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- Title
Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells.
- Authors
Ingram, Alistair J.; James, Leighton; Ly, Hao; Thai, Kerri; Cai, Lu; Scholey, James W.
- Abstract
Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells. Background. In vivo, intraglomerular hypertension results in resident cell hypertrophy, proliferation and matrix protein production, leading to glomerulosclerosis. Mesangial cells (MCs) exposed to in vitro stretch also proliferate and produce matrix. We have shown activation of Jun N-terminal kinase/stress-activated protein kinase (SAPK) and p42/44 mitogen-activated protein kinase (MAPK) in stretched MCs and have also demonstrated that l-arginine decreases resident cell proliferation and protects against glomerulosclerosis in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/44 MAPK activation in stretched MCs. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 0 to 30 minutes of cyclic strain (60 cycles per minute) by computer-driven generation of vacuum of -27 kPa, inducing 28% elongation in the diameter of the surface. Control MCs were grown on coated, flexible-bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK and p42/44 MAPK were performed under these conditions. As maximal activation was at 10 minutes, with decay by 30 minutes, the effect of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by preincubation with 70 μmol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO donor) or 1 mmol/L 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Downstream events in response to stretch and NO were studied at the time of maximal response (10 minutes) by examining nuclear translocation of SAPK with immunofluorescence microscopy and transcription factor activator protein-1 nuclear protein binding by gel mobility shift assay. The effect of kinase inhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). Results....
- Subjects
NITRIC oxide; PROTEIN kinases; ARGININE; IMMUNOFLUORESCENCE
- Publication
Kidney International, 2000, Vol 58, Issue 3, p1067
- ISSN
0085-2538
- Publication type
Article
- DOI
10.1046/j.1523-1755.2000.00264.x