. We demonstrate that oxoproline, an intracellular product of the γ-glutamyl cycle, stimulates the sodium-dependent systems A and Bo,+ by 70 and 20%, respectively. Study of system A showed that 2 mM oxoproline increased the affinity for its specific substrate N-methylaminoisobutyrate by 50%. This relationship between the activity of the γ-glutamyl cycle and system A transport may provide a short term regulatory mechanism by which the entry of potentially deleterious amino acids (i.e. neurotransmitters or their precursors) may be retarded and their removal from brain accelerated.

      INTRODUCTION

      The endothelial cells of cerebral capillaries are joined by tight junctions forming the blood-brain barrier (BBB).
      The abbreviations used are: BBB
      blood-brain barrier
      GGT
      γ-glutamyl transpeptidase
      MeAIB
      N-(methylamino)isobutyric acid
      PSA
      permeability-surface area.
      Thus, hydrophilic nutrients, such as amino acids, require the presence of carriers in the respective luminal and abluminal membranes to reach the brain. Using isolated plasma membrane vesicles from cerebral endothelial cells it has been shown that system L1, characterized by affinity for a broad spectrum of neutral amino acids (especially large neutral amino acids) (
      • Christensen H.N.
      • Oxender D.L.
      • Liang M.
      • Vatz K.A.
      ), is equally distributed between luminal and abluminal membranes (
      • Sánchez
      • Pino M.M.
      • Peterson D.R.
      • Hawkins R.A.
      ). Therefore, system L1 is in a position to facilitate neutral amino acid movement between blood and brain. Two sodium-dependent transport systems, A and Bo,+, are also present (
      • Sánchez
      • Pino M.M.
      • Peterson D.R.
      • Hawkins R.A.
      ,
      • Sánchez
      • Pino M.M.
      • Hawkins R.A.
      • Peterson D.R.
      ), but they exist only in abluminal membranes (
      • Sánchez
      • Pino M.M.
      • Peterson D.R.
      • Hawkins R.A.
      ). The sodium-dependent systems are, therefore, in a position to pump amino acids out of the brain extracellular fluid. Although system A preferentially transports small neutral amino acids (
      • Christensen H.N.
      • Oxender D.L.
      • Liang M.
      • Vatz K.A.
      ), and system Bo,+ favors the transport of neutral and basic amino acids (
      • Kilberg M.S.
      • Stevens B.R.
      • Novak D.A.
      ), there is a considerable overlap in the spectrum of amino acids that can be transported by each of the systems: L1, A, and Bo,+ (
      • Oxender D.L.
      • Christensen H.N.
      ). This overlap in transport activity coupled with the asymmetrical distribution of the amino acid transporters at the BBB provides a potential means for the regulation of amino acid delivery to the brain.
      γ-Glutamyl transpeptidase (GGT) is located on the luminal membrane of cerebral endothelial cells (
      • Sánchez
      • Pino M.M.
      • Hawkins R.A.
      • Peterson D.R.
      ). A role for GGT in amino acid transport was suggested by Meister and co-workers (
      • Orlowski M.
      • Meister A.
      ,
      • Meister A.
      • Anderson M.E.
      ,
      • Meister A.
      ), as part of the γ-glutamyl cycle. However, the idea that the cycle is involved directly in amino acid translocation into cells is controversial, having received support (
      • Lauterburg B.H.
      • Mitchell J.R.
      ,
      • Viña J.
      • Puertes I.R.
      • Estrela J.M.
      • Viña J.R.
      • Galbis J.L.
      ,
      • Viña J.R.
      • Puertes
      • Viña J.
      ,
      • Viña J.R.
      • Blay P.
      • Ramirez A.
      • Castells A.
      • Viña J.
      ,
      • Cancilla P.A.
      • DeBault L.E.
      ,
      • Smith Q.R.
      ,
      • Cotgreave I.A.
      • Schuppe-Koistinen I.
      ,
      • Sweiry J.H.
      • Sastre J.
      • Viña J.
      • Elasser H.P.
      • Mann G.E.
      ) and criticism (
      • Pellefigue F.
      • Butler J.D.
      • Spielberg S.P.
      • Hollenberg M.D.
      • Goodman S.I.
      • Schulman J.D.
      ,
      • Sepulveda F.V.
      • Burton K.A.
      • Pearson J.D.
      ,
      • Hsu B.Y.L.
      • Foreman J.W.
      • Corcoran S.M.
      • Ginkinger K.
      • Segal S.
      ,
      • Payne G.M.
      • Payne J.W.
      ,
      • Bell J.G.
      • Buddington R.K.
      • Walton M.J.
      • Cowey C.B.
      ).
      Studies using mammary glands from lactating rats and placenta of pregnant rats showed that oxoproline, an intermediate of the γ-glutamyl cycle, serves as an intracellular signal to stimulate amino acid transport (
      • Viña J.R.
      • Puertes I.R.
      • Montoro J.B.
      • Saez G.T.
      • Viña J.
      ,
      • Viña J.R.
      • Palacin M.
      • Puertes I.R.
      • Hernandez R.
      • Viña J.
      ). To determine whether such a regulatory mechanism is present in the BBB, amino acid transport was measured in isolated luminal- and abluminal-enriched plasma membrane vesicles derived from bovine cerebral capillary endothelial cells.

      DISCUSSION

      The principal findings were as follows. 1) Oxoproline accelerated the initial rate of substrate transport by the two sodium-dependent amino acid carriers known to be at the abluminal membrane of the BBB (systems A and Bo,+). 2) System A, which was stimulated to the greatest degree, manifested an increased affinity for its substrate. 3) There was no effect of oxoproline on the facilitated transport of phenylalanine. These observations suggest that oxoproline, produced as part of the γ-glutamyl cycle (
      • Orlowski M.
      • Meister A.
      ,
      • Meister A.
      • Anderson M.E.
      ,
      • Meister A.
      ), may be an important link in the short term control of sodium-dependent amino acid transport by the BBB.
      System L1 is distributed symmetrically between the luminal and abluminal membrane domains of cerebral endothelial cells (
      • Sánchez
      • Pino M.M.
      • Peterson D.R.
      • Hawkins R.A.
      ) and serves to facilitate the diffusion of neutral amino acids between blood and brain. Although L1 is characterized by a high affinity for large neutral amino acids, it has a measurable affinity for almost all neutral amino acids (
      • Smith Q.R.
      • Momma S.
      • Aoyagi M.
      • Rapoport S.I.
      ). There is also a nonsaturable component that may allow an additional influx of neutral amino acids, accounting for about 5% of carrier-mediated transport (
      • Smith Q.R.
      • Momma S.
      • Aoyagi M.
      • Rapoport S.I.
      ). The combined effect of these two pathways could provide a supply of both essential and nonessential amino acids, some of which may be neurotransmitters or modulators of neurotransmission.
      Two sodium-dependent amino acid transport systems (A and Bo,+) are present exclusively on the abluminal membrane of the BBB (
      • Sánchez
      • Pino M.M.
      • Peterson D.R.
      • Hawkins R.A.
      ). The capacity of these transporters is an order of magnitude greater than the facilitative transporter L1 (
      • Sánchez
      • Pino M.M.
      • Peterson D.R.
      • Hawkins R.A.
      ). Because the electrochemical gradient for sodium is oriented to flow from the extracellular fluid into the endothelial cells, these sodium-dependent transport systems are in a position to export amino acids from the brain extracellular fluid. Thus, amino acids that pass both endothelial cell membranes and enter the basement membrane space could be actively, and selectively, pumped back across the abluminal membrane. This asymmetrical distribution of sodium-dependent carriers has the potential, therefore, to restrict the availability of amino acids to the brain.
      The γ-glutamyl cycle has been shown to influence amino acid transport in diverse tissues. The first reaction of the cycle occurs extracellularly and is catalyzed by GGT (
      • Meister A.
      • Anderson M.E.
      ). Glutathione is exported across the luminal membrane, and transpeptidation occurs in the presence of extracellular amino acids. The γ-glutamyl amino acids that result enter cells by a transport system that is not shared by free amino acids. Intracellularly, γ-glutamyl amino acids are substrates of γ-glutamyl cyclotransferase, which converts the γ-glutamyl amino acids into oxoproline and the corresponding free amino acids. The subsequent conversion of oxoproline to glutamate by oxoprolinase is the rate-limiting step of the γ-glutamyl cycle (
      • Van Der Werf P.
      • Meister A.
      ).
      Irrespective of whether the γ-glutamyl cycle directly mediates significant amino acid transport, it does seem to influence amino acid transport systems. This has been shown in mammary glands of lactating rats and placenta of pregnant rats in which the activity of the γ-glutamyl cycle was correlated with active amino acid transport (
      • Viña J.R.
      • Puertes I.R.
      • Montoro J.B.
      • Saez G.T.
      • Viña J.
      ,
      • Viña J.R.
      • Palacin M.
      • Puertes I.R.
      • Hernandez R.
      • Viña J.
      ). Specifically, it appears that oxoproline, produced intracellularly as an intermediary metabolite of the γ-glutamyl cycle, acts as a signal to activate the translocation of amino acids into these tissues.
      The presence of GGT in the BBB has been an enigma. GGT activity is high in tissues that actively transport amino acids (
      • Meister A.
      • Anderson M.E.
      ), such as the brush border of the proximal convoluted tubules of the kidney (
      • Curto K.A.
      • Sweeney W.E.
      • Avner E.D.
      • Piesco N.P.
      • Curthoys N.P.
      ), the lactating mammary gland (
      • Puente I.R.
      • Saez G.T.
      ), and the apical portion of the intestinal epithelium (
      • Garvey T.Q.
      • Hyman P.E.
      • Isselbacher K.J.
      ). The BBB differs from these tissues in that it is not associated with active amino acid uptake from plasma. Although brain requires essential amino acids for its function and growth, their supply is not much greater than the demand, and it is difficult to detect arteriovenous differences of amino acids across the brain (
      • Hawkins R.A.
      ,
      • Sacks W.
      • Sacks S.
      • Brebbia D.R.
      • Fleischer A.
      ). It has, therefore, been puzzling why brain capillaries have such high GGT activity.
      Our data support the hypothesis that the γ-glutamyl cycle and GGT serve to monitor the availability of amino acids to the brain and constitute the first step in a control mechanism that influences the accessibility and content of brain amino acids (Fig. 4). The question arises whether the oxoproline concentrations that exist in vivo are sufficient to stimulate sodium-dependent transport. Although we are unaware of measurements of oxoproline in cerebral microvessels, the concentrations in normal human plasma and various tissue extracts are between 20 and 50 µM (
      • Van Der Werf P.
      • Meister A.
      ,
      • Van Der Werf P.
      • Stephani R.A.
      • Meister A.
      ) and as high as 6 mM in plasma and cerebrospinal fluid in pathological conditions (
      • Meister A.
      ). Stimulation of sodium-dependent transport of MeAIB was a linear function of the oxoproline concentration up to 2 mM, a range that does not seem unreasonable.
      Figure thumbnail gr4
      Fig. 4Scheme of the oxoproline role on amino acid transport across the blood-brain barrier. γ-Glutamyl amino acids are formed at the outer surface of luminal membranes of the endothelial cells by transfer of the γ-glutamyl moiety of glutathione to amino acids, a reaction catalyzed by GGT. The γ-glutamyl amino acids enter endothelial cell where oxoproline is formed. The sodium-dependent transport systems A and Bo,+, located on the abluminal side, are activated by oxoproline. The transport system L1 is distributed symmetrically between luminal and abluminal membrane and is not affected by oxoproline.
      The transpeptidation activity of GGT is a function of the plasma concentration and spectrum of amino acids (
      • Allison R.D.
      • Meister A.
      ), both of which may vary considerably, depending on nutritional status. This provides a feedback mechanism in which the γ-glutamyl amino acids produced by GGT enter cerebral capillary endothelial cells and are converted to oxoproline, which in turn activates the A system at the abluminal membrane. Since the A system is oriented to remove amino acids from the brain in an energy-dependent fashion, its up-regulation could provide a control mechanism to guard against elevations of amino acids in brain when their availability is excessive. This is of particular interest with regard to smaller nonessential amino acids for which system A has a relatively high affinity. Thus, this process may serve to modulate entry of amino acids that serve as neurotransmitters or their precursors.

      REFERENCES

        • Christensen H.N.
        • Oxender D.L.
        • Liang M.
        • Vatz K.A.
        J. Biol. Chem. 1965; 240: 3609-3616
        • Sánchez
        • Pino M.M.
        • Peterson D.R.
        • Hawkins R.A.
        J. Biol. Chem. 1995; 270: 14913-14918
        • Sánchez
        • Pino M.M.
        • Hawkins R.A.
        • Peterson D.R.
        J. Biol. Chem. 1992; 267: 25951-25957
        • Kilberg M.S.
        • Stevens B.R.
        • Novak D.A.
        Annu. Rev. Nutr. 1993; 13: 137-165
        • Oxender D.L.
        • Christensen H.N.
        J. Biol. Chem. 1963; 238: 3686-3699
        • Sánchez
        • Pino M.M.
        • Hawkins R.A.
        • Peterson D.R.
        J. Biol. Chem. 1995; 270: 14907-14912
        • Orlowski M.
        • Meister A.
        Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1248-1255
        • Meister A.
        • Anderson M.E.
        Annu. Rev. Biochem. 1983; 52: 711-760
        • Meister A.
        Science. 1973; 180: 33-39
        • Lauterburg B.H.
        • Mitchell J.R.
        Gastroenterology. 1979; 77: A24
        • Viña J.
        • Puertes I.R.
        • Estrela J.M.
        • Viña J.R.
        • Galbis J.L.
        Biochem. J. 1981; 194: 99-102
        • Viña J.R.
        • Puertes
        • Viña J.
        Biochem. J. 1981; 200: 705-708
        • Viña J.R.
        • Blay P.
        • Ramirez A.
        • Castells A.
        • Viña J.
        FEBS Lett. 1990; 269: 86-88
        • Cancilla P.A.
        • DeBault L.E.
        J. Neuropathol. Exp. Neurol. 1983; 42: 191-199
        • Smith Q.R.
        Adv. Exp. Med. Biol. 1991; 291: 55-71
        • Cotgreave I.A.
        • Schuppe-Koistinen I.
        Biochim. Biophys. Acta. 1994; 1222: 375-382
        • Sweiry J.H.
        • Sastre J.
        • Viña J.
        • Elasser H.P.
        • Mann G.E.
        J. Physiol. 1995; 485: 167-177
        • Pellefigue F.
        • Butler J.D.
        • Spielberg S.P.
        • Hollenberg M.D.
        • Goodman S.I.
        • Schulman J.D.
        Biochem. Biophys. Res. Commun. 1976; 73: 997-1102
        • Sepulveda F.V.
        • Burton K.A.
        • Pearson J.D.
        Biochem. J. 1982; 208: 509-512
        • Hsu B.Y.L.
        • Foreman J.W.
        • Corcoran S.M.
        • Ginkinger K.
        • Segal S.
        J. Membr. Biol. 1984; 80: 167-173
        • Payne G.M.
        • Payne J.W.
        Biochem. J. 1984; 218: 147-155
        • Bell J.G.
        • Buddington R.K.
        • Walton M.J.
        • Cowey C.B.
        J. Comp. Physiol. B. 1987; 157: 161-169
        • Viña J.R.
        • Puertes I.R.
        • Montoro J.B.
        • Saez G.T.
        • Viña J.
        Biol. Neonate. 1985; 48: 250-256
        • Viña J.R.
        • Palacin M.
        • Puertes I.R.
        • Hernandez R.
        • Viña J.
        Am. J. Physiol. 1989; 257: E916-E922
        • Betz A.L.
        • Firth J.A.
        • Goldstein G.W.
        Brain Res. 1980; 192: 17-28
        • Mayer L.D.
        • Hope M.J.
        • Cullis P.R.
        Biochim. Biophys. Acta. 1986; 858: 161-168
        • Strauss G.
        • Schurtenberger P.
        • Hauser H.
        Biochim. Biophys. Acta. 1986; 858: 169-180
        • MacDonald R.C.
        • Jones F.D.
        • Qiu R.
        Biochim. Biophys. Acta. 1994; 1191: 362-370
        • Lash L.H.
        • Jones D.P.
        J. Biol. Chem. 1984; 259: 14508-14514
        • Christensen H.N.
        Methods Enzymol. 1989; 173: 576-616
        • Grammas P.
        • Kwaiser T.M.
        • Caspers M.L.
        Neuropharmacology. 1992; 31: 409-412
        • Kilberg M.S.
        Trends Biochem. Sci. 1986; 11: 183-186
        • Schenerman M.A.
        • Kilberg M.S.
        Biochim. Biophys. Acta. 1986; 856: 428-436
        • Munck L.K.
        • Munck B.G.
        Biochim. Biophys. Acta. 1992; 1116: 91-96
        • Barker G.A.
        • Ellory J.C.
        Exp. Physiol. 1990; 75: 3-26
        • Shotwell M.A.
        • Jayme D.W.
        • Kilberg M.S.
        • Oxender D.L.
        J. Biol. Chem. 1981; 256: 5422-5427
        • Bradford M.M.
        Anal. Biochem. 1976; 72: 248-254
        • Smith Q.R.
        • Momma S.
        • Aoyagi M.
        • Rapoport S.I.
        J. Neurochem. 1987; 49: 1651-1658
        • Van Der Werf P.
        • Meister A.
        Adv. Enzymol. 1975; 43: 519-554
        • Van Der Werf P.
        • Stephani R.A.
        • Meister A.
        Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1026-1029
        • Meister A.
        Laufer R.S. Warren E. McIvor D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1983: 348
        • Curto K.A.
        • Sweeney W.E.
        • Avner E.D.
        • Piesco N.P.
        • Curthoys N.P.
        J. Histochem. Cytochem. 1988; 36: 159-166
        • Puente I.R.
        • Saez G.T.
        FEBS Lett. 1979; 126: 250-252
        • Garvey T.Q.
        • Hyman P.E.
        • Isselbacher K.J.
        Gastroenterology. 1976; 71: 778-785
        • Hawkins R.A.
        Fed. Proc. 1986; 45: 2055-2059
        • Sacks W.
        • Sacks S.
        • Brebbia D.R.
        • Fleischer A.
        J. Neurosci. Res. 1982; 7: 431-436
        • Allison R.D.
        • Meister A.
        J. Biol. Chem. 1981; 256: 2988-2992