Amna Al-Shamali1, Islam Khan. 1. Department of Biochemistry, Faculty of Medicine, Kuwait University, Kuwait.
Abstract
OBJECTIVES: Mechanism of the apical transporter Na-H exchanger-8 (NHE-8) regulation was investigated by examining the effects of anti-inflammatory dexamethasone in experimental colitis. In addition, its localization was investigated in the lipid rich membrane domain called membrane rafts. MATERIAL AND METHODS: Colitis was induced by trinitrobenzene sulfonic acid (TNBS) and colon segments were removed from 5 day post-TNBS and used to estimate the levels of NHE-8 protein and mRNA using ECL western blot analysis and a competitive RT-PCR method. Myeloperoxidase activity, malondialdehyde levels and histologic changes were evaluated. RESULTS: NHE-8 protein level was decreased in inflamed colon and was not reversed by dexamethasone. However, mRNA levels remained unchanged in inflamed colon. The levels of NHE-8 protein and mRNA were not significantly different in non-colitic control as compared to dexamethasone treated non-colitis. Elevation of myeloperoxidase activity, malondialdehyde and infiltration of inflammatory cells in inflamed colon were suppressed by dexamethasone treatment of colitis significantly. Furthermore, NHE-8 protein was not detected in the detergent resistant membrane (DRM) or lipid rafts, but was present in the detergent sensitive membrane (DRS) fractions. Actin showed its partition similar to NHE-8. On the contrary, NHE-3 was present in both DRM and DRS fractions. Flotillin-1 and caveolin were enriched in the fractions designated as lipid rafts. CONCLUSIONS: These findings demonstrate the suppression of NHE-8 protein in inflamed adult rat colon, which seems to be regulated post-transcriptionally. Furthermore, the absence of NHE-8 in lipid rafts suggests its regulation independent of cAMP or recycling through endocytosis unlike NHE-3 isoform.
OBJECTIVES: Mechanism of the apical transporter Na-H exchanger-8 (NHE-8) regulation was investigated by examining the effects of anti-inflammatory dexamethasone in experimental colitis. In addition, its localization was investigated in the lipid rich membrane domain called membrane rafts. MATERIAL AND METHODS:Colitis was induced by trinitrobenzene sulfonic acid (TNBS) and colon segments were removed from 5 day post-TNBS and used to estimate the levels of NHE-8 protein and mRNA using ECL western blot analysis and a competitive RT-PCR method. Myeloperoxidase activity, malondialdehyde levels and histologic changes were evaluated. RESULTS:NHE-8 protein level was decreased in inflamed colon and was not reversed by dexamethasone. However, mRNA levels remained unchanged in inflamed colon. The levels of NHE-8 protein and mRNA were not significantly different in non-colitic control as compared to dexamethasone treated non-colitis. Elevation of myeloperoxidase activity, malondialdehyde and infiltration of inflammatory cells in inflamed colon were suppressed by dexamethasone treatment of colitis significantly. Furthermore, NHE-8 protein was not detected in the detergent resistant membrane (DRM) or lipid rafts, but was present in the detergent sensitive membrane (DRS) fractions. Actin showed its partition similar to NHE-8. On the contrary, NHE-3 was present in both DRM and DRS fractions. Flotillin-1 and caveolin were enriched in the fractions designated as lipid rafts. CONCLUSIONS: These findings demonstrate the suppression of NHE-8 protein in inflamed adult rat colon, which seems to be regulated post-transcriptionally. Furthermore, the absence of NHE-8 in lipid rafts suggests its regulation independent of cAMP or recycling through endocytosis unlike NHE-3 isoform.