Biochemical and Biophysical Research Communications
5-Lipoxygenase is involved in the angiotensin II-induced NAD(P)H-oxidase activation☆
Section snippets
Materials and methods
Reagents. Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and medium additives were from Life Technologies, Invitrogen. LTB4, ANG II, and 2′,7′-dichloroflourescein-diacetate (DCFH-DA) were obtained from Sigma. DPI, a flavoprotein inhibitor, was obtained from Calbiochem, MK886, a 5-LOX inhibitor, was obtained from Biomol, and leukotriene EIA was from Cayman Chemicals Company.
Cell culture. Smooth muscle cells (SMC) were isolated from the aorta of 200–250 g male Sprague–Dawley
Results
5-LOX, involved in LTB4-formation, was detected in SMC by RT-PCR. A product of a predicted length of 597 bp was obtained (Fig. 1A). Alveolar macrophages were used as control. Western Blot analysis revealed the presence of 5-LOX protein in SMC (Fig. 1B).
Formation of LTA4 required for LTB4-formation was previously demonstrated in SMC [18] and subsequently in aortic tissue [19]. Therefore, LTB4-formation was investigated. ANG II enhances the formation of LTB4 (5-fold ± 0.98) at 2.5 min compared to
Discussion
The present study demonstrates that ANG II enhances AA-derived LTB4-formation in a 5-LOX dependent manner. 5-LOX, detected in SMC, was shown to be critically involved in ANG II-induced NAD(P)H-oxidase-derived ROS-formation and ANG II-induced IL-6 formation.
The synthesis of LTB4 is initiated by the conversion of AA to the unstable intermediates 5-HPETE and LTA4 by 5-LOX. 5-LOX in leukocytes exhibits both, lipoxygenase and LTA4 synthase activities [21], [22]. In this regard, it is tempting to
Acknowledgements
The authors are indebted to Karsten Grote, PhD, and Tina Selle, PhD, for critical discussion. Furthermore, the authors are grateful to Tanja Sander and Silke Pretzer for excellent technical assistance.
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This work was supported by the Deutsche Forschungsgemeinschaft Grants SFB 566/B9 and SFB-TR02/B4.