Application of Rapid Dual Tracer Dilution Techniques for the Study of Endothelial Cell Amino Acid Transport in Perfused Microcarrier Cultures

  • G. E. Mann
  • C-J. Sheriff
  • V. J. Toothill
  • J. D. Pearson


Endothelial cells play an important role in the regulation of vascular tone, blood coagulation, platelet aggregation and leukocyte traffic by means of cell surface reactions and secreted mediators such as, prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF) (Moncada et al., 1976; Furchgott & Zawadzki, 1980; Pearson & Gordon, 1984; Gordon et al., 1986; Gordon & Pearson, 1987; Van de Voorde et al., 1987; Luscher et al., 1990; see reviews Furchgott, 1990 and Moncada & Higgs, 1990). EDRF induces vascular relaxation and inhibits platelet aggregation and adhesion by activating soluble guanylate cyclase in vascular smooth muscle (see Griffiths et al., 1985). Recent studies with perfused aortic endothelial cell microcarrier cultures have now identified EDRF as nitric oxide (Palmer et al., 1987), and suggested that a novel NADPH-dependent enzyme may be involved in the generation of nitric oxide from the terminal nitrogen atom(s) of L-arginine (Palmer & Moncada, 1989).


Nitric Oxide Human Umbilical Vein Endothelial Cell Amino Acid Transport Aortic Endothelial Cell Bovine Aortic Endothelial Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Allen, L.A. & Gerritsen, M.E. 1986. Regulation of glucose transport in cultured bovine retinal microvessel endothelium by insulin. Exp.o Res. 43: 679–686.Google Scholar
  2. Aisaka, K., Gross, S.S., Griffith, O.W. & Levi, R. 1990. Role of L-arginine in blood pressure regulation. Arch. Intern. Pharmacody. Therap. 305: 226 P.Google Scholar
  3. Bassingthwaighte, J.B. & Sparks, H.V. 1986. Indicator dilution estimation of capillary endothelial transport. Ann. Rev. Physiol. 48: 321–334.CrossRefGoogle Scholar
  4. Baydoun, A.R., Emery, P.W., Pearson, J.D. & Mann, G.E. (1990). Modulation of intracellular amino acid concentrations in cultured bovine aortic endothelial cells deprived of exogenous L-arginine. J. Physiol. (abstract in press).Google Scholar
  5. Betz, A.L., Gilboe, D.D. & Drewes, L.R. 1975. Kinetics of unidirectional leucine transport into brain: effects of isoleucine, valine and anoxia. Am. J. Physiol. 228, 895–900.PubMedGoogle Scholar
  6. Betz, A.L., Gibloe, D.D., Yudilevich, D.L. & Drewes, L.R. 1973. Kinetics of unidirectional glucose transport into the isolated dog brain. Am. J. Physiol. 225: 586–592.PubMedGoogle Scholar
  7. Betz, A.L. & Goldstein, G.W. 1986. Specialized properties and solute transport in brain capillaries. Ann. Rev. Physiol. 48: 241–250.CrossRefGoogle Scholar
  8. Boje, K.M. & Fung, H-L. 1990. Endothelial cell nitric oxide generating enzyme(s) in the bovine aorta: Subcellular location and metabolic characterization. J. Pharmacol. Therap. 253: 20–26.Google Scholar
  9. Boyd, C.A.R. & Parsons, D.S. 1979. Movement of monosaccharides between blood and tissue of vascularly perfused small intestine. J. Physiol. 287: 371–391.PubMedGoogle Scholar
  10. Busse, R., Trogisch, G. & Bassenge, E. 1985. The role of endothelium in the control of vascular tone. Basic Res. Cardiol. 80: 475–490.PubMedCrossRefGoogle Scholar
  11. Cancilla, M.D. & Debault, L.E. 1983. Neutral amino acid transport properties of cerebral endothelial cells in vitro. J. Neuropathol. Exp. Neurol, 42: 191–199.PubMedCrossRefGoogle Scholar
  12. Cardelli-Cangiano, P., Fiori, A., Cangiano, C., Barberini, F., Allegra, P., Peresemmpio, V. & Strom, R. 1987. Isolated brain microvessels as in vitro equivalents of the blood-brain barrier: Selective removal by collagenase of the A-system of neutral amino acid transport. J. Neurochem. 49: 1667–1675.PubMedCrossRefGoogle Scholar
  13. Christensen, H.N. & Kilberg, M. 1987. Amino acid transport across the plasma membrane: role of regulation in interorgan flows. In: Amino Acid Transport in Animal Cells, ed. Christensen, H.N. & Kilberg, M, pp. 1–46. Manchester University Press.Google Scholar
  14. Corkey, R.F., Corkey, B.E. & Gimbone, M.A. 1981. Hexose transport in normal and SV-40 transformed human endothelial cells in culture. J. Cell Physiol, 106: 425–434.PubMedCrossRefGoogle Scholar
  15. Deneke, S.M., Steiger, V, Fanburg, B.L. 1987. Effect of hyperoxia on glutathione levels and glutamic acid uptake in endothelial cells. J. Appl. Physiol. 63: 1966–1971.PubMedGoogle Scholar
  16. Ewadh, M.J.A., Tuball, N. & Rose, F.A. 1988. Transport of L-cystine in human umbilical vein endothelial cells in culture. Bioscience Reports 8: 449–453.PubMedCrossRefGoogle Scholar
  17. Furchgott, R.F. 1990. Studies on the endothelium-dependent vasodilatation and the endothelium-derived relaxing factor. Acta Physiol. Scand. 139: 257–270.PubMedCrossRefGoogle Scholar
  18. Furchgott, R.F. & Zawadski J.V. 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373–376.PubMedCrossRefGoogle Scholar
  19. Garthwaite, J., Charles, G.L. & Chess-Williams, R. 1988. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intracellular messenger in the brain. Nature 336: 385–388.PubMedCrossRefGoogle Scholar
  20. Gerritsen, M.E., Burke, T.M. & Allen, L.A. 1988. Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells. Microvase. Res. 35: 153–166.CrossRefGoogle Scholar
  21. Gerritsen, M.E. & Cheli, C.D. 1983. Arachidonic acid and prostaglandin endoperoxide metabolism in isolated rabbit coronary microvessels and isolated and cultivated coronary microvessel endothelial cells. J. Clin. Invest. 72: 1658–1671.PubMedCrossRefGoogle Scholar
  22. Gillis, C.N. 1986. Pharmacological aspects of metabolic processes in the pulmonary circulation. Ann. Rev. Pharmacol. Toxicol. 26: 183–200.CrossRefGoogle Scholar
  23. Gold, M.E., Bush, P.A. & Ignarro, L.J. 1989. Depletion of arterial L-arginine causes reversible tolerance to endothelium-derived relaxation. Biochem. Biophys. Res. Commun. 164: 714–721.PubMedCrossRefGoogle Scholar
  24. Gordon, J.L. & Pearson, J.D. 1987. Biology of the vascular endothelium. In: Haemostasis and Thrombosis, eds. Gordon, J.L. & Pearson, J.D, pp. 303–311, Churchill Livingstone.Google Scholar
  25. Gordon, E.L., Pearson, J.D. & Slakey, L.L. 1986. The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface. J. Biol. Chem. 261: 15496–15504.PubMedGoogle Scholar
  26. Griffiths, T.M., Edwards, D.H., Lewis, M.J. & Henderson, A.H. 1985. Evidence that cyclic guanosine monophosphate (cGMP) mediates endothelium-dependent relaxation. Eur. J. Pharmacol. 112: 195–202.CrossRefGoogle Scholar
  27. Hibbs, J.B., Taintor, R.R. & Vavrin, Z. 1987. Macrophage cytotoxicity: role of L-arginine deiminase and imino nitrogen oxidation to nitrate. Science 235: 473–476.PubMedCrossRefGoogle Scholar
  28. Ignarro, L.J., Gold, M.E., Buga, G.M., Byrns, R.E., Wood, K.S., Chaudhuri, G. & Frank, G. 1989. Basic polyamino acids rich in arginine, lysine or ornithine cause both enhancement of and refractoriness to formation of endothelium-derived nitric oxide in pulmonary artery and vein. Circ. Res. 64: 315–329.PubMedCrossRefGoogle Scholar
  29. Iyengar, R., Stuehr, D.J. & Marletta, M.A. 1987. Macrophage synthesis of nitrite, nitrate and N-nitrosamines: precursors and role of respiratory burst. Proc. Natl. Acad. Sci. USA 84: 6369–6373.PubMedCrossRefGoogle Scholar
  30. Jaffe, E.A., Nachman, R.L., Becker, C.G. & Minick, C.R. 1973. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 52: 2745–2756.PubMedCrossRefGoogle Scholar
  31. King, G.L., Buzney, S.M., Kahn, C.R., Hetu, N., Buchwald, S., Macdonald, S.G. & Rand, L.I. 1983. Differential responsiveness to insulin of endothelial and support cells from micro-and macrovessels. J Clin. Invest. 71: 974–979.PubMedCrossRefGoogle Scholar
  32. Knowles, R.G., Palacios, M., Palmer, R.M.J. & Moncada, S. 1989. Formation of nitric oxide from L-arginine in the central nervous system: A transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Natl. Acad. Sci. USA 86: 5159–5162.PubMedCrossRefGoogle Scholar
  33. Luckhoff, A., Pohl, U., Mulsch, A. & Busse, R. 1988. Differential role of extra-and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br. J. Pharmacoj. 95: 189–196.CrossRefGoogle Scholar
  34. Luscher, T.F., Richard, V., Tschudi, M., Yang, Z. & Boulanger, C. 1990. Endothelial control of vascular tone in large and small coronary arteries. J. Am. Coll. Cardiol. 15: 519–527.PubMedCrossRefGoogle Scholar
  35. Mann, G.E., Munoz, M. & Peran, S. 1986. Fasting and refeeding modulate neutral amino acid transport activity in the basolateral membrane of the rat exocrine pancreatic epithelium: fasting-induced insulin insensitivity. Biochim. Biophys. Acta 862: 119–126.PubMedCrossRefGoogle Scholar
  36. Mann, G.E., Norman, P.S.R. & Smith, I.C.H. 1989a. Amino acid efflux in the isolated perfused rat exocrine pancreas: Trans-stimulation by extracellular amino acids. J. Physiol. 416: 485–502.PubMedGoogle Scholar
  37. Mann, G.E., Pearson, J.D., Sheriff, C-J. & Toothill, V.J. 1988. Characterization of amino acid and glucose transport in cultured endothelial cells. Pflugers Arch. 411: R42.CrossRefGoogle Scholar
  38. Mann, G.E., Pearson, J.D., Sheriff, C-J. & Toothill, V.J. 1989b. Expression of amino acid transport systems in cultured human umbilical vein endothelial cells. J. Physiol. 410: 325–339.PubMedGoogle Scholar
  39. Mann, G.E. & Peran, S. 1986. Basolateral amino acid transport systems in the perfused exocrine pancreas: sodium-dependency and kinetic interactions between influx and efflux mechanisms. Biochim. Biophys. Acia 858: 263–274.Google Scholar
  40. Mann, G.E., Sheriff, C-J. & Pearson, J.D. 1990. N°-monomethyl-L-arginine and cationic amino acids inhibit L-arginine transport in perfused microcarrier cultures of aortic endothelial cells. In: Nitric Oxide from L-Arginine: A Bioregulatory System, ed. Moncada, S. & Higgs, E.A., pp. 331–339. Amsterdam: Excerpta Medica.Google Scholar
  41. Maruka, C., Spatz, M., Ueki, Y., Nagatsu, I. & Bembray, J. 1984. Cerebrovascular endothelial cell culture: Metabolism and synthesis of 5-hydroxytryptamine. L Neurochem. 43: 316–319.CrossRefGoogle Scholar
  42. Moncada, S. & Higgs, E.A. 1990. Nitric Oxide from L-Arginine: A Bioregulatory System. pp. 1–512 Excerpta Medica, Amsterdam.Google Scholar
  43. Moncada, S., Gryglewski, R.J., Bunting, S. & Vane, J.R. 1976. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263: 663–665.PubMedCrossRefGoogle Scholar
  44. Myers, P.R., Guerra, R. & Harrison, D.G. 1989. Release of NO and EDRF from cultured bovine aortic endothelial cells. Am. J. Physiol. 256: H1030 - H1037.PubMedGoogle Scholar
  45. Needham, L., Cusack, N.J., Pearson, J.D. & Gordon, J.L. 1987. Characteristics of the PZ purinoceptor that mediates prostacyclin production by pig aortic endothelial cells. Eur. J. Ph. rmacol. 134: 199–209.Google Scholar
  46. Norman, P.S.R., Habara, Y. & Mann, G.E. 1989. Paradoxical effects of endogenous and exogenous insulin on amino acid transport activity in the isolated rat pancreas: somatostatin-14 inhibits insulin action. Diabetologia 32: 177–184.PubMedCrossRefGoogle Scholar
  47. Norman, P.S.R. & Mann, G.E. 1987. Ionic dependence of amino acid transport in the exocrine pancreatic epithelium: calcium dependence of insulin action. L Membrane Biol. 96: 153–163.CrossRefGoogle Scholar
  48. Palmer, R.M.J. & Moncada, S. 1989. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem. Biophys. Res. Comm. 158: 348–353.PubMedCrossRefGoogle Scholar
  49. Palmer, R.M.J., Ferrige, A.G. & Moncada, S. 1987.. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524–526.Google Scholar
  50. Palmer, R.M.J., Rees, D.R., Ashton, D.S. & Moncada, S. 1988. L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem. Biophys. Res. Comm. 153: 1251–1256.PubMedCrossRefGoogle Scholar
  51. Pardridge, W.M. 1983. Brain metabolism: a perspective from the blood-brain barrier. Physiol. Rev. 63: a1481–1535.Google Scholar
  52. Pearson, J.D. & Gordon, J.L. 1984. Metabolism of serotonin and adenosine. In: Biology of Endothelial Cells, ed. Pearson, J.D. & Gordon, J.L, pp. 330–342. Martinus Nijholt.Google Scholar
  53. Peran, S. & McGee, M.P. 1986. Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures. Biochim. Biophys. Acta 856: 231–236.PubMedCrossRefGoogle Scholar
  54. Schmidt, H.H.H.W., Klein, M.M., Niroomand, F. & Bohme, E. 1988. Is arginine a physiological precursor of endothelium-derived nitric oxide? Eur. J. Pharmacol. 148: 293–295.PubMedCrossRefGoogle Scholar
  55. Shepro, D. & Dunham, B. 1986. Endothelial cell metabolism of biogenic amines. Ann. Rev. Physiol. 48, 335–345.CrossRefGoogle Scholar
  56. Sheriff, C-J., Pearson, J.D. & Mann, G.E. 1990. Characteristics of amino acid transporters in perfused endothelial cell microcarrier cultures: Role of L-arginine in nitric oxide synthesis. J. Physiol. 423: 6 P.Google Scholar
  57. Smith, Q.R., Momma, S., Aoyagi, M. & Rapoport, S.I. 1987. Kinetics of neutral amino acid transport across the blood-brain barrier. J. Neurochem, 49: 1651–1658.PubMedCrossRefGoogle Scholar
  58. Sneddon, J.M., Bearpark, T.M., Galton, S.A. & Vane, J.R. 1990. Transport and metabolism of L-arginine by bovine aortic endothelial cells. In: Nitric Oxide from L-Arginine: A Bioregulatory System, eds. Moncada, S. & E.A. Higgs, pp. 457–461. Amsterdam: Excerpta Medica.Google Scholar
  59. Steiger, V., Deneke, S.M. & Fanburg, B.L. 1987. Characterization of glutamic acid uptake by bovine pulmonary arterial endothelial cells. J. Appl. Physiol. 63: 1961–1965.PubMedGoogle Scholar
  60. Strum, J.M. & Junod, A.F. 1972. Radioautographic demonstration of 5hydroxytryptamine-[3H] uptake by pulmonary endothelial cells. J. Cell Biol. 54: 456–467.PubMedCrossRefGoogle Scholar
  61. Syrota, A., Girault, M., Pocidalo, J-J. & Yudilevich, D.L. 1982. Endothelial uptake of amino acids, sugars, lipids and prostaglandins in rat lung. Am. J. Physiol. 243: C20 - C26.PubMedGoogle Scholar
  62. Takasoto, Y., Momma, S. & Smith, Q.R. 1985. Kinetic analysis of cerebrovascular isoleucine transport from saline and plasma. J. Neurochem. 45: 1013–1020.CrossRefGoogle Scholar
  63. Thomas, G., Hecker, M. & Ramwell, P.W. 1989. Vascular activity of polycations and basic amino acids: L-arginine does not specifically elicit endothelium-dependent relaxation. Biochem. Biophys. Res. Comm. 158: 177–180.PubMedCrossRefGoogle Scholar
  64. Van de Voorde, J., Vanderstichele, H. & Leusen, I. 1987. Release of endothelium-derived relaxing factor from human umbilical vessels. Circ. Res. 60: 517–522.PubMedCrossRefGoogle Scholar
  65. Vinters, H.V., Beck, D.W., Bready, J.V., Maxwell, K., Berliner, J.A., Hart, M.N. & Cancilla, P.A. 1985. Uptake of glucose analogues into cultured cerebral microvessel endothelium. J. Neuropathol. Exp. Neurol. 5: 445–458.CrossRefGoogle Scholar
  66. Wade, L.A. & Katzman, R. 1975. Synthetic amino acids and the nature of L-DOPA transport at the blood-brain barrier. J. Neurochem. 25: 837–842.PubMedCrossRefGoogle Scholar
  67. Yudilevich, D.L. & Boyd, C.A.R. 1987. Amino Acid Transport in Animal Cells, Manchester: Manchester Univ. Press.Google Scholar
  68. Yudilevich, D.L. & Mann, G.E. 1982. Unidirectional uptake of substrates at the blood side of secretory epithelia: stomach, salivary gland and pancreas. Fed. Proc. 41: 3045–3053.PubMedGoogle Scholar
  69. Zetter, B.R. 1980. Migration of capillary endothelial cells is stimulated by tumour-derived factors. Nature 285: 41–43.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1991

Authors and Affiliations

  • G. E. Mann
    • 1
  • C-J. Sheriff
    • 1
  • V. J. Toothill
    • 2
  • J. D. Pearson
    • 2
  1. 1.Biomedical Sciences DivisionKing’s College LondonLondonUK
  2. 2.Section of Vascular BiologyMRC Clinical Research CentreHarrowUK

Personalised recommendations