Molecular mechanism of cGMP-mediated smooth muscle relaxation

Carvajal, Jorge; Germaín, A; Huidobro‐Toro, J. Pablo; Weiner, Carl P.

doi:10.1002/1097-4652(200009)184:3<409::aid-jcp16>3.0.co;2-k

Cited by 339 publications

(148 citation statements)

References 137 publications

(191 reference statements)

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“…In the present study we have used mass spectrometry to study structural and biochemical aspects of the cGMP-dependent protein kinase (PKG) I␣. PKG is involved in the nitric oxide/cGMPsignaling pathway, serves as a major receptor protein for intracellular cGMP and controls a variety of cellular responses, including smooth muscle relaxation [5,6]. PKG is the closest homolog of PKA and in particular the catalytic domains of the two enzymes show a remarkable high sequence homology [7].…”

mentioning

confidence: 99%

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Pinkse

Heck

Rumpel

et al. 2004

J. Am. Soc. Mass Spectrom.

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Nanoflow electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) was used to study activation properties of the cGMP-dependent protein kinase (PKG). Our nanoflow ESI-TOF-MS analysis confirms that PKG mainly occurs as a 153 kDa homodimer and is able to bind four cGMP molecules, which is in agreement with the known stoichiometry. Binding order and stoichiometry of cGMP, the non-hydrolysable ATP analog ␤,␥-imidoadenosine 5=-triphosphate (AMPPNP) and Mn 2ϩ for PKG were characterized as model for the active PKG-cGMP-ATP/Mg 2ϩ complex. Already in the absence of cGMP, a noncovalent complex between PKG and two molecules of AMPPNP could be observed by ESI-TOF-MS. Binding of AMPPNP to PKG was strongly enhanced by the addition of MnCl 2 to the spray solution. This is in agreement with binding of AMPPNP/Mn 2ϩ in the ATP binding pocket of PKG since all protein kinases require a metal ion to accompany ATP in the ATP-binding pocket for proper positioning of the ␤ and ␥ phosphates. Additionally, this finding could imply that within the inactive conformation of PKG, the autoinhibition-domain, when in contact with the substratedocking domain, does not block the entrance to the ATP-binding site. In the presence of cGMP, less of the fully saturated PKG-(cGMP) 4 (AMPPNP/Mn 2ϩ ) 2 complex was observed, suggesting that the PKG-ATP interaction is weakened in the active conformation of PKG. Additionally, limited proteolysis in combination with native-ESI MS showed to be a useful tool to study the contact regions on the PKG-dimer and also allowed the rapid determination of the overall autophosphorylation status of the protein. These measurements indicated that autophosphorylation mainly occurs within the first 80 aminoterminal residues and involves in total 3-4 phosphates per subunit. (J Am Soc Mass Spectrom 2004, 15, 1392-1399

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confidence: 99%

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Pinkse

Heck

Rumpel

et al. 2004

J. Am. Soc. Mass Spectrom.

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“…NO leads to pulmonary vascular smooth muscle (VSM) relaxation by stimulating soluble guanylyl cyclase (sGC) (7). The subsequent elevation in cGMP activates protein kinase G-1 (PKG-1), causing relaxation through a variety of mechanisms involving decreased VSM intracellular calcium and desensitization of the contractile apparatus to calcium (24).…”

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confidence: 99%

Pulmonary PKG-1 is upregulated following chronic hypoxia

Jernigan

Walker

Resta

2003

American Journal of Physiology-Lung Cellular and Molecular Phys

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. Pulmonary PKG-1 is upregulated following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 285: L634-L642, 2003. First published May 23, 2003 10.1152/ ajplung.00328.2002.-Recent studies from our laboratory indicate that pulmonary vasodilatory responses to exogenous nitric oxide (NO) are attenuated following chronic hypoxia (CH) and that this NO-dependent vasodilation is mediated by cGMP. Similarly, we have demonstrated that CH attenuates vasodilatory responses to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). We hypothesized that attenuated pulmonary vasodilation to 8-BrcGMP following CH is mediated by decreased protein kinase G-1 (PKG-1) expression/activity. Therefore, we examined vasodilatory responses to 8-BrcGMP (1 M) in isolated, saline-perfused lungs from control and CH (4 wk at barometric pressure of 380 mmHg) rats in the presence of the competitive PKG inhibitor Rp-␤-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (30 M) or the highly specific PKG inhibitor KT-5823 (10 M). PKG-1 expression and activity were determined in whole lung homogenates from each group, and vascular PKG-1 levels were assessed by quantitative immunohistochemistry. PKG inhibition with either Rp-8-Br-PET-cGMPS or KT-5823 diminished vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats, thus indicating a role for PKG in mediating reactivity to 8-BrcGMP in each group. However, in contrast to our hypothesis, PKG-1 levels were approximately twofold greater in lungs from CH rats vs. controls, and furthermore, this upregulation was localized to the vasculature. This correlates with an increase in PKG activity following CH. We conclude that PKG-1 is involved in 8-BrcGMPmediated vasodilation; however, attenuated pulmonary vasodilation following CH is not associated with decreased expression/activity of PKG-1. isolated rat lung; nitric oxide; pulmonary hypertension; 8-bromoguanosine 3',5'-cyclic monophosphate; KT-5823; Rp-␤-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothioate CHRONIC EXPOSURE TO HYPOXIA has been shown to augment pulmonary vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent vasodilators (18, 26, 28, 32-34, 36, 37). EDNO is synthesized in the vasculature by endothelial nitric oxide synthase (eNOS), and several studies have demonstrated that chronic hypoxia (CH) is associated with increased pulmonary eNOS levels, gene expression, and activity (18,21,26,33,34,39,46). Consistent with elevated eNOS levels, nitric oxide (NO) synthesis appears to be greater in lungs isolated from CH rats compared with controls (18,26,44). In contrast to enhanced reactivity to our laboratory (19,34) and others (35) have demonstrated impaired responsiveness to exogenous NO following CH. However, the mechanism(s) responsible for this diminished reactivity is not fully understood.NO leads to pulmonary vascular smooth muscle (VSM) relaxation by stimulating soluble guanylyl cyclase (sGC) (7). The subsequent elevation in cGMP activates prot...

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“…On the one hand PKG could infl uence directly the contractile machinery of smooth muscles 14 , on the other hand it could block L-type Ca 2+ channels (in terms of Ca 2+ infl ux reduction, necessary for muscle contraction) 15 and on third hand PKG could activate calcium pumps whish also leads to reduction of intracellular calcium. The overall processes cumulation leads to 5-HT 1B and α 1D activation and subsequent smooth muscle relaxation at a concentration of 5×10 -5 mol/l of frovatriptan.…”

Section: Effects Of 5×10 -5 Mol/l Frovatriptan On the Con-tractile Acmentioning

confidence: 99%

Effects of the Novel High-affinity 5-HT(1B/1D)-receptor Ligand Frovatriptan on the Rat Carotid Artery

et al. 2017

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Citation: Saracheva KE, Prissadova NA, Turiiski VI, Slavchev VI, Krastev AD, Getova DP. Effects of the novel high-affi nity 5-HT(1B/1D)-receptor ligand frovatriptan on the rat carotid artery.Folia Medica 2017;59(1): 31-36. doi: 10.1515/folmed-2017-0006 Background: In blood vessels 5-HT stimulates sympathetic nerves, the endothelium and vascular smooth muscle cells. Triptans are specifi c anti-migraine drugs and they activate the serotoninergic 5HT1b/d receptors causing vasoconstriction of the cerebral vessels. Aim: To evaluate the eff ect of frovatriptan on isolated rat carotid artery. Methods: Contractile activity of the preparations was registered isometrically. Krebs solution (pH = 7.4) was used for washing smooth muscle (SM) preparations aerated with 95% O 2 and 5% CO 2 at 37°C. The 60-minute adaptation of tone level of preparations was taken as a starting tone and the changes such as contraction or relaxation were calculated using it. Results: Frovatriptan (1×10 -6 mol/l -1×10 -5 mol/l) induced a contraction, but at higher concentrations it caused relaxation of the carotid artery. The L-norepinephrine contractile reaction was enhanced in the presence of frovatriptan. In the presence of 5-HT2 receptor antagonist, methysergide, frovatriptan increased the relaxation. In the presence of the specifi c α-1 receptor antagonist, prazosin, the frovatriptan-induced relaxation decreased. Conclusion:The observed contractile eff ect of frovatriptan is probably associated with the main eff ect of the drug -activation of the serotoninergic 5HT 1B /1D receptors causing vasoconstriction of the cerebral vessels and their anti-migraine eff ect. At higher concentrations, frovatriptan, most likely via some non-specifi c mechanism, could activate the following intracellular chain reaction: stimulation of α 1D could activate eNOS which may increase in the concentration of NO which results in the fi nal eff ect of relaxation. BACKGROUND

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Molecular mechanism of cGMP-mediated smooth muscle relaxation

Cited by 339 publications

References 137 publications

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Pulmonary PKG-1 is upregulated following chronic hypoxia

Effects of the Novel High-affinity 5-HT(1B/1D)-receptor Ligand Frovatriptan on the Rat Carotid Artery

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