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Activity and expression of the Na+/H+ exchanger in human endothelial cells cultured in high glucose

Diabetologia volume 38, pages 785–791 (1995)Cite this article

Summary

Establishing whether high ambient glucose affects the plasma membrane Na+/H+ exchanger is relevant to understanding the adverse effects of high glucose on cell replication and the mechanisms of the increased exchanger activity encountered in diabetic patients with nephropathy. In 8 primary and 15 first-passage isolates of human endothelial cells cultured in 30 mmol/l glucose for 8.7±2.3 and 15.8±2.3 days, respectively, we determined Na+/H+ exchanger activity and mRNA levels. Activity was determined by measuring 22Na+ influx in the presence or absence of dimethylamiloride (DMA) after intracellular acidification. We also measured fibronectin mRNA because fibronectin provides signals for cell replication through the Na+/H+ antiporter. Control cells grown in 5 mmol/l glucose showed at morphologic confluency a total Na+ influx (in nmol · mg protein-1 · min-1) of 10.1±3.2 in primary and 11.7±2.2 in first subculture, which was reduced to 5.3±0.3 in the presence of DMA. Paired cultures exposed to 30 mmol/l glucose and exhibiting pHi and cell densities identical to controls showed in both primary and first subculture a reduction in total Na+ influx (A = -0.98±0.93 nmol · mg protein-1 · min-1 p < 0.005) whereas DMA-resistant Na+ influx was identical to that of control. Neither chronic hypertonicity nor acute exposure to high glucose mimicked the effects of chronic high glucose. The level of the Na+/H+ exchanger isoform 1 (NHE-1) mRNA was unchanged by high glucose whereas fibronectin mRNA levels were increased 1.5-fold. These studies indicate that in endothelial cells exposed to elevated ambient glucose the regulation of the Na+/H+ exchanger is altered at the post-transcriptional level; decreased activity of the antiporter is concomitant with fibronectin overexpression and may contribute to the decreased replication caused by high glucose.

Abbreviations

DMA:

dimethylamiloride

pHi:

intracellular pH

BSA:

bovine serum albumin

PCR:

polymerase chain reaction

PKC:

protein kinase C

NHE-1:

Na+/H+ exchanger isoform 1

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Affiliations

  1. Endocrine-Hypertension Division, Brigham and Women's Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    G. Zerbini & M. Canessa

  2. Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA

    T. Roth, E. Podestá, E. Cagliero & M. Lorenzi

  3. Epidemiology and Genetics Section, Research Division of the Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA

    A. Doria

Authors
  1. G. Zerbini
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  2. T. Roth
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  3. E. Podestá
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  4. E. Cagliero
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  5. A. Doria
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  6. M. Canessa
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  7. M. Lorenzi
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Zerbini, G., Roth, T., Podestá, E. et al. Activity and expression of the Na+/H+ exchanger in human endothelial cells cultured in high glucose. Diabetologia 38, 785–791 (1995). https://doi.org/10.1007/s001250050353

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  • DOI: https://doi.org/10.1007/s001250050353

Key words

  • Na+/H+ exchanger
  • endothelial cells
  • cell replication
  • fibronectin
  • diabetic nephropathy