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. 2007 Dec;152(7):1060-9.
doi: 10.1038/sj.bjp.0707479. Epub 2007 Sep 24.

Protein kinase G regulates the basal tension and plays a major role in nitrovasodilator-induced relaxation of porcine coronary veins

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

Protein kinase G regulates the basal tension and plays a major role in nitrovasodilator-induced relaxation of porcine coronary veins

H Qi et al. Br J Pharmacol. 2007 Dec.

Abstract

Background and purpose: Coronary venous activity is modulated by endogenous and exogenous nitrovasodilators. The present study was to determine the role of protein kinase G (PKG) in the regulation of the basal tension and nitrovasodilator-induced relaxation of coronary veins.

Experimental approach: Effects of a PKG inhibitor on the basal tension and responses induced by nitroglycerin, DETA NONOate, and 8-Br-cGMP in isolated porcine coronary veins were determined. Cyclic cGMP was measured with radioimmunoassay. PKG activity was determined by measuring the incorporation of 32P from gamma-32P-ATP into the specific substrate BPDEtide.

Key results: Rp-8-Br-PET-cGMPS, a specific PKG inhibitor, increased the basal tension of porcine coronary veins and decreased PKG activity. The increase in tension was 38% of that caused by nitro-L-arginine. Relaxation of the veins induced by nitroglycerin and DETA NONOate was accompanied with increases in cGMP content and PKG activity. These effects were largely eliminated by inhibiting soluble guanylyl cyclase with ODQ. The increase in PKG activity induced by the nitrovasodilators was abolished by Rp-8-Br-PET-cGMPS. The relaxation caused by these dilators and by 8-Br-cGMP at their EC50 was attenuated by the PKG inhibitor by 51-66%.

Conclusions and implications: These results suggest that PKG is critically involved in nitric oxide-mediated regulation of the basal tension in porcine coronary veins and that it plays a primary role in relaxation induced by nitrovasodilators. Since nitric oxide plays a key role in modulating coronary venous activity, augmentation of PKG may be a therapeutic target for improving coronary blood flow.

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Figures

Figure 1
Figure 1
Upper panel: original traces of the effects of Rp-8-Br-PET-cGMPS (3 × 10−5 M) and nitro-L-arginine (10−4 M) on the basal tension of porcine coronary veins. Lower panel: concentration-dependent response of porcine coronary veins to Rp-8-Br-PET-cGMPS in the presence or absence of nitro-L-arginine (NLA, 10−4 M). Data are shown as means±s.e. mean; n=4–9 for each group. *Significantly different from vessels treated with NLA (P<0.05). Rp-8-Br-PET-cGMPS, β-phenyl-1, N2-etheno-8-bromoguanosine-3′5′-cyclic monophosphorothioate, Rp-isomer.
Figure 2
Figure 2
Relaxation of porcine coronary veins to nitroglycerin and DETA NONOate. Vessels were preconstricted to the same tension with U46619 (Table 2). Data are shown as means±s.e. mean; n=4–10 for each group. *Significant difference between control and those treated with Rp-8-Br-PET-cGMPS (3 × 10−5 M); †significant difference between control and those treated with ODQ (3 × 10−5 M) (P<0.05). Rp-8-Br-PET-cGMPS, β-phenyl-1, N2-etheno-8-bromoguanosine-3′5′-cyclic monophosphorothioate, Rp-isomer; ODQ, 1H-[1,24]oxadiazolo[4,3-a]quinoxalin-1-one.
Figure 3
Figure 3
Relaxation of porcine coronary veins to 8-Br-cGMP and 8-Br-cAMP. Vessels were preconstricted with U46619 (Table 2). Data are shown as means±s.e. mean; n=4–13 for each group. *Significant difference between control and treated with Rp-8-Br-PET-cGMPS (3 × 10−5 M) (left panel) and treated with myristoylated PKI (6 × 10−6 M) (right panel) (P<0.05). 8-Br-cAMP, 8-bromo-adenosine 3′,5′-cyclic monophosphate; 8-Br-cGMP, 8-bromo-guanosine 3′,5′-cyclic monophosphate; Rp-8-Br-PET-cGMPS, β-phenyl-1, N2-etheno-8-bromoguanosine-3′5′-cyclic monophosphorothioate, Rp-isomer.
Figure 4
Figure 4
Upper panel: the intracellular content of cGMP of porcine coronary veins with and without endothelium under basal conditions, treated with nitro-L-arginine (NLA, 10−4 M) or ODQ (3 × 10−5 M). Data are shown as means±s.e. mean; n=4–9 for each group. *Significantly different from the basal value of vessels with endothelium (P<0.05). Lower panel: the intracellular content of cGMP of porcine coronary veins without endothelium stimulated with nitroglycerin (3 × 10−5 M) or DETA NONOate (3 × 10−5 M) in the absence or presence of ODQ (3 × 10−5 M). Data are shown as means±s.e. mean; n=6–10 for each group. *Significantly different from the basal value (P<0.05). ODQ, 1H-[1,24]oxadiazolo[4,3-a]quinoxalin-1-one.
Figure 5
Figure 5
Effects of nitro-L-arginine (NLA, 10−4 M), ODQ (3 × 10−5 M), Rp-8-Br-PET-cGMPS (3 × 10−5 M) and myristoylated PKI (6 × 10−6 M) on the activity of PKG and PKA of porcine coronary veins with endothelium under basal conditions. Data are shown as means±s.e. mean; n=10–25 for each group. *Significantly different from control (P<0.05). Rp-8-Br-PET-cGMPS, β-phenyl-1, N2-etheno-8-bromoguanosine-3′5′-cyclic monophosphorothioate, Rp-isomer; ODQ, 1H-[1,24]oxadiazolo[4,3-a]quinoxalin-1-one.
Figure 6
Figure 6
Effects of nitroglycerin (3 × 10−5 M) and DETA NONOate (3 × 10−5 M) on the activity of PKG and PKA of porcine coronary veins without endothelium in the absence or presence of Rp-8-Br-PET-cGMPS (3 × 10−5 M). Data are shown as means±s.e. mean; n=7–8 for each group. *Significantly different from basal (P<0.05). Rp-8-Br-PET-cGMPS, β-phenyl-1, N2-etheno-8-bromoguanosine-3′5′-cyclic monophosphorothioate.
Figure 7
Figure 7
Left panel: PKG activity of the homogenates of porcine coronary veins using different concentrations of protein. Right panel: the concentration-dependent effects of cGMP and cAMP on PKG activity of the homogenates of porcine coronary veins. The inset shows the effects of cAMP and cGMP on PKA activity. −cGMP and −cAMP, without cGMP and cAMP, respectively; +cGMP and +cAMP, in the presence of cGMP and cAMP at 3.16 μM, respectively. Data are shown as means±s.e. mean; n=4 for each group.
Figure 8
Figure 8
Effects of Rp-8-Br-PET-cGMPS (3 × 10−5 M) and myristoylated PKI (6 × 10−6 M) on PKG and PKA activity of the homogenate preparations of porcine coronary veins. −cGMP and −cAMP, without cGMP and cAMP, respectively; +cGMP and +cAMP, in the presence of cGMP and cAMP at 3.16 μM, respectively. Data are shown as means±s.e. mean; n=4 for each group. *Significantly different from activity without cGMP or cAMP (P<0.05). †Significantly different from control (P<0.05). Rp-8-Br-PET-cGMPS, β-phenyl-1, N2-etheno-8-bromoguanosine-3′5′-cyclic monophosphorothioate.
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