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Prolonged incubation with elevated glucose inhibits the regulatory response to shrinkage of cultured human retinal pigment epithelial cells

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Abstract

Transport defects by retinal pigment epithelial (RPE) and other cells are observed in experimental models of diabetes mellitus. Recent studies have established that glucose concentration, per se, is the critical risk factor in the pathogenesis of diabetic complications. This study was designed to test whether transport alterations could be produced in the simplest model of diabetes, sustained exposure of cultured cells to a high-glucose environment. The regulatory transport responses to acute changes in cell volume were measured in order to assess the effects of glucose on a range of transport processes. Continuous lines of nontransformed human retinal pigment epithelial (hRPE) cells were grown for two weeks with either 5.6 low glucose (LG) or 26.0 high glucose (HG) mm in paired experiments. The cell volumes of suspended cells were studied in hypo-, iso- and hypertonic solutions containing the same ionic composition. Hypotonic swelling triggered a regulatory volume decrease (RVD), inhibited by reducing the chemical driving force for K+ efflux, or blocking K+ channels (with Ba2+) or Cl− channels (with NPPB). Thus, the RVD of the hRPE cells likely reflects efflux of K+ and Cl− through parallel channels. Shrinkage caused a regulatory volume increase (RVI), which was inhibited by blocking Na+/H+ (with dimethylamiloride) or Cl−/HCO −3 exchange (with DIDS). Bumetanide inhibited the RVI significantly only when the K+ concentration was increased above the baseline level. Therefore, the RVI under our baseline conditions likely reflects primarily Na+/H+ and Cr/HCO −3 antiport exchange. Growth in high-glucose medium had no substantial effect on the RVD, but reduced the rate constant of the RVI by ≈ 50%. The RVI was unaffected by growth in high-mannitol medium. Stimulation of protein kinase C (PKC) with DiC8 increased the RVI of HG-cells, but not of LG-cells. The DiC8-induced stimulation was bumetanide insensitive and abolished by 1 mM amiloride. Other transport effects of PKC (on the RVD) were unaltered in the HG-cells. We conclude that sustained elevation of extracellular glucose, per se, can downregulate the Na+/H+ antiport of target cells, an effect noted in streptozotocin-treated rats, and that this downregulation does not reflect interruption of the PKC-signaling pathway.

We are grateful to Dr. Monte Del Monte (Kellogg Eye Institute, Ann Arbor, MI) for making cell lines of human retinal pigmented epithelium available to us, and Prof. Rainer Greger (Albert-Ludwigs-Universität, Freiburg, FRG) for providing us with a generous sample of NPPB. We also thank Drs. Thomas Kleyman (University of Pennsylvania) and George M. Fanelli, Jr. (Merck Institute for Therapeutic Research, West Point, PA), and Hoffmann-La Roche for gifts of dimethylamiloride, amiloride and bumetanide, respectively. We are also grateful to Dr. Carol Deutsch (University of Pennsylvania) for permitting us to use her Coulter counter during the initial phase of this work. We thank Dr. Robert D. Purves (University of Otago) and David A. Carré and Michael L. Chalfant (University of Pennsylvania) for their critical readings of the manuscript.

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Authors and Affiliations

  1. Department of Physiology, University of Pennsylvania, 19104, Philadelphia, Pennsylvania

    M. M. Civan & K. Peterson-Yantorno

  2. Department of Medicine, University of Pennsylvania, Richards Building, 19104, Philadelphia, Pennsylvania

    M. M. Civan

  3. Department of Biochemistry and Biophysics, University of Pennsylvania, 19104, Philadelphia, Pennsylvania

    C. W. Marano & F. W. Matschinsky

  4. Diabetes Research Center, University of Pennsylvania, 19104, Philadelphia, Pennsylvania

    F. W. Matschinsky

Authors
  1. M. M. Civan
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  2. C. W. Marano
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  3. F. W. Matschinsky
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  4. K. Peterson-Yantorno
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Supported in part by research grants from the National Institutes of Health [EYO-8343 and EY01583 (for core facilities) (to M.M.C.), 2P30DK19525 (to the University of Pennsylvania Diabetes Center), DK07314 and HL07614 (to C.W.M.), and NIDK19525 (to F.M.M.)]. This work was also supported by a JDF Postdoctoral Fellowship (to C.W.M.), and a JDF research grant (to F.M.M.).

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Civan, M.M., Marano, C.W., Matschinsky, F.W. et al. Prolonged incubation with elevated glucose inhibits the regulatory response to shrinkage of cultured human retinal pigment epithelial cells. J. Membarin Biol. 139, 1–13 (1994). https://doi.org/10.1007/BF00232670

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  • DOI: https://doi.org/10.1007/BF00232670

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