Chronic hypoxia changes gene expression profile of primary rat carotid body cells: consequences on the expression of NOS isoforms and ET-1 receptors
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE118544 | GEO | 2019/05/22
REPOSITORIES: GEO
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Project description:Background: Hypoxia plays an important role in the vascular tone of pulmonary circulation via the vasculature and parenchymal tissue. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, plays a role in inflammation in mononuclear cells. Nitric oxide synthase (NOS), which generates nitric oxide (NO)/cyclic 3', 5'-monophosphate (cGMP), is coexpressed with ET-1 in many cell types. The aim of this study was to assess whether hypoxia induces the production of ET-1 and associated expression of NOS, NO/cGMP and chemokines in rat alveolar macrophages (AMs). Methods: NR8383 cells were cultured under hypoxic (1% oxygen) conditions for 0, 2, 4, 8 and 12 hours. Levels of ET-1, inducible NOS (iNOS), phosphorylated iNOS (p-iNOS), nitrite/nitrate (NOx), cGMP and monocyte chemoattractant protein-1 (MCP-1) were measured. Results: ET-1, p-iNOS, NOx, and cGMP increased significantly in AMs after 4 hours of hypoxia (p < 0.05). ET-1 and MCP-1 mRNA increased after 8 hours (p < 0.05). The protein expression of ET-1, MCP-1, and p-iNOS increased in a time-dependent manner, while iNOS expression decreased with time. Conclusions: The changes in ET-1, p-iNOS, and the NO/cGMP pathway in AMs may help elucidate the mechanisms in the hypoxic lung. Understanding changes in the endothelin axis in hypoxic AMs is a crucial first step to unravel its role in pulmonary circulation.
| S-EPMC6428981 | BioStudies
Project description:<h4>Background and purpose</h4>Obesity, an important risk factor for developing chronic kidney disease (CKD), affects the kidneys by two main molecular signalling pathways: the endocannabinoid/CB<sub>1</sub> receptor system, whose activation in obesity promotes renal inflammation, fibrosis, and injury, and the inducible NOS (iNOS), which generates ROS resulting in oxidative stress. Hence, a compound that inhibits both peripheral CB<sub>1</sub> receptors and iNOS may serve as an effective therapeutic agent against obesity-induced CKD.<h4>Experimental approach</h4>Here, we describe the effect of a novel peripherally restricted, orally bioavailable dual CB<sub>1</sub> receptor/iNOS antagonist, MRI-1867 (3 mg·kg<sup>-1</sup> ), in ameliorating obesity-induced CKD, and compared its metabolic and renal efficacies to a stand-alone peripheral CB<sub>1</sub> receptor antagonist (JD5037; 3 mg·kg<sup>-1</sup> ), iNOS antagonist (1400W; 10 mg·kg<sup>-1</sup> ), and pair feeding. Mice with high-fat diet-induced obesity were treated orally with these compounds or vehicle (Veh) for 28 days. Standard diet-fed mice treated with Veh served as controls.<h4>Key results</h4>Enhanced expression of CB<sub>1</sub> receptors and iNOS in renal tubules was found in human kidney patients with obesity and other CKDs. The hybrid inhibitor ameliorated obesity-induced kidney morphological and functional changes via decreasing kidney inflammation, fibrosis, oxidative stress, and renal injury. Some of these features were independent of the improved metabolic profile mediated via inhibition of CB<sub>1</sub> receptors. An additional interesting finding is that these beneficial effects on the kidney were partially associated with modulating renal adiponectin signalling.<h4>Conclusions and implications</h4>Collectively, our results highlight the therapeutic relevance of blocking CB<sub>1</sub> receptors and iNOS in ameliorating obesity-induced CKD.
| S-EPMC6976880 | BioStudies
Project description:1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression.
| S-EPMC1573919 | BioStudies
Project description:Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN-heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808-3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 +/- 5 and 35 +/- 3 s(-1), respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS.
| S-EPMC2596912 | BioStudies
Project description:INTRODUCTION:Early allograft dysfunction following lung transplantation is mainly an ischemia/reperfusion (IR) injury. We showed that relaxin-2 (relaxin) exerts a protective effect in lung IR, attributable to decreases in endothelin-1 (ET-1) production, leukocyte recruitment, and free radical generation. Here, we summarize our investigations into relaxin's signalling. MATERIALS AND METHODS:Isolated rat lungs were perfused with vehicle or 5 nM relaxin (n?=?6-10 each). Thereafter, experiments were conducted in the presence of relaxin plus vehicle, the protein kinase A inhibitors H-89 and KT-5720, the NO synthase (NOS) inhibitor L-NAME, the iNOS inhibitor 1400W, the nNOS inhibitor SMTC, the extracellular signal-regulated kinase-1/2 (ERK-1/2) inhibitor PD-98059, the phosphatidylinositol-3 kinase (PI3K) inhibitor wortmannin, the endothelin type-B (ETB) antagonist A-192621, or the glucocorticoid receptor (GR) antagonist RU-486. After 90 min ischemia and 90 min reperfusion we determined wet-to-dry (W/D) weight ratio, mean pulmonary arterial pressure (MPAP), vascular release of ET-1, neutrophil elastase (NE), myeloperoxidase (MPO), and malondialdehyde (MDA). Primary rat pulmonary vascular cells were similarly treated. RESULTS:IR lungs displayed significantly elevated W/D ratios, MPAP, as well as ET-1, NE, MDA, and MPO. In the presence of relaxin, all of these parameters were markedly improved. This protective effect was completely abolished by L-NAME, 1400W, PD-98059, and wortmannin whereas neither PKA and nNOS inhibition nor ETB and GR antagonism were effective. Analysis of NOS gene expression and activity revealed that the relaxin-induced early and moderate iNOS stimulation is ERK-1/2-dependent and counter-balanced by PI3K. Relaxin-PI3K-related phosphorylation of a forkhead transcription factor, FKHRL1, paralleled this regulation. In pulmonary endothelial and smooth muscle cells, FKHRL1 was essential to relaxin-PI3K signalling towards iNOS. CONCLUSION:In this short-time experimental setting, relaxin protects against IR-induced lung injury via early and moderate iNOS induction, dependent on balanced ERK-1/2 and PI3K-FKHRL1 stimulation. These findings render relaxin a candidate drug for lung preservation.
| S-EPMC3787055 | BioStudies
Project description:Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.
2008-01-01 | S-EPMC2570888 | BioStudies
Project description:A series of hybrid uranocenes consisting of uranium(iv) sandwiched between cyclobutadienyl (Cb) and cyclo-octatetraenyl (COT) ligands has been synthesized, structurally characterized and studied computationally. The dimetallic species [(η<sup>4</sup>-Cb'''')(η<sup>8</sup>-COT)U(μ:η<sup>2</sup>:η<sup>8</sup>-COT)U(THF)(η<sup>4</sup>-Cb'''')] (<b>1</b>) forms concomitantly with, and can be separated from, monometallic [(η<sup>4</sup>-Cb'''')U(THF)(η<sup>8</sup>-COT)] (<b>2</b>) (Cb'''' = 1,2,3,4-tetrakis(trimethylsilyl)cyclobutadienyl, COT = cyclo-octatetraenyl). In toluene solution at room temperature, <b>1</b> dissociates into <b>2</b> and the unsolvated uranocene [(η<sup>4</sup>-Cb'''')U(η<sup>8</sup>-COT)] (<b>3</b>). By applying a high vacuum, both <b>1</b> and <b>2</b> can be converted directly into <b>3</b>. Using bulky silyl substituents on the COT ligand allowed isolation of base-free [(η<sup>4</sup>-Cb'''')U{η<sup>8</sup>-1,4-(<sup>i</sup>Pr<sub>3</sub>Si)<sub>2</sub>C<sub>8</sub>H<sub>6</sub>}] (<b>4</b>), with compounds <b>3</b> and <b>4</b> being new members of the bis(annulene) family of actinocenes and the first to contain a cyclobutadienyl ligand. Computational studies show that the bonding in the hybrid uranocenes <b>3</b> and <b>4</b> has non-negligible covalency. New insight into actinocene bonding is provided by the complementary interactions of the different ligands with uranium, whereby the 6d orbitals interact most strongly with the cyclobutadienyl ligand and the 5f orbitals do so with the COT ligands. The redox-neutral activation of diethyl ether by [(η<sup>4</sup>-Cb'''')U(η<sup>8</sup>-C<sub>8</sub>H<sub>8</sub>)] is also described and represents a uranium-cyclobutadienyl cooperative process, potentially forming the basis of further small-molecule activation chemistry.
| S-EPMC8179396 | BioStudies
Project description:The reformulation of chocolates seeks to find innovative alternatives to cocoa butter (CB) that are more economical and adhere to nutritional recommendations to replace saturated fats with unsaturated ones. In this research, chocolates were elaborated by substituting CB with an oleogel (OG) formulated with hydroxypropyl methylcellulose (HPMC) as an entrapper of sunflower oil by using the foam-templated approach. Four different CB/OG blends were prepared and characterized as potential CB substitutes (100/0 control), at replacement levels of 30%, 50%, 70% and 100% (70/30, 50/50, 30/70 and 0/100 blends), and subsequently, CB/OG-based chocolates (CB/OG-Ch) were formulated (100/0-Ch, 70/30-Ch, 50/50-Ch, 30/70-Ch and 0/100-Ch). Both the CB/OG blends and the CB/OG-Ch counterparts were characterized by dynamic and stationary rheology, hardness, thermal parameters, microstructure, and oil-binding capacity; in addition, the lipid profile of the chocolates was analyzed, and a sensory analysis was performed. Increasing the OG proportion in the CB/OG blend weakens the rigidity and strength of the fat-crystal network conferred by the CB, and decreases both its viscoelasticity and thermal parameters, but the differences between all the different properties and parameters of the CB/OG-Ch samples diminished in presence of the other ingredients used in the chocolate formulation. Sensory analysis evidenced that it is possible to replace up to 70% of CB with the OG, although from a technological point of view a replacement level of 50% would seem more appropriate. As compared to 100/0-Ch, 50/50-Ch and 30/70-Ch involve saturated fat reductions of 55% and 37%, respectively.
| S-EPMC8628694 | BioStudies
Project description:<h4>Background</h4>The combination of the nutraceuticals, Paullinia cupana, ginger rhizome, muira puama, and the amino acid L-citrulline (COMP-4) has been shown to stimulate the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and cGMP in rat corpora cavernosa smooth muscle cells (CSMC). When administered to middle-aged rats, long-term treatment with COMP-4 resulted in both an increase in the number of CSMC and an improvement in erectile function. We, therefore, aimed to determine whether a commercial formulation of COMP-4, Revactin<sup>®</sup>, could have a similar stimulatory effect on human CSMC.<h4>Methods</h4>Primary human CSMC cultures (HCSMC) were grown and incubated with Revactin® for up to 24 hours. cGMP generation and nitrite formation were determined by ELISA and Griess reaction, respectively. IBMX (1 mM), sildenafil (0.4 mM), and L-NIL (4 µM) were utilized as modulators of the NO-cGMP pathway. iNOS, endothelial NOS (eNOS), and neuronal NOS (nNOS) expressions were determined by Western blot.<h4>Results</h4>Revactin<sup>®</sup> up-regulated both nitrite formation and cGMP expression, achieving the highest expression at 24 hours in the HCSMC. These effects were completely blocked by L-NIL. Revactin<sup>®</sup> up-regulated iNOS expression, but not that of eNOS or nNOS.<h4>Conclusions</h4>The results presented in this study confirmed that Revactin<sup>®</sup> activated the iNOS-NO-cGMP pathway intracellularly in HCSMC. It still needs to be determined whether the upregulation of this pathway would be an effective approach for counteracting the fibrosis and apoptosis of the corporal smooth muscle cells associated with aging.
| S-EPMC8350259 | BioStudies
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by degeneration of motor neurons throughout the central nervous system. Mutations of the free radical scavenging enzyme superoxide dismutase-1 (SOD1) are a cause of familial ALS but it is not known how mutations lead to cell death. Free radicals such as nitric oxide (NO) are thought to play a key pathogenic role. NO is synthesized by NO synthases (NOSs) from arginine, which is a rate-limiting factor for NO production. We found that neuronal NOS (nNOS)-positive motor neurons were depleted while inducible NOS (iNOS)-positive activated glial cells were increased in transgenic mtSOD1 (G93A) ALS mice. iNOS expression was up regulated consistent with the increases of motor neuron loss and glial activation and citrulline and NO levels while nNOS expression was decreased in G93A ALS mice. Administration of l-arginine to G93A mice reduced the severity of motor neuron depletion and glial activation. In treated animals, nNOS expression was preserved while citrulline and NO were reduced, possibly due to reduced activation of glia expressing iNOS. Our findings show that high concentrations of NO correlate with iNOS expression rather than nNOS expression in G93A ALS mice. This suggests that therapy focused on iNOS inhibition might be a fruitful direction for future ALS therapeutic trials.
| S-EPMC2742676 | BioStudies