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Am J Physiol Lung Cell Mol Physiol (October 26, 2001). doi:10.1152/ajplung.00320.2000
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Articles in PresS, published online ahead of print October 26, 2001
Am J Physiol Lung Cell Mol Physiol, 10.1152/ajplung.00320.2000
Submitted on September 19, 2000
Accepted on October 25, 2001

Different Pattern of NOS2 and NOS3 mRNA Expression in Human A549 Cells and Primary Cultured Alveolar Epithelial Cells Type II

Dmitri V Pechkovsky1, Gernot Zissel1*, Torsten Goldmann1, Martin Einhaus2, Christian Taube2, Helgo Magnussen2, Max Schlaak1, and Joachim Mueller-Quernheim1

1 Medical Hospital, Research Center Borstel, Borstel, Schleswig-Holstein, Germany
2 Zentrum fur Pneumologie und Thoraxchirurgie, Krankenhaus Grosshansdorf, Grosshansdorf, Schleswig-Holstein, Germany

* To whom correspondence should be addressed. E-mail: gzissel{at}fz-borstel.de.

The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS type 3 (NOS3), and produce nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of human alveolar epithelial cells type II (AEC-II) in primary culture. Therefore we investigated the expression of NOS isoforms in human AEC-II in comparison with the AEC-II-like cell line A549 on mRNA and protein levels. NOS3 mRNA was detected in freshly isolated AEC-II and after 24 h of culture. We found that NOS3 mRNA levels were much higher in AEC-II, cultured for 24 h with or without proinflammatory cytokines such as interferon{gamma} (IFN{gamma}), interleukin 1ß (IL-1ß) and tumor necrosis factor {alpha} (TNF{alpha}), compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC-II in comparison with unstimulated cells. NOS3 protein expression was verified by Western blot technique and measuring nitrite/nitrate revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC-II in 24 h or 72 h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC-II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC-II and A549 cells in NOS mRNA expression pattern, and that NOS2 and NOS3 mRNA levels are dependent on the cell culture conditions of A549 and AEC-II respectively.




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