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Am J Physiol Lung Cell Mol Physiol 275: L461-L468, 1998;
1040-0605/98 $5.00
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Vol. 275, Issue 3, L461-L468, September 1998

Hypoxia-specific upregulation of calpain activity and gene expression in pulmonary artery endothelial cells

Jianliang Zhang, Jawaharlal M. Patel, and Edward R. Block

Department of Medicine, University of Florida, and Medical Research Service, Veterans Affairs Medical Center, Gainesville, Florida 32608-1197

The effects of exposure to hypoxia on the catalytic activity and mRNA expression of calpain, a calcium-regulated neutral cysteine protease, were examined in porcine pulmonary artery endothelial cells (PAECs). Specificity of the response to hypoxia was determined by comparing the effects of hypoxic exposure with exposure to oxidants such as nitrogen dioxide (NO2) and nitric oxide (NO), as well as to the sulfhydryl reactive chemical acrolein. Exposure of cells to hypoxia (0% O2) for 1 and 12 h significantly increased catalytic activity (P < 0.01 for both 1 and 12 h vs. control cells), as well as mRNA expression (P < 0.01 for 1 h and P < 0.05 for 12 h vs. control cells) of calpain. With more prolonged exposure to 24 h of hypoxia, calpain activity remained significantly elevated, whereas calpain mRNA expression returned to the control level. Calpain activities in cells exposed to NO2 [5 parts/million (ppm)] or NO (7.5 ppm) for 1 h or to acrolein (5 µM) for 1 and 24 h were unchanged. However, calpain activities in cells exposed to NO2 or NO for 24 h were significantly (P < 0.05) reduced compared with control cells. The hypoxia-induced increases in calpain mRNA content were prevented by the transcriptional inhibitor actinomycin D and by calpain inhibitor I. In addition, hypoxia increased the degradation of nuclear factor-kappa B (NF-kappa B) inhibitor Ikappa B and enhanced the translocation of the p50 subunit of NF-kappa B to the nuclear membrane. Pretreatment with the calpain-specific inhibitor E-64d prevented hypoxia-induced mRNA expression and degradation of Ikappa Balpha , as well as translocation of p50 subunit to the nuclear membrane. These results demonstrate for the first time that hypoxia upregulates calpain activity and mRNA expression in PAECs and that the upregulation is specific to hypoxia. Upregulation appears to involve activation of the transcription factor NF-kappa B.

endothelium


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