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Acta Physiologica 2012; Volume 204, Supplement 689
91st Annual Meeting of The German Physiological Society
3/22/2012-3/25/2012
Dresden, Germany
RELATIONSHIP BETWEEN PHOSPHOLIPASE A2 AND SGK1 IN MURINE COLLECTING DUCT
Abstract number: P254
Grussel1 *S., Himmerkus1 N., Steels2 P., Bleich1 M.
1CAU Kiel, Physiologisches Institut, Kiel, Germany
2University Hasselt, Biomed, Diepenbeek, Belgium
Objective:
The serum- and glucocorticoid-inducible kinase 1(SGK1), an important regulator of epithelial ion transport, interferes with several biological processes such as sodium homeostasis, osmoregulation and cell growth. Phospholipase A2 (PLA2) is one of the key enzymes in arachidonic acid metabolism. We investigated the possible linkage of both regulatory pathways by testing the effects of two different PLA2 inhibitors on mammalian cortical collecting duct cells regarding changes in SGK1 activity.
Methods:
Cultured M-1 cells were detached and treated in suspension in a thermo shaker (37°C, 350rpm) with the PLA2 inhibitors quinacrine (Quin [100mM]) or aristolochic acid (ArA [50mM]) for different time periods. NDRG1 (n-mycdownstream-regulatedgene1) served as a biomarker for SGK1 activity since its threonine residues 346, 356 and 366 are specifically phosphorylated by activated SGK1. The state of NDRG1 phosphorylation was measured by Western blot and flow cytometry. In addition, freshly isolated mouse cortical collecting ducts were treated likewise and NDRG1 phosphorylation was analysed by Western Blotting. A specific SGK1 inhibitor was used as a control.
Results:
PLA2 inhibition by Quin or ArA in M-1 cells reduced NDRG1 phosphorylation by 65% and 77.7% respectively, with a maximum effect after 35 min. Persistent Quin treatment up to 30 min showed an inhomogeneous trend with a partially or total recovery of Phospho-NDRG1 after 20 - 30 min. Analogous treatment with ArA in M-1 cells revealed a fast reduction of phospho NDRG1 with a total recovery after 20 min. The suppressing effect of ArA on NDRG1 phosphorylation could be verified with flow cytometry. M-1 cells showed a high fluorescence intensity of phospho NDRG1 which was reduced by 40% at the maximum effect. Moreover, both PLA2 inhibiting compounds were able to decrease the activity of SGK1 in freshly isolated mouse collecting ducts. The potency of Quin which inhibited by 78% was higher than that of ArA with a max. suppression by 16.6%.
Conclusion:
These data indicate an association of the arachidonic acid pathway and SGK1 pathway. This offers a new perspective on the regulation of epithelial ion transport.
To cite this abstract, please use the following information:
Acta Physiologica 2012; Volume 204, Supplement 689 :P254