Soluble guanylyl cyclase expression is reduced in LPS-induced lung injury

2007 ◽  
Vol 292 (4) ◽  
pp. R1448-R1455 ◽  
Author(s):  
Constantinos Glynos ◽  
Anastasia Kotanidou ◽  
Stylianos E. Orfanos ◽  
Zongmin Zhou ◽  
Davina C. M. Simoes ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Lung Injury ◽  
Guanylyl Cyclase ◽  
Muscle Tone ◽  
Soluble Guanylyl Cyclase ◽  
Mrna Levels ◽  
Subunit Protein ◽  
Protein Levels ◽  
Lps Challenge ◽  
Tnf Α

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC α1 and β1 subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the β1 subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in β1 mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased α1 and β1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-α was required for the LPS-triggered reduction in sGC protein levels, as no change in α1 and β1 levels was observed in TNF-α knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-l-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI.

Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms

2001 ◽  
Vol 280 (2) ◽  
pp. L272-L278 ◽  
Author(s):  
Masao Takata ◽  
Galina Filippov ◽  
Heling Liu ◽  
Fumito Ichinose ◽  
Stefan Janssens ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
No Production ◽  
Mrna Levels ◽  
Protein Levels ◽  
Subunit Mrna ◽  
Tnf Α ◽  
Pulmonary Artery Smooth Muscle

Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of α1- and β1-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1β and TNF-α for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity.l- N 6-(1-iminoethyl)lysine (NOS2 inhibitor) or 1 H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.

Soluble guanylyl cyclase expression is reduced in allergic asthma

2006 ◽  
Vol 290 (1) ◽  
pp. L179-L184 ◽  
Author(s):  
Andreas Papapetropoulos ◽  
Davina C. M. Simoes ◽  
Georgia Xanthou ◽  
Charis Roussos ◽  
Christina Gratziou
Keyword(s):  
Smooth Muscle ◽  
Allergic Asthma ◽  
Guanylyl Cyclase ◽  
Muscle Relaxation ◽  
Soluble Guanylyl Cyclase ◽  
Airway Hyperreactivity ◽  
Smooth Muscle Relaxation ◽  
Mrna Levels ◽  
Bronchial Smooth Muscle Cells ◽  
Biochemical Analyses

Soluble guanylyl cyclase (sGC) is an enzyme highly expressed in the lung that generates cGMP contributing to airway smooth muscle relaxation. To determine whether the bronchoconstriction observed in asthma is accompanied by changes in sGC expression, we used a well-established murine model of allergic asthma. Histological and biochemical analyses confirmed the presence of inflammation in the lungs of mice sensitized and challenged with ovalbumin (OVA). Moreover, mice sensitized and challenged with OVA exhibited airway hyperreactivity to methacholine inhalation. Steady-state mRNA levels for all sGC subunits (α1, α2, and β1) were reduced in the lungs of mice with allergic asthma by 60–80%, as estimated by real-time PCR. These changes in mRNA were paralleled by changes at the protein level: α1, α2, and β1 expression was reduced by 50–80% as determined by Western blotting. Reduced α1 and β1 expression in bronchial smooth muscle cells was demonstrated by immunohistochemistry. To study if sGC inhibition mimics the airway hyperreactivity seen in asthma, we treated naïve mice with a selective sGC inhibitor. Indeed, in mice receiving ODQ the methacholine dose response was shifted to the left. We conclude that sGC expression is reduced in experimental asthma contributing to the observed airway hyperreactivity.

scholarly journals GMP-compliant fully automated radiosynthesis of [18F]FEPPA for PET/MRI imaging of regional brain TSPO expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chi-Wei Chang ◽  
Chuang-Hsin Chiu ◽  
Ming-Hsien Lin ◽  
Hung-Ming Wu ◽  
Tsung-Hsun Yu ◽  
...  
Keyword(s):  
Mr Imaging ◽  
Western Blotting ◽  
Second Generation ◽  
Mrna Levels ◽  
Automated Radiosynthesis ◽  
Lps Challenge ◽  
Tnf Α ◽  
The Brain ◽  
Tspo Expression

Abstract Background Expression of translocator protein (TSPO) on the outer mitochondrial membrane of activated microglia is strongly associated with neuroinflammation. The second-generation PET ligand [18F]FEPPA specifically binds TSPO to enable in vivo visualization and quantification of neuroinflammation. We optimized a fully automated radiosynthesis method and evaluated the utility of [18F]FEPPA, the second-generation PET ligand specifically binds TSPO, in a mouse model of systemic LPS challenge to detect TSPO-associated signals of central and peripheral inflammation. In vivo dynamic PET/MR imaging was performed in LPS-induced and control mice after [18F]FEPPA administration. The relationship between the [18F]FEPPA signal and the dose of LPS was assessed. The cytokine levels (i.e., TNF-α, Il-1β, Il-6) in LPS-induced mice were measured by RT-PCR. Standard uptake value (SUV), total volume of distribution (VT) and area under the curve (AUC) were determined based on the metabolite-uncorrected plasma input function. Western blotting and immunostaining were used to measure TSPO expression in the brain. Results The fully automated [18F]FEPPA radiosynthesis produced an uncorrected radiochemical yield of 30 ± 2% within 80 min, with a radiochemical purity greater than 99% and specific activity of 148.9‒216.8 GBq/µmol. Significant differences were observed in the brain after [18F]FEPPA administration: SUV, VT and AUC were 1.61 ± 0.1, 1.25 ± 0.12 and 1.58 ± 0.09-fold higher in LPS-injected mice than controls. TNF-α, Il-1β and Il-6 mRNA levels were also elevated in the brains of LPS-injected mice. Western blotting revealed TSPO (p < 0.05) and Iba-1 (p < 0.01) were upregulated in the brain after LPS administration. In LPS-injected mice, TSPO immunoactivity colocalized with Iba-1 in the cerebrum and TSPO was significantly overexpressed in the hippocampus and cerebellum. The peripheral organs (heart, lung) of LPS-injected mice had higher [18F]FEPPA signal-to-noise ratios than control mice. Conclusions Based on the current data on ligand specificity and selectivity in central tissues using 7 T PET/MR imaging, we demonstrate that [18F]FEPPA accumulations significant increased in the specific brain regions of systemic LPS-induced neuroinflammation (5 mg/kg). Future investigations are needed to determine the sensitivity of [18F]FEPPA as a biomarker of neuroinflammation as well as the correlation between the PET signal intensity and the expression levels of TSPO.

scholarly journals Glutamate regulates Ca2+ signals in smooth muscle cells of newborn piglet brain slice arterioles through astrocyte- and heme oxygenase-dependent mechanisms

2010 ◽  
Vol 298 (2) ◽  
pp. H562-H569 ◽  
Author(s):  
Qi Xi ◽  
Edward Umstot ◽  
Guiling Zhao ◽  
Damodaran Narayanan ◽  
Charles W. Leffler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Heme Oxygenase ◽  
Guanylyl Cyclase ◽  
Brain Slices ◽  
Soluble Guanylyl Cyclase ◽  
Muscle Cells ◽  
Newborn Piglet ◽  
Signal Modulation ◽  
Cerebral Arterioles

Glutamate is the principal cerebral excitatory neurotransmitter and dilates cerebral arterioles to match blood flow to neural activity. Arterial contractility is regulated by local and global Ca2+ signals that occur in smooth muscle cells, but modulation of these signals by glutamate is poorly understood. Here, using high-speed confocal imaging, we measured the Ca2+ signals that occur in arteriole smooth muscle cells of newborn piglet tangential brain slices, studied signal regulation by glutamate, and investigated the physiological function of heme oxygenase (HO) and carbon monoxide (CO) in these responses. Glutamate elevated Ca2+ spark frequency by ∼188% and reduced global intracellular Ca2+ concentration ([Ca2+]i) to ∼76% of control but did not alter Ca2+ wave frequency in brain arteriole smooth muscle cells. Isolation of cerebral arterioles from brain slices abolished glutamate-induced Ca2+ signal modulation. In slices treated with l-2-α-aminoadipic acid, a glial toxin, glutamate did not alter Ca2+ sparks or global [Ca2+]i but did activate Ca2+ waves. This shift in Ca2+ signal modulation by glutamate did not occur in slices treated with d-2-α-aminoadipic acid, an inactive isomer of l-2-α-aminoadipic acid. In the presence of chromium mesoporphyrin, a HO blocker, glutamate inhibited Ca2+ sparks and Ca2+ waves and did not alter global [Ca2+]i. In isolated arterioles, CORM-3 [tricarbonylchloro(glycinato)ruthenium(II)], a CO donor, activated Ca2+ sparks and reduced global [Ca2+]i. These effects were blocked by 1 H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one, a soluble guanylyl cyclase inhibitor. Collectively, these data indicate that glutamate can modulate Ca2+ sparks, Ca2+ waves, and global [Ca2+]i in arteriole smooth muscle cells via mechanisms that require astrocytes and HO. These data also indicate that soluble guanylyl cyclase is involved in CO activation of Ca2+ sparks in arteriole smooth muscle cells.

Abstract 556: The Soluble Guanylyl Cyclase Activator Bay 60-2770 Inhibits Arterial Smooth Muscle Cell Migration in Protein Kinase G-dependent Manner

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Andrew Holt ◽  
Danielle Martin ◽  
Patti Shaver ◽  
Shaquria Adderley ◽  
Joshua Stone ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Protein Kinase ◽  
Cyclic Gmp ◽  
Guanylyl Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
Protein Kinase G ◽  
Growth Disorders ◽  
Arterial Smooth Muscle ◽  
Cyclase Activator

Atherosclerotic lower extremity peripheral artery disease (PAD) is among the most prevalent, morbid and mortal of all cardiovascular disorders. Pathologic arterial smooth muscle (ASM) cell migration is a major component of atherogenic PAD and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of PAD and other vascular growth disorders. In this study we hypothesized that the novel, heme-independent soluble guanylyl cyclase activator BAY 60-2770 (BAY) inhibits ASM cell migration through phosphorylation of the protein kinase G (PKG) target and actin-binding protein vasodilator-stimulated phosphoprotein (VASP). In a rat model of injury-induced arterial growth, BAY significantly reduced neointima formation and luminal narrowing compared to vehicle (Veh)-treated control arteries after 2 weeks. Using rat and human ASM cells BAY significantly attenuated cell migration, reduced G:F actin, and increased cyclic GMP content, PKG activity and phosphorylated VASP at Ser239 (pVASP.S239) compared to Veh controls. Using site-directed mutagenesis, both full-length VASP-overexpressing (wild type, WT) and VASP.S239 phosphorylation-resistant mutants showed significantly reduced cell migration compared to naïve controls, however, there was no effect on cell migration between either VASP transfected group in the presence of BAY. Interestingly, both VASP mutants showed significantly increased PKG activity compared to naïve cells, and in turn pharmacologic PKG blockade in the presence of BAY fully reversed the inhibitory effect of BAY alone on cell migration. These data suggest BAY has capacity to inhibit ASM cell migration through cyclic GMP/PKG/VASP signaling yet through mechanisms independent of pVASP.S239. Findings from this study implicate BAY via cyclic GMP/PKG/VASP as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as PAD.

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

1995 ◽  
Vol 269 (2) ◽  
pp. F212-F217 ◽  
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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