Nitric oxide decreases IGF-1 receptor function in vitro; glutathione depletion enhances this effect in vivo.
Insulin-like growth factor-1 (IGF) helps maintain healthy articular cartilage; however, arthritic cartilage becomes less responsive to the anabolic actions of IGF. We previously showed that high concentrations of nitric oxide (NO) decrease IGF receptor tyrosine phosphorylation and response to IGF in intact chondrocytes. The current studies evaluate direct effects of NO on IGF receptor kinase (IGF-RK) in vitro. NO from S-nitroso-N-acetyl-d,l-penicillamine (SNAP) or 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) inhibits IGF-RK auto- and substrate phosphorylation in a dose and time dependent manner. There is a linear correlation between inhibition of auto- and substrate phosphorylation (r(2)=0.98). Increasing either dithiothreitol or reduced glutathione (GSH) content of the phosphorylation buffer to protect thiol groups blocks NO inhibition of IGF-RK substrate phosphorylation. Increased S-nitrosylation of cysteines in IGF-RK after exposure to SNAP suggests that NO may react with sulfhydryl groups, form S-nitrosothiols, which may result in functional modifications. NO blockade of IGF-1 stimulated proteoglycan synthesis in intact cells is enhanced when chondrocyte glutathione is depleted. The in vitro system shows that there can be direct effects of NO on IGF-RK that modify receptor function; the intact cell studies suggest that the mechanisms identified in vitro may be important in intact chondrocyte insensitivity to IGF-1 in cells exposed to NO.
Studer
R K
RK
VA Pittsburgh Healthcare System, University of Pittsburgh School of Medicine, Department of Orthopaedic Surgery, Pittsburgh, PA 15240, USA. rstuder@pitt.edu
D000818:Animals; D001168:Arthritis / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes; D004229:Dithiothreitol / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors; D010766:Phosphorylation; D011817:Rabbits; D017526:Receptor, IGF Type 1 / Q000187:drug effects* / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D026403:S-Nitrosothiols / Q000378:metabolism
C115239:3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine; D016166:Free Radical Scavengers; D006834:Hydrazines; D020030:Nitric Oxide Donors; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D026423:S-Nitroso-N-Acetylpenicillamine; D017526:Receptor, IGF Type 1; D005978:Glutathione; D004229:Dithiothreitol