Role of cGMP‐dependent protein kinase in development of tolerance to nitroglycerine in porcine coronary arteries

Background and purpose: The cGMP‐dependent protein kinase (PKG) is a key enzyme for nitrovasodilator‐induced vasodilation. The present study was to determine its role in nitrate tolerance. Experimental approach: isolated porcine coronary arteries were incubated for 24 h with nitroglycerin (NTG) and their relaxant responses were determined. PKG activity was assayed by measuring the incorporation of 32 P into BPDEtide. PKG protein was determined by Western blotting and PKG mRNA by real‐time PCR. Key results: A 24 h incubation with NTG attenuated relaxation of coronary arteries to NTG, which was associated with decreased PKG activity. The nitrate tolerance induced with NTG at 10 −7  M was affected by a scavenger of reactive oxygen species and the tolerance induced with NTG at 10 −6 and 10 −5  M showed cross‐tolerance to DETA NONOate and 8‐Br‐cGMP (a cell permeable cGMP analogue). PKG protein and mRNA were down‐regulated by a 24 h incubation with NTG at 10 −5  M but not at 10 −7  M. Acute exposure to exogenous superoxide inhibited PKG activity stimulated by NTG at 10 −7  M but not at 10 −5  M. Superoxide had no effect on PKG activity stimulated with exogenous cGMP. Conclusions and implications: Nitrate tolerance induced by NTG at low concentrations may result from an increased production of reactive oxygen species acting on sites upstream of PKG. The tolerance induced by NTG at higher concentrations may be in part due to suppression of PKG expression resulting from sustained activation of the enzyme. These distinct mechanisms of nitrate tolerance may be of clinical significance. British Journal of Pharmacology (2008) 153 , 497–507; doi: 10.1038/sj.bjp.0707600 ; published online 26 November 2007