Abstract 227: Activity of Soluble Guanylyl Cyclase, the Nitric Oxide Receptor, is Modulated by Cysteines Redox

The enzyme soluble guanylyl cyclase (sGC) increases cellular level of the second messenger cGMP upon stimulation by nitric oxide (NO), whose role is exemplified in the cardiovascular system where it governs smooth muscle cell proliferation, vascular relaxation and platelet aggregation. Various pathologies like diabetes, hypertension and hypercholesterolemia are associated with enhanced oxidative stress with increased formation of reactive oxygen (ROS) and nitrogen species (RNS). We recently showed that cysteines beta1C243 and beta1C122 are S-nitrosylated in sGC leading to its desensitization. We have now identified by Mass Spectrometry, two additional cysteines (beta1C571 and alpha1C516) as potentially modified. Our hypothesis is that loss in vascular reactivity in oxidative pathologies is mediated, in part, by desensitization of sGC via thiols oxidation. We studied the effect of oxidative stress on sGC in primary vascular smooth muscle cells (VSMC) by directly exposing them to hydrogen peroxide (H2O2) in the absence or presence of a catalase inhibitor Amino-Triazole (AT). H2O2 induced inhibition of NO-stimulated sGC activity in VSMC and in COS 7 cells transiently transfected with sGC. Desensitization of sGC was further increased in VSMC treated with both H2O2 and AT. Next, we conducted mutational analysis of the 4 cysteines to assess their involvement in redox sensing. Replacement of C571 and C122 eliminated the oxidative-dependent desensitization observed in the wild-type. In addition, peroxynitrite, a strong oxidant, inhibited the NO-stimulated sGC activity in a purified system without oxidation of the heme. In conclusion, our results suggest that sGC activity is directly modulated by the redox state of the cells. Moreover, the identified 4 cysteines can be modified by both ROS and RNS suggesting a complex modulation. Ultimately, our goal is to determine whether loss of vascular reactivity in oxidative pathophysiologies is in part mediated by desensitization of sGC and what are the involved cysteines. This work was supported by NIH GM067640, HL089771.

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Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00138.2012 ◽

2012 ◽

Vol 303 (5) ◽

pp. H597-H604 ◽

Cited By ~ 30

Author(s):

Pierre-Antoine Crassous ◽

Samba Couloubaly ◽

Can Huang ◽

Zongmin Zhou ◽

Padmamalini Baskaran ◽

...

Keyword(s):

Oxidative Stress ◽

Smooth Muscle ◽

Angiotensin Ii ◽

Guanylyl Cyclase ◽

Vascular Reactivity ◽

Nitrosative Stress ◽

Vascular Dysfunction ◽

Soluble Guanylyl Cyclase ◽

Smooth Muscle Relaxation ◽

Ang Ii

Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.

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TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1995.269.2.f212 ◽

1995 ◽

Vol 269 (2) ◽

pp. F212-F217 ◽

Cited By ~ 3

Author(s):

K. S. Lau ◽

O. Nakashima ◽

G. R. Aalund ◽

L. Hogarth ◽

K. Ujiie ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Nitric Oxide Synthase ◽

Vascular Smooth Muscle ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Tnf Alpha ◽

No Production ◽

Ifn Gamma ◽

Oxide Synthase

Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription-polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN-gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC.

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Evaluation of the relaxant effect of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in isolated detrusor smooth muscle

European Journal of Pharmacology ◽

10.1016/j.ejphar.2010.04.008 ◽

2010 ◽

Vol 637 (1-3) ◽

pp. 171-177 ◽

Cited By ~ 16

Author(s):

Fernando R. Báu ◽

Fabiola Z.T. Mónica ◽

Fernanda B.M. Priviero ◽

Lineu Baldissera ◽

Gilberto de Nucci ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Detrusor Smooth Muscle ◽

Relaxant Effect

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Selective sequestration of nitric oxide by subcellular components of vascular smooth muscle and platelets: relationship to nitric oxide stimulation of the soluble guanylyl cyclase

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y93-142 ◽

1993 ◽

Vol 71 (12) ◽

pp. 938-945 ◽

Cited By ~ 8

Author(s):

Zhenguo Liu ◽

Kanji Nakatsu ◽

James F. Brien ◽

E. Danielle Beaton ◽

Gerald S. Marks ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Catalytic Activity ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Subcellular Fractions ◽

Cyclase Activity ◽

Cytosolic Fraction ◽

Headspace Gas ◽

Guanylyl Cyclase Activity

Sequestration of nitric oxide (NO) by subcellular fractions isolated from bovine pulmonary arterial medial layer (BPA) and rabbit platelets (RP) was studied utilizing a novel chemiluminescence – headspace gas technique. Sequestration in all fractions was similarly rapid (5 min) and remained constant for at least 30 min. When incubated with 108 pmol of NO, the BPA mitochondrial, microsomal, and nuclear fractions sequestered 22.8 ± 1.9, 20.5 ± 2.2 and 15.2 ± 3.6% of the NO, respectively (n = 14). However, significantly more of the 108 pmol of NO, 36.8 ± 2.8 and 32.9 ± 3.6%, respectively, was sequestered by the BPA homogenate (about 2 mg protein/mL) and BPA cytosolic fraction (about 1 mg protein/mL) (n = 19). Also, RP cytosolic fraction (about 3 mg protein/mL) sequestered a greater amount of NO than any BPA fraction when incubated with 108 pmol of NO (83.0 ± 1.0%; n = 3). Analysis of the binding data obtained for the BPA homogenate and cytosolic fraction was consistent with the existence of two binding sites, one site with a Kd of approximately 100 nM and another with a Kd of approximately 1 μM. Both the BPA homogenate fraction and the cytosolic fraction as well as the RP cytosolic fraction were shown to have soluble guanylyl cyclase activity. The nitrovasodilator sodium nitroprusside (SNP) caused a concentration-dependent increase in the activity of this enzyme in all these fractions. Maximum stimulations caused by 1 mM SNP in BPA homogenate fraction, BPA cytosolic fraction, and RP cytosolic fraction were equivalent to 2-, 4- and 3-fold increases in catalytic activity, respectively. No effect of SNP was observed in BPA mitochondrial, microsomal, or nuclear fraction. Prior incubation of BPA and RP cytosolic fractions with authentic NO significantly stimulated the soluble guanyiyl cyclase activity. In both the BPA and RP cytosolic fractions, maximal stimulation brought about by prior incubation with authentic NO was equivalent to approximately 60% of that caused by 100 μM SNP. Thus, incubation of subcellular fractions from two nitrovasodilator-sensitive tissues with authentic NO resulted in significant sequestration of the free radical in these fractions and a concentration-dependent activation of the soluble guanylyl cyclase. In conclusion, the chemiluminescence – headspace gas technique is a suitable method for the study of NO sequestration in subcellular fractions of various tissues. Also, this study demonstrates that NO is sequestered preferentially by subcellular fractions of BPA and RP that contain soluble guanylyl cyclase activity, and that the sequestration of NO in these fractions stimulates the catalytic activity of this enzyme.Key words: nitric oxide, vascular smooth muscle, platelets, soluble guanylyl cyclase.

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W1756 Association of Soluble Guanylyl Cyclase with 90-Kda Heat Shock Protein (Hsp90) Regulates sGC Expression and Activity and Nitric Oxide-Mediated Smooth Muscle Relaxation

Gastroenterology ◽

10.1016/s0016-5085(08)63311-9 ◽

2008 ◽

Vol 134 (4) ◽

pp. A-709

Author(s):

Karnam S. Murthy ◽

Sunila Mahavadi

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Heat Shock ◽

Heat Shock Protein ◽

Guanylyl Cyclase ◽

Muscle Relaxation ◽

Soluble Guanylyl Cyclase ◽

Smooth Muscle Relaxation ◽

Expression And Activity

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Nitric oxide-independent activation of soluble guanylyl cyclase contributes to endotoxin shock in rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.4.h1148 ◽

1998 ◽

Vol 275 (4) ◽

pp. H1148-H1157 ◽

Cited By ~ 1

Author(s):

Chin-Chen Wu ◽

Shiu-Jen Chen ◽

Mao-Hsiung Yen

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Guanylyl Cyclase ◽

Ex Vivo ◽

Specific Inhibitor ◽

Soluble Guanylyl Cyclase ◽

Treated Group ◽

Cgmp Level ◽

Vascular Hyporeactivity

We investigated whether a complete inhibition of nitric oxide (NO) formation caused by bacterial endotoxin (lipopolysaccharide, LPS) in vivo prevents the hypotension and restores the vascular hyporeactivity to normal in vivo and ex vivo. The combination of dexamethasone (Dex; 3 mg/kg at 30 min before LPS) plus aminoguanidine (AG; 15 mg/kg at 2 h after LPS) inhibited the overproduction of nitrate (an indicator of NO) in the plasma and aortic smooth muscle and also prevented the development of the delayed hypotension in rats treated with LPS for 6 h. However, the vascular hyporeactivity to norepinephrine (NE) was only partially improved either in vivo or ex vivo in endotoxemic rats treated with Dex plus AG. Pretreatment of aortic rings with N ω-nitro-l-arginine methyl ester (l-NAME) or 1 H-[1,2,4]oxidazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced the contraction to NE in rings obtained from LPS-treated rats, but not in those from Dex plus AG-treated endotoxemic rats. Methylene blue, an inhibitor of soluble guanylyl cyclase (GC), completely restored contractions to NE and aortic cGMP levels to normal either in LPS-treated rats or in Dex plus AG-treated endotoxemic rats, whereas the cGMP level was partially inhibited by ODQ in LPS-treated rats only. These results suggest that non-NO mediator(s) also activates soluble GC during endotoxemia. Interestingly, we found that in the presence of tetraethylammonium (an inhibitor of K+ channels) plusl-NAME or charybdotoxin [a specific inhibitor of large-conductance Ca2+-activated K+(KCa) channels] plus ODQ, the vascular hyporeactivity to NE in the LPS-treated group was also completely restored to normal. In addition, in the presence ofl-NAME or ODQ, the vascular hyporeactivity to high K+ was abolished in rings from the LPS-treated group. These results suggest that LPS causes the production of other mediator(s), in addition to NO, which also stimulates soluble GC (i.e., increases the formation of cGMP) and then activates the large-conductance KCa channels in the vascular smooth muscle causing vascular hyporeactivity.

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Loss of Expression of the β Subunit of Soluble Guanylyl Cyclase Prevents Nitric Oxide–Mediated Inhibition of DNA Synthesis in Smooth Muscle Cells of Old Rats

Circulation Research ◽

10.1161/01.res.86.5.520 ◽

2000 ◽

Vol 86 (5) ◽

pp. 520-525 ◽

Cited By ~ 25

Author(s):

Lihua Chen ◽

Günter Daum ◽

Jens W. Fischer ◽

Suzanne Hawkins ◽

Marie-Luce Bochaton-Piallat ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Dna Synthesis ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Muscle Cells ◽

Β Subunit ◽

Old Rats ◽

Inhibition Of Dna Synthesis

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Modulation of nitric oxide-stimulated soluble guanylyl cyclase activity by cytoskeleton-associated proteins in vascular smooth muscle

Biochemical Pharmacology ◽

10.1016/j.bcp.2018.08.009 ◽

2018 ◽

Vol 156 ◽

pp. 168-176 ◽

Cited By ~ 2

Author(s):

Alexander Kollau ◽

Bernd Gesslbauer ◽

Michael Russwurm ◽

Doris Koesling ◽

Antonius C.F. Gorren ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Cyclase Activity ◽

Associated Proteins ◽

Guanylyl Cyclase Activity

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Exercise training induces eNOS coupling and restores relaxation in coronary arteries of heart failure rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00624.2017 ◽

2018 ◽

Vol 314 (4) ◽

pp. H878-H887 ◽

Cited By ~ 14

Author(s):

Gisele K. Couto ◽

Suliana M. Paula ◽

Igor L. Gomes-Santos ◽

Carlos Eduardo Negrão ◽

Luciana V. Rossoni

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Heart Failure ◽

Exercise Training ◽

Coronary Arteries ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

No Production ◽

Gtp Cyclohydrolase ◽

No Bioavailability

Exercise training (ET) has emerged as a nonpharmacological therapy for cardiovascular diseases because of its helpful milieu for improving vascular function. The aim of the present study was to assess whether ET reverses the alterations in vascular reactivity observed in heart failure (HF)-related coronary arteries and to elucidate the molecular mechanisms involved in these adjustments. Male Wistar rats were subjected to either coronary artery ligation or sham operation. Four weeks after the surgery, rats were divided into two groups: untrained HF (UHF) and exercise-trained HF (THF). ET was conducted on a treadmill for 8 wk. An untrained SO group was included in the study as a normal control. ET restored the impaired acetylcholine (ACh)- and sodium nitroprusside-induced relaxation in coronary arteries to levels of the control. Oxidative stress and reduced nitric oxide (NO) production were observed in UHF, whereas ET restored both parameters to the levels of the control. Expression levels of endothelial NO synthase (eNOS) and soluble guanylyl cyclase subunits were increased in coronary arteries of UHF rats but reduced in THF rats. Tetrahydrobiopterin restored ACh-induced NO production in the UHF group, indicating that eNOS was uncoupled. ET increased the eNOS dimer-to-monomer ratio and expression of GTP cyclohydrolase 1, thus increasing NO bioavailability. Taken together, these findings demonstrate that ET reverses the dysfunction of the NO/soluble guanylyl cyclase pathway present in coronary arteries of HF rats. These effects of ET are associated with increased GTP cyclohydrolase 1 expression, restoration of NO bioavailability, and reduced oxidative stress through eNOS coupling. NEW & NOTEWORTHY The present study provides a molecular basis for the exercise-induced improvement in coronary arteries function in heart failure. Increasing the expression of GTP cyclohydrolase 1, the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin, exercise training couples endothelial nitric oxide synthase, reduces oxidative stress, and increases nitric oxide bioavailability and sensitivity in coronary arteries of heart failure rats.

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