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. 2008 Feb;153(3):497-507.
doi: 10.1038/sj.bjp.0707600. Epub 2007 Nov 26.

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

D Dou  1 , X Zheng, X Qin, H Qi, L Liu, J U Raj, Y Gao
Affiliations

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

D Dou et al. Br J Pharmacol. 2008 Feb.

Abstract

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.

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Figures

Figure 1
Figure 1
Relaxations of porcine coronary arteries to NTG (left panel) and DETA NONOate (right panel) following 24h incubation with NTG. Vessels were constricted with U46619 (10−7 to 3 × 10−7 M) before testing their relaxant responses. Data are shown as means±s.e.mean; n=7–10 for each group. *Significantly different from vessels incubated with NTG from 10−7 to 10−5 M (left panel) or with NTG at 10−6 and 10−5 M (right panel) (P<0.05); †significantly different from vessels incubated with NTG at 10−7 and 10−6 M (P<0.05). DETA NONOate, [2,2′-(hydroxynitrosohydrazono) bis(ethanamine)]; NTG, nitroglycerine; U46619, (9,11)-dideoxy-(11α,9α)-epoxymethanoprostaglandin F2α.
Figure 2
Figure 2
Relaxations of porcine coronary arteries to NTG (left panel) and DETA NONOate (right panel) following 24 h incubation with DETA NONOate at 10−5 M. Vessels were constricted with U46619 (10−7 to 3 × 10−7 M) before testing their relaxant responses. Data are shown as means±s.e.mean; n=6 for each group. *Significant difference between vessels incubated with solvent and those with DETA NONOate at 10−5 M (P<0.05). DETA NONOate, [2,2′-(hydroxynitrosohydrazono) bis(ethanamine)]; NTG, nitroglycerine; U46619, (9,11)-dideoxy-(11α,9α)-epoxymethanoprostaglandin F2α.
Figure 3
Figure 3
Relaxations of porcine coronary arteries to NTG (left panel) and DETA NONOate (right panel) following 24 h incubation with the respective nitrovasodilators plus NAC (10−2 M). Vessels were constricted with U46619 (10−7 to 3 × 10−7 M) before testing their relaxant responses. Data are shown as means±s.e.mean; n=6–8 for each group. *Significant difference between vessels incubated with NAC and those with NTG at 10−6 M plus NAC (left panel) or between vessels incubated with NAC and DETA NONOate at 10−5 M plus NAC (right panel) (P<0.05); †significantly different from vessels incubated with NTG at 10−6 M plus NAC (left panel) (P<0.05). DETA NONOate, [2,2′-(hydroxynitrosohydrazono) bis(ethanamine)]; NTG, nitroglycerine; NAC, N-acetyl-L-cysteine; U46619, (9,11)-dideoxy-(11α,9α)-epoxymethanoprostaglandin F2α.
Figure 4
Figure 4
Relaxations of porcine coronary arteries to 8-Br-cGMP following 24 h incubation with NTG (left panel) or DETA NONOate (right panel). Vessels were constricted with U46619 (10−7 to 3 × 10−7 M) before testing their relaxant responses. Data are shown as means±s.e.mean; n=6–9 for each group. *Significant difference between vessels incubated with solvent and those with NTG at 10−5 M (left panel) or between vessels incubated with solvent and those with DETA NONOate at 10−5 M (right panel) (P<0.05). 8-Br-cGMP, 8-bromo-guanosine 3′5′-cyclic monophosphate; DETA NONOate, [2,2′-(hydroxynitrosohydrazono) bis(ethanamine)]; NTG, nitroglycerine.
Figure 5
Figure 5
PKG activity of porcine coronary arteries after 24 h incubation with NTG (10−5 M; left panel) or DETA NONOate (DETA NO, 10−5 M; right panel). (−) cGMP, without cGMP; (+) cGMP, cGMP at 3 × 10−6 M. Data are shown as means±s.e.; n=6 for each group. *Significantly different from those without cGMP (P<0.05); †significantly different from the control group (P<0.05). cGMP, cyclic GMP; DETA NONOate, [2,2′-(hydroxynitrosohydrazono) bis(ethanamine)]; NTG, nitroglycerine; PKG, cGMP-dependent protein kinase.
Figure 6
Figure 6
PKG activity of porcine coronary arteries activated with NTG (control, 10−7 or 10−5 M; left panel) or cGMP (control, 10−7 or 3 × 10−7 M; right panel). XO (10 mU ml−1) and pterine (10−4 M). Data are shown as means±s.e.; n=6–10 for each group. *Significantly different from those the control group (P<0.05). cGMP, cyclic GMP; NTG, nitroglycerine; PKG, cGMP-dependent protein kinase; XO, xanthine oxidase.
Figure 7
Figure 7
PKG protein of porcine coronary arteries following 24 h incubation with NTG (a, b), DETA NO (c) or 8-Br-cGMP or 8-bromo-adenosine 3′5′-cyclic monophosphate (d). The upper panels are western blots of PKG. The lower panels are the densitometric scanning of PKG protein normalized to actin. NTG, 10−5 M unless otherwise stated; DETA NO,10−5 M; 8-Br-cGMP or 8- 8-bromo-adenosine 3′5′-cyclic monophosphate, 10−5 M; ODQ, 3 × 10−5 M; PKG-I, (Rp-8-Br-PET-cGMPS), 3 × 10−5 M. Data shown as means±s.e.mean; n=4–6 for each group. *Significantly different from the control group; †significantly different from vessels incubated with NTG (b) or incubated with DETA NO (c) (P<0.05). 8-Br-cGMP, 8-bromo-guanosine 3′5′-cyclic monophosphate; DETA NONOate, [2,2′-(hydroxynitrosohydrazono) bis(ethanamine)]; NTG, nitroglycerine; ODQ, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; PKG, cGMP-dependent protein kinase.
Figure 8
Figure 8
Real-time PCR of mRNA fragments of PKG Iα and Iβ of porcine coronary arteries incubated for 24 h with NTG (10−5 M) or DETA NONOate (DETA NO, 10−5 M (left panel) and with 8-Br-cGMP (10−4 M) (right panel). The densitometric scanning of mRNA fragments of PKG is normalized to β-actin. PKG-I (Rp-8-Br-PET-cGMPS), 3 × 10−5 M. Data shown as means±s.e.mean; n=4–8 for each group. *Significantly different from vessels incubated with solvent (P<0.05); †significantly different from vessels not treated with PKG-I (P<0.05). 8-Br-cGMP, 8-bromo-guanosine 3′5′-cyclic monophosphate; DETA NONOate, [2,2′-(hydroxynitrosohydrazono) bis(ethanamine)]; NTG, nitroglycerin; ODQ, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; PCR, polymerase chain reaction; PKG, cGMP-dependent protein kinase.
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References

    1. Abou-Mohamed G, Kaesemeyer WH, Caldwell RB, Caldwell RW. Role of L-arginine in the vascular actions and development of tolerance to nitroglycerin. Br J Pharmacol. 2000;130:211–218. - PMC - PubMed
    1. Anderson PG, Boerth NJ, Liu M, McNama DB, Cornwell TL, Lincoln TM. Cyclic CMP-dependent protein kinase expression in coronary arterial smooth muscle in response to balloon catheter injury. Arterioscler Thromb Vasc Biol. 2000;20:2192–2197. - PubMed
    1. Atlante A, Valenti D, Gagliardi S, Passarella S. A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells. Brain Res Brain Res Protoc. 2000;6:1–5. - PubMed
    1. Browner NC, Sellak H, Lincoln TM. Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells. Am J Physiol Cell Physiol. 2004;287:C88–C96. - PubMed
    1. Butt E, Pohler D, Genieser HG, Huggins JP, Bucher B. Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS. Br J Pharmacol. 1995;116:3110–3116. - PMC - PubMed

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