NFkappaB in the mechanism of ammonia-induced astrocyte swelling in culture

Abstract

Astrocyte swelling and brain edema are major neuropathological findings in the acute form of hepatic encephalopathy (fulminant hepatic failure), and substantial evidence supports the view that elevated brain ammonia level is an important etiological factor in this condition. Although the mechanism by which ammonia brings about astrocyte swelling remains to be determined, oxidative/nitrosative stress and mitogen-activated protein kinases (MAPKs) have been considered as important elements in this process. One factor known to be activated by both oxidative stress and MAPKs is nuclear factor kappaB (NFkappaB), a transcription factor that activates many genes, including inducible nitric oxide synthase (iNOS). As the product of iNOS, nitric oxide (NO), is known to cause astrocyte swelling, we examined the potential involvement of NFkappaB in ammonia-induced astrocyte swelling. Western blot analysis of cultured astrocytes showed a significant increase in NFkappaB nuclear translocation (a measure of NFkappaB activation) from 12 h to 2 days after treatment with NH(4)Cl (5 mM). Cultures treated with anti-oxidants, including superoxide dismutase, catalase, and vitamin E as well as the MAPKs inhibitors, SB239063 (an inhibitor of p38-MAPK) and SP600125 (an inhibitor of c-Jun N-terminal kinase), significantly diminished NFkappaB activation by ammonia, supporting a role of oxidative stress and MAPKs in NFkappaB activation. The activation of NFkappaB was associated with increased iNOS protein expression and NO generation, and these changes were blocked by BAY 11-7082, an inhibitor of NFkappaB. Additionally, ammonia-induced astrocyte swelling was inhibited by the NFkappaB inhibitors, BAY 11-7082 and SN-50, thereby implicating NFkappaB in the mechanism of astrocyte swelling. Our studies indicate that cultured astrocytes exposed to ammonia display NFkappaB activation, which is likely to be a consequence of oxidative stress and activation of MAPKs. NFkappaB activation appears to contribute to the mechanism of ammonia-induced astrocyte swelling, apparently through its up-regulation of iNOS protein expression and the subsequent generation of NO.

Figure 1

Increased nuclear NFκB was identified in cultured astrocytes treated with 5 mM NH₄Cl at 12–48 h. A. Western blots of nuclear fraction; B. Quantification of Western blots. No significant increase in nuclear NFκB was detected at 1, 3 and 6 h (data not shown). NFκB content was normalized against lamin. ANOVA, n=5. *p<0.05 vs. control.

Figure 2

NFκB inhibitors diminish ammonia-induced translocation of NFκB to nuclei in cultured astrocytes as detected by immunoblots. A. Cultures exposed to 5 mM NH₄Cl by 1 day significantly increased NFκB translocation, while co-treatment with BAY 11-7082 (BAY, 5 μM) and SN50 (3.5 μM) significantly diminished such translocation. B. Quantification of ammonia-induced NFκB content after treatment with BAY 11-7082. NFκB content was normalized against lamin. ANOVA, n=5. *p<0.05 vs. control; † p<0.05 vs. NH₄Cl.

Figure 3

Immunocytochemical staining for NFκB in ammonia-treated cultured astrocytes. A. Control. B. After 24 h of ammonia treatment. C. Quantification of immunocytochemical staining of ammonia-treated cultured astrocytes. Ammonia progressively increased the percentage of NFκB-possitive nuclei. n=4. *p<0.05 vs. control.

Figure 4

Antioxidants attenuate the ammonia-induced NFκB nuclear translocation. A. Cultured astrocytes exposed to 5 mM NH₄Cl significantly increased nuclear NFκB content at 24 h. Co-treatment with SOD (25 units/ml), vitamin E (100 μM) and catalase (250 units/ml) significantly diminished ammonia-induced NFκB translocation, while desferioxamine (DFX) and PBN showed minimal effect. B. Quantification of ammonia-induced NFκB translocation after treatment with antioxidants. NFκB content was normalized against lamin. ANOVA, n=4. *p<0.05 vs. control; †p<0.05 vs. NH₄Cl.

Figure 5

MAPK inhibitors prevent ammonia-induced NFκB translocation. A. Western blots reveal a slight, but not significant, reduction in NFκB activation at 1 day when cultures were exposed to the ERK1/2 inhibitor UO126 (10 μM). However, a significant inhibition in NFκB activation was observed when cultures were co-treated with inhibitors of p38-MAPK, SB239063 (10 μM) and JNK, SP600125 (1 μM). B. Quantification of ammonia-induced NFκB translocation after treatment with MAPK inhibitors. NFκB content was normalized against lamin. ANOVA, n=4. *p<0.05 vs. control; †p<0.05 vs. NH₄Cl. SB, SB239063; UO, UO126; SP, SP600125.

Figure 6

NFκB inhibitors prevent ammonia-induced astrocyte swelling. Cultured astrocytes exposed to 5 mM NH₄Cl significantly increased cell swelling (47.19% by 24 h). Co-treatment with BAY 11-7082 (5 μM) and SN50 (3.5 μM) significantly diminished the extent of astrocyte swelling. ANOVA, n=7. *p<0.05 vs. control; † p<0.05 vs. NH₄Cl.

Figure 7

NFκB inhibitors prevent ammonia-induced iNOS protein expression and NO generation. A. Cultured astrocytes exposed to 5 mM NH₄Cl for 24 h increased iNOS protein expression and co-treatment with BAY 11-7082 (5 μM) significantly diminished this effect. B. Quantification of ammonia-induced iNOS protein expression. C. Cultures exposed to 5 mM NH₄Cl for 24 h increased nitric oxide generation, and such effect was significantly diminished by co-treatment with BAY 11-7082 (5 μM). iNOS expression was normalized against α-tubulin ANOVA, n=5. *p<0.05 vs. control; † p<0.05 vs. NH₄Cl.

Figure 8

Inhibition of iNOS prevents ammonia-induced astrocyte swelling. Cultured astrocytes exposed to 5 mM NH₄Cl significantly increased cell swelling (40.2% by 24 h). Co-treatment with the iNOS inhibitor aminoguanidine (AGN) blocked such swelling. ANOVA, n=3. *p<0.05 vs. control; † p<0.05 vs. NH₄Cl.