5-Lipoxygenase is involved in the angiotensin II-induced NAD(P)H-oxidase activation

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Abstract

Angiotensin II (ANG II)-induced interleukin (IL)-6 synthesis requires NAD(P)H-oxidase-derived superoxide anions. Since NAD(P)H-oxidase activation by cytokines involves 5-lipoxygenase (LOX)-derived leukotriene B4 (LTB4) formation, we postulated that LTB4 is involved in the ANG II-dependent NAD(P)H-oxidase activation. Therefore, 5-LOX expression and LTB4 formation following ANG II (100 nM) stimulation were determined in rat aortic smooth muscle cells (SMC). Reactive oxygen species (ROS)-formation and IL-6 mRNA expression were analyzed following ANG II and LTB4 (0.6 μM) stimulation. 5-LOX mRNA and protein were detected in SMC. ANG II-induced LTB4 formation at 2.5 min and was followed by an increase in ROS-formation and IL-6 mRNA expression. Blockade of 5-LOX by MK886 (200 nM) abrogated LTB4-formation, ROS-formation, and IL-6 mRNA expression. Moreover, LTB4-induced ROS-formation and IL-6 mRNA expression was abolished by NAD(P)H-oxidase inhibition using diphenyleneiodonium chloride (DPI 10 μM). In conclusion, the present study demonstrates that ANG II enhances LTB4-formation in an 5-LOX dependent manner. LTB4 activates the vascular type NAD(P)H-oxidase, leading to an increase in IL-6 transcripts.

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.

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