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cGMP-mediated antioxidant signaling: a role for the c-Abl tyrosine kinase
BMC Pharmacology volume 11, Article number: P70 (2011)
Oxidant injury to the pulmonary endothelium contributes to acute lung injury. We have shown that activation of PKGI by cGMP increases protein levels of the antioxidant enzymes catalase and glutathione peroxidase-1 (GPx-1) and ameliorates oxidant injury in mouse lung endothelium . Catalase and GPx-1 mRNA was not increased. The pathway downstream of PKGI that leads to increases in catalase and GPx-1 is unknown. The c-Abl tyrosine kinase has been reported to regulate catalase and GPx-1; fibroblasts deficient in c-Abl have increased levels of catalase and resist oxidant injury [2–4]. We hypothesized that 1) activation of PKGI would decrease c-Abl protein levels; and 2) inhibition of c-Abl with imatinib would increase antioxidant proteins and hydrogen peroxide (H2O2) degradation, attenuate H2O2-induced endothelial permeability, and decrease H2O2-induced cell death in mouse lung microvascular endothelial cells (MLVMEC).
MLMVEC were isolated from wild-type (wt) and PKGI knock-out (PKGI -/-) C57Bl/6 mice. MLMVEC were treated for 4 hours with 8pCPT-cGMP (50 μM) or imatinib. H2O2 scavenging was measured with a H2O2 electrode after addition of known concentrations of H2O2 to cells in suspension. Nuclear condensation was assessed using fluorescence microscopy. Transendothelial resistance (TER) was measured using electric cell-substrate impedance sensing (ECIS). Proteins were quantified by Western blotting.
Treatment of wt MLMVEC with 8pCPT-cGMP significantly decreased protein levels of c-Abl by 29% (p=0.02). 8pCPT-cGMP had no effect on cAbl in PKGI -/- MLMVEC. 7 days of sildenafil treatment (100mg/kg/day)  significantly decreased whole lung c-Abl protein by 37 % and increased whole lung catalase protein by 40% (p<0.05 for both). Treatment of wt MLMVEC with 10 and 20 µM imatinib increased catalase protein by 16% and 37% respectively, and GPx-1 protein levels by 24% and 36%, respectively (p<0.05 for all values). Imatinib (10 µM) decreased the measured peak H2O2 after addition of extracellular H2O2 (20, 50, and 100 µM) by 18%, 23%, and 9%, respectively (p ≤0.05 at all concentrations). Imatinib (10 µM) significantly attenuated 100 and 250 µM H2O2-induced nuclear condensation by 15% (p<0.05). Finally, imatinib (20 µM) attenuated the H2O2-induced TER decrease in monolayers of MLMVEC (p<0.05 by ANOVA).
These data suggest that cGMP, through PKGI, increases MLMVEC antioxidant activity by down regulating the cAbl tyrosine kinase. Inhibition of c-Abl with imatinib attenuates oxidant-induced MLMVEC death and dysfunction, mimicking the antioxidant effects of cGMP-PKGI signalling. The mechanism linking activated PKGI and cAbl expression is unknown.
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Stephens, R.S., Servinsky, L.E. & Pearse, D.B. cGMP-mediated antioxidant signaling: a role for the c-Abl tyrosine kinase. BMC Pharmacol 11, P70 (2011). https://doi.org/10.1186/1471-2210-11-S1-P70
- Oxidant Injury
- Nuclear Condensation
- Antioxidant Enzyme Catalase
- Catalase Protein