HYPOXIA-SPECIFIC UP-REGULATION OF CALPAIN ACTIVITY AND GENE-EXPRESSION IN PULMONARY-ARTERY ENDOTHELIAL-CELLS

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). S pecificity 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 rea ctive chemical acrolein. Exposure of cells to hypoxia (0% O-2) for 1 a nd 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 fo r 1 h add P < 0.05 for 12 h vs. control cells) of calpain. With more p rolonged exposure to 24 h of hypoxia, calpain activity remained signif icantly elevated, whereas calpain mRNA expression returned to the cont rol level. Calpain activities in cells exposed to NO2 [5 parts/million (ppm)] or NO (7.5 ppm) for 1 h or to acrolein (5 mu M) for 1 and 24 h were unchanged. However, calpain activities in cells exposed to NO2 o r NO for 24 h were significantly (P < 0.05) reduced compared with cont rol cells. The hypoxia-induced increases in calpain mRNA content were prevented by the transcriptional inhibitor actinomycin D and by calpai n inhibitor I. In addition, hypoxia increased the degradation of nucle ar factor-kappa B (NF-kappa B) inhibitor I kappa B and enhanced the tr anslocation of the p50 subunit of NF-kappa B to the nuclear membrane. Pretreatment with the calpain-specific inhibitor E-64d prevented hypox ia-induced mRNA expression and degradation of I kappa B alpha, as well as translocation of p50 subunit to the nuclear membrane. These result s demonstrate for the first time that hypoxia upregulates calpain acti vity and mRNA expression in PAECs and that the upregulation is specifi c to hypoxia. Upregulation appears to involve activation of the transc ription factor NF-kappa B.