Nitric oxide supports carbon monoxide-induced protection from apoptosis in isolated primary mouse hepatocytes

Abstract

Background and Aims: Heme oxygenase 1 (HO–1) induction or over-expression has been shown to interfere with acute and chronic hepatitis in mice. On the other hand, HO–1 is over-expressed in tumors and there interferes with apoptosis-induction via its product carbon monoxide (CO), inducing cytoprotective mechanisms in tumor cells. Our aim is to identify signaling pathways and mediators of CO-induced cytoprotection as specific targets for tumor therapy. Methods: Freshly isolated primary mouse hepatocytes (PH) were incubated in the presence of the CO donor methylene chloride (MC) for 2 to 6 hours. Nitric oxide (NO) was provided by incubation with the NO donor SNAP up to 20 hours. Cellular damage was induced by actinomycin (ActD)/TNF incubation. Cytotoxicity was measured by LDH release assay. Wild type, HO–1- and iNOS-knockout mice were used. Changes in protein expression and distribution due to MC incubation were determined by 2D-PAGE and mass spectrometry. Results: Evaluation of mass spectrometry identified the NO pathway as a target for CO-induced cytoprotection. Incubation with CO (2–6 hours) or NO (>20 hours) dose-dependently reduced ActD/TNF-induced cytotoxicity in wild type hepatocytes. CO also protected iNOSko- or HO–1ko-hepatocytes, while NO did not protect HO–1ko-hepatocytes. Inhibition of soluble guanylate cyclase (sGC) of cGMP-dependent protein kinase (PKG) partly abolished the protective effects of CO. Conclusions: Although CO induces expression of NO-pathway components, and CO as well as NO protected PH from induced cellular damage, CO was able to protect independently of NO production. Protection by NO, but not CO, was found to dependent on HO–1 induction and therefore represents a feedback loop supporting CO-induced protection. Mediators of CO-induced protection are sGC and PKG. Inhibitors of downstream molecules of CO signaling might be useful targets for anti-tumor therapy.