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 glomeru losclerosis. Mesangial cells (MCs) exposed to in vitro stretch also prolife rate 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 glomeruloscleros is in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/4 4 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 coat ed, flexible-bottom plates. Protein levels (by Western blot) and activity a ssays for SAPK/JNK and p42/44 MAPK were performed under these conditions. A s maximal activation was at 10 minutes, with decay by 30 minutes, the effec t of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by prei ncubation with 70 mu mol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO don or) or 1 mmol/L X-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Down stream events in response to stretch and NO were studied at the time of max imal 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 i nhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). Results. Cyclic MC stretch led to prompt SAPK and p42/44 MAPK activation, w hich was maximal at 10 minutes. Preincubation with either SNAP or 8-bromo-c GMP decreased this by 50 and 70%, respectively (N = 4), suggesting that the effect of NO was through cGMP generation. Nuclear translocation of both ph osphorylated kinases was seen after 10 minutes of stretch and was largely p revented by 8-bromo-cGMP, Increased DNA binding of activator protein-1 prot eins was observed in the nuclei of stretched MCs at 10 minutes by mobility shift assay (N = 4), which was also largely prevented by X-bromo-cGMP. Stre tch increased PCNA expression by MCs, and this was inhibited by 8-bromo-cGM P. Conclusions. Stretch-induced activation of SAPK and p42/44 MAPK in MCs can be inhibited by NO. The effect of NO is mediated by the generation of cGMP. These mechanisms may be responsible, at least in part, for the protective effect of NO in animal models of glomerular injury characterized by glomeru lar capillary hypertension.