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Demonstration of functional dipeptide transport with expression of PEPT2 in guinea pig cardiomyocytes

  • Hua Lin
  • Nicola KingEmail author
Transporters

Abstract

The transporters PEPT1 and PEPT2 accept a broad spectrum of substrates including small, naturally occurring peptides and peptidomimetic drugs. This study aimed to investigate for the first time whether these transporters are expressed and active in isolated cardiomyocytes. PEPT1/PEPT2 expression in rat kidney (positive control), guinea pig kidney and cardiomyocytes were investigated by reverse transcription polymerase chain reaction. l-Glycyl-l-[14C]sarcosine (Gly-sar) uptake was characterised using freshly isolated suspensions of adult male guinea pig cardiomyocytes. PEPT2-specific primers recognised mRNA of appropriate size and sequence in cardiomyocytes and kidney, whilst PEPT1 was expressed in the kidney only. The initial uptake (30 s) of 200 μM Gly-sar was dependent on extracellular pH with a maximum at pH 6.0 (237.8 ± 12.2 pmol/μl) and a minimum at pH 8.0 (72.1 ± 13.4 pmol/μl, n = 6 ± SE, p < 0.01, T test). The K m and V max of Gly-sar uptake at pH 6.0 were 495.5 ± 69.6μM and 1470.5 ± 69.6 pmol μl−1 min−1. The addition of 10 mM fosinopril, cefadroxil, carnosine, cyclacillin or a variety of l-amino acid containing dipeptides/tripeptides significantly reduced Gly-sar uptake. Gly-sar uptake was not affected by 10 mM d-ala-d-ala, glycine or sarcosine. These results support the presence of a functional dipeptide transporter in isolated cardiomyocytes, with accompanying expression of PEPT2.

Keywords

PEPT2 Membrane transport Isolated cardiomyocytes Dipeptides Peptidomimetic drugs 

Notes

Acknowledgements

This work was funded by the British Heart Foundation. We would also like to thank Mrs. Valerie Buswell for her excellent technical assistance.

References

  1. 1.
    Baños G (1978) The influx of amino acids into the heart of the rat. J Physiol 280:471–486PubMedGoogle Scholar
  2. 2.
    Boll M, Herget M, Wagener M, Weber WM, Markovich D, Biber J, Clauss W, Murer H, Daniel H (1996) Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter. Proc Natl Acad Sci USA 93:284–289PubMedCrossRefGoogle Scholar
  3. 3.
    Brandsch M, Brandsch C, Prasad PD, Ganapathy V, Hopfer U, Leibach FH (1995) Identification of a renal cell line that constitutively expresses the kidney-specific high-affinity H+/peptide cotransporter. FASEB J 9:1489–1496PubMedGoogle Scholar
  4. 4.
    Daniel H, Kottra G (2004) The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology. Pflügers Arch 447:610–618PubMedCrossRefGoogle Scholar
  5. 6.
    Dringen R, Kranich O, Löschmann PA, Hamprecht B (1997) Use of dipeptides for the synthesis of glutathione by astroglia-rich primary cultures. J Neurochem 69:868–874PubMedCrossRefGoogle Scholar
  6. 5.
    Dringen R, Hamprecht B, Bröer S (1998) The peptide transporter PepT2 mediates the uptake of the glutathione precursor CysGly in astroglia-rich primary cultures. J Neurochem 71:388–393PubMedCrossRefGoogle Scholar
  7. 7.
    Fei Y-J, Kanai Y, Nussberger S, Ganapathy V, Leibach FH, Romero MF, Singh SK, Boron WF, Hediger MA (1994) Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368:563–566PubMedCrossRefGoogle Scholar
  8. 8.
    Ganapathy ME, Brandsch M, Prasad PD, Ganapathy V, Leibach FH (1995) Differential recognition of b-lactam antibiotics by intestinal and renal peptide transporters, PEPT 1 and PEPT 2. J Biol Chem 270:25672–25677PubMedCrossRefGoogle Scholar
  9. 9.
    King N, Korolchuk S, McGivan JD, Suleiman MS (2004) A new method of quantifying glutathione levels in freshly isolated single superfused rat cardiomyocytes. J Pharmacol Toxicol Methods 50:215–222PubMedCrossRefGoogle Scholar
  10. 10.
    King N, McGivan JD, Griffiths EJ, Halestrap AP, Suleiman MS (2003) Glutamate loading protects freshly isolated and perfused adult rat cardiomyocytes from intracellular ROS generation. J Mol Cell Cardiol 35:975–984PubMedCrossRefGoogle Scholar
  11. 11.
    King N, Suleiman MS (2001) L-leucine transport in rat heart under normal conditions and effect of simulated hypoxia. Mol Cell Biochem 221:99–108PubMedCrossRefGoogle Scholar
  12. 12.
    King N, Williams H, McGivan JD, Suleiman MS (2001) Characteristics of L-aspartate transport and expression of EAAC-1 in sarcolemmal vesicles and isolated cells from rat heart. Cardiovasc Res 52:84–94PubMedCrossRefGoogle Scholar
  13. 13.
    Matsui T, Imamura M, Oka H, Osajima K, Kimoto K, Kawasaki T, Matsumoto K (2004) Tissue distribution of antihypertensive dipeptide, Val-tyr, after its single dose administration to spontaneously hypertensive rats. J Pept Sci 10:535–545PubMedCrossRefGoogle Scholar
  14. 14.
    Pisarenko OI (1996) Mechanisms of myocardial protection by amino acids: facts and hypothesis. Clin Exp Pharmacol Physiol 23:627–633PubMedGoogle Scholar
  15. 15.
    Rodrigo GC, Chapman RA (1991) The calcium paradox in isolated guinea-pig ventricular myocytes—effects of membrane-potential and intracellular sodium. J Physiol 434:627–645PubMedGoogle Scholar
  16. 16.
    Rubio-Aliaga I, Daniel H (2002) Mammalian peptide transporters as targets for drug delivery. Trends Pharmacol Sci 23:434–440PubMedCrossRefGoogle Scholar
  17. 17.
    Rühl A, Hoppe S, Frey I, Daniel H, Schemann M (2005) Functional expression of the peptide transporter PEPT2 in the mammalian enteric nervous system. J Comp Neurol 490:1–11PubMedCrossRefGoogle Scholar
  18. 18.
    Safer B (1975) The metabolic significance of the malate-aspartate cycle in heart. Circ Res 36:527–533Google Scholar
  19. 19.
    Shu C, Shen H, Hopfer U, Smith DE (2001) Mechanism of intestinal absorption and renal reabsorption of an orally active ACE inhibitor: uptake and transport of fosinopril in cell cultures. Drug Metab Dispos 29:1307–1315PubMedGoogle Scholar
  20. 20.
    Shu C, Shen H, Teuscher NS, Lorenzi PJ, Keep RF, Smith DE (2002) Role of PEPT2 in peptide/mimetic trafficking at the blood–cerebrospinal fluid barrier: studies in rat choroid plexus epithelial cells in primary culture. J Pharmacol Exp Ther 301:820–829PubMedCrossRefGoogle Scholar
  21. 21.
    Takahashi K, Nakamura N, Terada T, Okano T, Futami T, Saito H, Inui K (1998) Interaction of b-lactam antibiotics with H+/peptide cotransporters in rat renal brush-border membranes. J Pharmacol Exp Ther 286:1037–1042PubMedGoogle Scholar
  22. 22.
    Terada T, Sawada K, Irie M, Saito H, Hashimoto Y, Inui K-I (2000) Structural requirements for determining the substrate affinity of peptide transporters PEPT1 and PEPT2. Pflügers Arch 440:679–684PubMedCrossRefGoogle Scholar
  23. 23.
    Thwaites DT, Brown CDA, Hirst BH, Simmons NL (1993) Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by expression of H+-coupled carriers at both apical and basal membranes. J Biol Chem 268:7640–7642PubMedGoogle Scholar
  24. 24.
    Thwaites DT, Hirst BH, Simmons NL (1994) Substrate specificity of the di/tripeptide transporter in human epithelia (Caco-2): identification of substrates that undergo H+-coupled absorption. Br J Pharmacol 113:1050–1056PubMedGoogle Scholar
  25. 25.
    Vaughan-Jones RD, Spitzer KW, Swietach P (2006) Spatial aspects of intracellular pH regulation in heart muscle. Prog Biophys Mol Biol 90:207–224PubMedCrossRefGoogle Scholar
  26. 26.
    Williams H, King N, Griffiths EJ, Suleiman MS (2001) Glutamate-loading stimulates metabolic flux and improves cell recovery following chemical hypoxia in isolated cardiomyocytes. J Mol Cell Cardiol 33:2109–2119PubMedCrossRefGoogle Scholar
  27. 27.
    Xiang J, Chiang P-P, Hu Y, Smith DE, Keep RF (2002) Role of PEPT2 in glycylsarcosine transport in astrocyte and glioma cultures. Neurosci Lett 396:225–229CrossRefGoogle Scholar
  28. 28.
    Zaloga GP, Roberts PR, Black KW, Lin M, Zapata-Sudo G, Sudo RT, Nelson TE (1997) Carnosine is a novel peptide modulator of intracellular calcium and contractility in cardiac cells. Am J Physiol 272:H462–H468PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Bristol Heart InstituteUniversity of BristolBristolUK

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