Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 2002 Mar 1;362(Pt 2):507–512. doi: 10.1042/0264-6021:3620507

Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification.

Melanie E Holmes 1, James Mwanjewe 1, Sue E Samson 1, James V Haist 1, John X Wilson 1, S Jeffrey Dixon 1, Morris Karmazyn 1, Ashok K Grover 1
PMCID: PMC1222413  PMID: 11853561

Abstract

Dehydroascorbic acid (DHAA) enters cells via Na(+)-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na(+) removal inhibited [(14)C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2-12 min at 10-200 microM DHAA in either the presence of 134 mM Na(+) or in its absence (N-methyl D-glucamine, choline or sucrose replaced Na(+)). This inhibition of DHAA uptake by Na(+) removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [(14)C]DHAA into [(14)C]ascorbate upon intracellular acidosis caused by the Na(+) removal. Consistent with this hypothesis: (i) the Na(+)/H(+)-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na(+) removal and Na(+)/H(+)-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12 min than in 2 min, and (iii) less of the cellular (14)C was present as ascorbate (determined by HPLC) in cells in Na(+)-free buffer than in those in Na(+)-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion.

Full Text

The Full Text of this article is available as a PDF (150.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aalkjaer C., Cragoe E. J., Jr Intracellular pH regulation in resting and contracting segments of rat mesenteric resistance vessels. J Physiol. 1988 Aug;402:391–410. doi: 10.1113/jphysiol.1988.sp017211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Apkon M., Weed R. A., Boron W. F. Motor responses of cultured rat cerebral vascular smooth muscle cells to intra- and extracellular pH changes. Am J Physiol. 1997 Jul;273(1 Pt 2):H434–H445. doi: 10.1152/ajpheart.1997.273.1.H434. [DOI] [PubMed] [Google Scholar]
  3. Bánhegyi G., Marcolongo P., Puskás F., Fulceri R., Mandl J., Benedetti A. Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles. J Biol Chem. 1998 Jan 30;273(5):2758–2762. doi: 10.1074/jbc.273.5.2758. [DOI] [PubMed] [Google Scholar]
  4. Carr A., Frei B. Does vitamin C act as a pro-oxidant under physiological conditions? FASEB J. 1999 Jun;13(9):1007–1024. doi: 10.1096/fasebj.13.9.1007. [DOI] [PubMed] [Google Scholar]
  5. Cornu M. C., Moore G. A., Nakagawa Y., Moldéus P. Ascorbic acid uptake by isolated rat hepatocytes. Stimulatory effect of diquat, a redox cycling compound. Biochem Pharmacol. 1993 Oct 19;46(8):1333–1338. doi: 10.1016/0006-2952(93)90096-f. [DOI] [PubMed] [Google Scholar]
  6. Daruwala R., Song J., Koh W. S., Rumsey S. C., Levine M. Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett. 1999 Nov 5;460(3):480–484. doi: 10.1016/s0014-5793(99)01393-9. [DOI] [PubMed] [Google Scholar]
  7. Del Bello B., Maellaro E., Sugherini L., Santucci A., Comporti M., Casini A. F. Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3 alpha-hydroxysteroid dehydrogenase. Biochem J. 1994 Dec 1;304(Pt 2):385–390. doi: 10.1042/bj3040385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elmoselhi A. B., Samson S. E., Grover A. K. SR Ca2+ pump heterogeneity in coronary artery: free radicals and IP3-sensitive and -insensitive pools. Am J Physiol. 1996 Nov;271(5 Pt 1):C1652–C1659. doi: 10.1152/ajpcell.1996.271.5.C1652. [DOI] [PubMed] [Google Scholar]
  9. Franceschi R. T., Wilson J. X., Dixon S. J. Requirement for Na(+)-dependent ascorbic acid transport in osteoblast function. Am J Physiol. 1995 Jun;268(6 Pt 1):C1430–C1439. doi: 10.1152/ajpcell.1995.268.6.C1430. [DOI] [PubMed] [Google Scholar]
  10. Frei B., England L., Ames B. N. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6377–6381. doi: 10.1073/pnas.86.16.6377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garewal H. S., Ahmann F. R., Schifman R. B., Celniker A. ATP assay: ability to distinguish cytostatic from cytocidal anticancer drug effects. J Natl Cancer Inst. 1986 Nov;77(5):1039–1045. [PubMed] [Google Scholar]
  12. Holmes M. E., Samson S. E., Wilson J. X., Dixon S. J., Grover A. K. Ascorbate transport in pig coronary artery smooth muscle: Na(+) removal and oxidative stress increase loss of accumulated cellular ascorbate. J Vasc Res. 2000 Sep-Oct;37(5):390–398. doi: 10.1159/000025755. [DOI] [PubMed] [Google Scholar]
  13. Kleyman T. R., Cragoe E. J., Jr Amiloride and its analogs as tools in the study of ion transport. J Membr Biol. 1988 Oct;105(1):1–21. doi: 10.1007/BF01871102. [DOI] [PubMed] [Google Scholar]
  14. Lam K. W., Yu H. S., Glickman R. D., Lin T. Sodium-dependent ascorbic and dehydroascorbic acid uptake by SV-40-transformed retinal pigment epithelial cells. Ophthalmic Res. 1993;25(2):100–107. doi: 10.1159/000267272. [DOI] [PubMed] [Google Scholar]
  15. Liebler D. C. The role of metabolism in the antioxidant function of vitamin E. Crit Rev Toxicol. 1993;23(2):147–169. doi: 10.3109/10408449309117115. [DOI] [PubMed] [Google Scholar]
  16. Maellaro E., Del Bello B., Sugherini L., Santucci A., Comporti M., Casini A. F. Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver. Biochem J. 1994 Jul 15;301(Pt 2):471–476. doi: 10.1042/bj3010471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Malo C., Wilson J. X. Glucose modulates vitamin C transport in adult human small intestinal brush border membrane vesicles. J Nutr. 2000 Jan;130(1):63–69. doi: 10.1093/jn/130.1.63. [DOI] [PubMed] [Google Scholar]
  18. May J. M. Is ascorbic acid an antioxidant for the plasma membrane? FASEB J. 1999 Jun;13(9):995–1006. doi: 10.1096/fasebj.13.9.995. [DOI] [PubMed] [Google Scholar]
  19. May J. M., Mendiratta S., Hill K. E., Burk R. F. Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase. J Biol Chem. 1997 Sep 5;272(36):22607–22610. doi: 10.1074/jbc.272.36.22607. [DOI] [PubMed] [Google Scholar]
  20. May J. M., Qu Z. C., Whitesell R. R. Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes. Biochemistry. 1995 Oct 3;34(39):12721–12728. doi: 10.1021/bi00039a031. [DOI] [PubMed] [Google Scholar]
  21. Neylon C. B., Little P. J., Cragoe E. J., Jr, Bobik A. Intracellular pH in human arterial smooth muscle. Regulation by Na+/H+ exchange and a novel 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)- and HCO3(-)-dependent mechanism. Circ Res. 1990 Oct;67(4):814–825. doi: 10.1161/01.res.67.4.814. [DOI] [PubMed] [Google Scholar]
  22. Ngkeekwong F. C., Ng L. L. Two distinct uptake mechanisms for ascorbate and dehydroascorbate in human lymphoblasts and their interaction with glucose. Biochem J. 1997 May 15;324(Pt 1):225–230. doi: 10.1042/bj3240225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olson A. L., Pessin J. E. Structure, function, and regulation of the mammalian facilitative glucose transporter gene family. Annu Rev Nutr. 1996;16:235–256. doi: 10.1146/annurev.nu.16.070196.001315. [DOI] [PubMed] [Google Scholar]
  24. Prasad P. D., Huang W., Wang H., Leibach F. H., Ganapathy V. Transport mechanisms for vitamin C in the JAR human placental choriocarcinoma cell line. Biochim Biophys Acta. 1998 Feb 2;1369(1):141–151. doi: 10.1016/s0005-2736(97)00215-0. [DOI] [PubMed] [Google Scholar]
  25. Rumsey S. C., Kwon O., Xu G. W., Burant C. F., Simpson I., Levine M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J Biol Chem. 1997 Jul 25;272(30):18982–18989. doi: 10.1074/jbc.272.30.18982. [DOI] [PubMed] [Google Scholar]
  26. Russ U., Balser C., Scholz W., Albus U., Lang H. J., Weichert A., Schölkens B. A., Gögelein H. Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes. Pflugers Arch. 1996 Nov-Dec;433(1-2):26–34. doi: 10.1007/s004240050244. [DOI] [PubMed] [Google Scholar]
  27. Spielholz C., Golde D. W., Houghton A. N., Nualart F., Vera J. C. Increased facilitated transport of dehydroascorbic acid without changes in sodium-dependent ascorbate transport in human melanoma cells. Cancer Res. 1997 Jun 15;57(12):2529–2537. [PubMed] [Google Scholar]
  28. Vera J. C., Rivas C. I., Velásquez F. V., Zhang R. H., Concha I. I., Golde D. W. Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid. J Biol Chem. 1995 Oct 6;270(40):23706–23712. doi: 10.1074/jbc.270.40.23706. [DOI] [PubMed] [Google Scholar]
  29. Vera J. C., Rivas C. I., Zhang R. H., Farber C. M., Golde D. W. Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid. Blood. 1994 Sep 1;84(5):1628–1634. [PubMed] [Google Scholar]
  30. Weissberg P. L., Little P. J., Cragoe E. J., Jr, Bobik A. Na-H antiport in cultured rat aortic smooth muscle: its role in cytoplasmic pH regulation. Am J Physiol. 1987 Aug;253(2 Pt 1):C193–C198. doi: 10.1152/ajpcell.1987.253.2.C193. [DOI] [PubMed] [Google Scholar]
  31. Welch R. W., Wang Y., Crossman A., Jr, Park J. B., Kirk K. L., Levine M. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol Chem. 1995 May 26;270(21):12584–12592. doi: 10.1074/jbc.270.21.12584. [DOI] [PubMed] [Google Scholar]
  32. Wells W. W., Xu D. P., Yang Y. F., Rocque P. A. Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol Chem. 1990 Sep 15;265(26):15361–15364. [PubMed] [Google Scholar]
  33. Wilson J. X., Dixon S. J. High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts. J Membr Biol. 1989 Oct;111(1):83–91. doi: 10.1007/BF01869211. [DOI] [PubMed] [Google Scholar]
  34. Wilson J. X., Dixon S. J., Yu J., Nees S., Tyml K. Ascorbate uptake by microvascular endothelial cells of rat skeletal muscle. Microcirculation. 1996 Jun;3(2):211–221. doi: 10.3109/10739689609148290. [DOI] [PubMed] [Google Scholar]
  35. Wilson J. X., Jaworski E. M., Dixon S. J. Evidence for electrogenic sodium-dependent ascorbate transport in rat astroglia. Neurochem Res. 1991 Jan;16(1):73–78. doi: 10.1007/BF00965831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Winkler B. S. Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid. Biochim Biophys Acta. 1992 Oct 27;1117(3):287–290. doi: 10.1016/0304-4165(92)90026-q. [DOI] [PubMed] [Google Scholar]
  37. Xu D. P., Washburn M. P., Sun G. P., Wells W. W. Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes. Biochem Biophys Res Commun. 1996 Apr 5;221(1):117–121. doi: 10.1006/bbrc.1996.0555. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES