Gaseous Mediators Nitric Oxide Research Articles

Gaseous mediators (H2S, NO and CO) a new approach in gastrointestinal protection

Gastrointestinal damage is generated by a wide range of harmful agents, and is the result of the activation of inflammatory, oxidant and cytotoxic mechanisms, that together overwhelm the “mucosa defense” of the gastrointestinal (GI) tract. It is currently known that the mechanisms by which gastric damage is generated are slightly different from those that generate intestinal damage. The treatments available on the market such as citoprotectors, the use of selective Ciclooxigenase 2 (COX-2) inhibitors, the co-prescription of acid suppressive agents and the use of prostaglandin analogues are mainly focused on the mechanisms of gastric damage, and their use has begun to be limited due to its adverse effects at intestinal and cardiovascular level. Due to its multifactorial origin, the prevention and treatment of GI damage requires effective therapies that can protect both gastric and intestinal level modulating more than one mechanism. Recently, has been reported the contribution of gaseous mediators nitric oxide (NO), hydrogen sulfide (H2S) and carbon monoxide (CO) in many physiological processes in the gastrointestinal tract, including the maintenance of GI mucosal barrier integrity. Experimental evidence has shown promising results regarding the gastric safety of these gasotransmitters, especially those coupled to NSAIDs. This review will try to give a general overview of the mechanisms of gastric and intestinal damage, their main differences, the existing therapies for their treatment and the information available on gas transmitters with a brief description of roles of each of these gaseous molecules.

Gaseous Mediators as a Key Molecular Targets for the Development of Gastrointestinal-Safe Anti-Inflammatory Pharmacology.

Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most widely used classes of drugs and play a pivotal role in the therapy of numerous inflammatory diseases. However, the adverse effects of these drugs, especially when applied chronically, frequently affect gastrointestinal (GI) tract, resulting in ulceration and bleeding, which constitutes a significant limitation in clinical practice. On the other hand, it has been recently discovered that gaseous mediators nitric oxide (NO), hydrogen sulfide (H2S) and carbon monoxide (CO) contribute to many physiological processes in the GI tract, including the maintenance of GI mucosal barrier integrity. Therefore, based on the possible therapeutic properties of NO, H2S and CO, a novel NSAIDs with ability to release one or more of those gaseous messengers have been synthesized. Until now, both preclinical and clinical studies have shown promising effects with respect to the anti-inflammatory potency as well as GI-safety of these novel NSAIDs. This review provides an overview of the gaseous mediators-based NSAIDs along with their mechanisms of action, with special emphasis on possible implications for GI mucosal defense mechanisms.

Increase of cystathionine-γ-lyase (CSE) during late wound repair: Hydrogen sulfide triggers cytokeratin 10 expression in keratinocytes

The gaseous mediators nitric oxide (NO), carbon monoxide (CO) and lately also hydrogen sulfide (H2S) have been described to contribute to the interplay of protein type- and lipid mediators in the regulation of wound healing. In particular, the recently reported role of H2S in skin repair remains largely unresolved. Therefore we assessed the expressional kinetics of potential H2S-producing enzymes during undisturbed skin repair: the cystathionine-γ-lyase (CSE), the cystathionine-β-synthase (CBS) and the 3-mercaptopyruvate sulfurtransferase (MPST). All three enzymes were not transcriptionally induced upon wounding and remained silent through the acute inflammatory and proliferative phase of skin repair. By contrast, CSE expression started to increase significantly at the later stages of healing, when cellular proliferation ceases within the granulation tissue and neoepidermis. The importance of H2S production in late healing phases was supported by a strong induction of otherwise not-induced CBS to complement the loss of CSE function in CSE-deficient mice. Immunohistochemistry revealed hair follicle keratinocytes and basal keratinocytes of the neo-epidermis covering the wound area as sources of CSE expression. Subsequent in vitro studies implicated a role of CSE-derived H2S for keratinocyte differentiation: the H2S-donor GYY4137 markedly increased the Ca2+-triggered expression of the early keratinocyte differentiation markers cytokeratin 10 (CK10) and involucrin (IVN) in cultured human keratinocytes. Here, GYY4137-derived H2S strongly enhanced CK10 expression by increasing the binding of RNA polymerase II to the CK10 promoter.

AMPK activation promotes gastroprotection through mutual interaction with the gaseous mediators H2S, NO, and CO

Activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) stimulates production of the gaseous mediators nitric oxide (NO) and carbon monoxide (CO), which are involved in mucosal defense and gastroprotection. As AMPK itself has gastroprotective effects against several gastric ulcer etiologies, in the present study, we aimed to elucidate whether AMPK may also prevent ethanol-induced injury and play a key role in the associated gastroprotection mediated by hydrogen sulfide (H2S), NO, and CO. Mice were pretreated with AICAR (20 mg/kg, an AMPK activator) alone or with 50% ethanol. Other groups were pretreated with respective gaseous mediator inhibitors PAG, l-NAME, or ZnPP IX 30 min prior to AICAR, or with gaseous mediator donors NaHS, Lawesson's reagent and l-cysteine (H2S), SNP, l-Arginine (NO), Hemin, or CORM-2 (CO) 30 min prior to ethanol with or without compound C (10 mg/kg, a non-selective AMPK inhibitor). H2S, nitrate/nitrite (NO3−/NO2−), bilirubin levels, GSH and MDA concentration were evaluated in the gastric mucosa. The gastric mucosa was also collected for histopathological analysis and AMPK expression assessment by immunohistochemistry. Pretreatment with AICAR attenuated the ethanol-induced injury and increased H2S and bilirubin levels but not NO3−/NO2− levels in the gastric mucosa. In addition, inhibition of H2S, NO, or CO synthesis exacerbated the ethanol-induced gastric damage and inhibited the gastroprotection by AICAR. Pretreatment with compound C reversed the gastroprotective effect of NaHS, Lawesson's reagent, l-cysteine, SNP, l-Arginine, CORM-2, or Hemin. Compound C also reversed the effect of NaHS on H2S production, SNP on NO3−/NO2− levels, and Hemin on bilirubin levels. Immunohistochemistry revealed that AMPK is present at basal levels mainly in the gastric mucosa cells, and was increased by pretreatment with NaHS, SNP, and CORM-2. In conclusion, our findings indicate that AMPK activation exerts gastroprotection against ethanol-induced gastric damage and mutually interacts with H2S, NO, or CO to facilitate this process.

A heterogeneous Ly-6B2+ leukocyte population consists of yet undescribed iNOS-expressing cell types in murine skin wounds

The gaseous mediator nitric oxide (NO) is a central regulatory molecule during the inflammatory phase of cutaneous tissue repair. The inducible NO-synthase (iNOS) represents the main isoform of the three NO producing enzymes at the wound site. In particular, keratinocytes and macrophages are described as main sources of iNOS-derived NO in skin wounds. Here we provide experimental evidence that Ly-6B2+ leukocytes are an additional cellular source of iNOS-derived NO in wounds. As wound iNOS protein expression temporally coincides with both macrophage and neutrophil infiltration, we used immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) to address iNOS expression in both macrophages and neutrophil subsets. IHC analyses excluded F4/80+ macrophages as iNOS producers, but indicated Ly-6G/C (Gr-1)+ neutrophils to express iNOS in wound granulation tissue. A subsequent FACS-based analysis from cellular wound tissue preparations revealed an iNOS-expressing fraction of Ly-6B2-determined leukocytes that consisted of Ly-6G+ and Ly-6G− cells, meaning that mainly mature neutrophils (Ly-6B2+/Ly-6G+) as well as inflammatory monocytes (Ly-6B2+/Ly-6G−) are dominant iNOS-expressing cell types in the developing granulation tissue of acute wounds.

Pathophysiological functions of gas mediators in neurodegeneration

Since the discovery of nitric oxide (NO) as gaseous signaling molecule, two other gaseous mediators, carbon monoxide (CO) and hydrogen sulfide (H2S) have been found to be also involved in many physiological and pathophysiological functions. This review will briefly summarize our recent progress in the pathophysiology of NO and H2S. In the photoreceptor cells, the level of intracellular Ca2+ is kept relatively low by H2S. Intraperitoneal injection of H2S donor to mice protected photoreceptor cells from light-induced retinal degeneration caused by oxidative stress and elevation of intracellular Ca2+. Another gaseous mediator NO induces Ca2+ release from the endoplasmic reticulum via S-nitrosylated type 1 ryanodine receptor (RyR1) Ca2+ release channel. NO-induced Ca2+ release (NICR) was abolished in primary cultured neurons from the knock-in mice, in which the S-nitrosylation site Cys-3636 of RyR1 was replaced by Ala (Ryr1C3636A). The neurons in hippocampal CA3 region of Ryr1C3636A mice were protected against seizure-induced neuronal cell death. The result indicates that NICR is critical for status epilepticus-induced neurodegeneration. The developments in the pathophysiology of gaseous mediators in the central nervous system will provide a better pharmacological advances for the treatment of neurodegenerative diseases.

Gaseous mediators nitric oxide and hydrogen sulfide in the mechanism of gastrointestinal integrity, protection and ulcer healing.

Nitric oxide (NO) and hydrogen sulfide (H2S) are known as biological messengers; they play an important role in human organism and contribute to many physiological and pathophysiological processes. NO is produced from l-arginine by constitutive NO synthase (NOS) and inducible NOS enzymatic pathways. This gaseous mediator inhibits platelet aggregation, leukocyte adhesion and contributes to the vessel homeostasis. NO is known as a vasodilatory molecule involved in control of the gastric blood flow (GBF) and the maintenance of gastric mucosal barrier integrity in either healthy gastric mucosa or that damaged by strong irritants. Biosynthesis of H2S in mammals depends upon two enzymes cystathionine-β-synthase and cystathionine γ-lyase. This gaseous mediator, similarly to NO and carbon monoxide, is involved in neuromodulation, vascular contractility and anti-inflammatory activities. For decades, H2S has been known to inhibit cytochrome c oxidase and reduce cell energy production. Nowadays it is generally considered to act through vascular smooth muscle ATP-dependent K+ channels, interacting with intracellular transcription factors and promote sulfhydration of protein cysteine moieties within the cell, but the mechanism of potential gastroprotective and ulcer healing properties of H2S has not been fully explained. The aim of this review is to compare current results of the studies concerning the role of H2S and NO in gastric mucosa protection and outline areas that may pose new opportunities for further development of novel therapeutic targets.

Upregulation and Mitochondrial Sequestration of Hemoglobin Occur in Circulating Leukocytes during Critical Illness, Conferring a Cytoprotective Phenotype.

The classical role of hemoglobin in the erythrocytes is to carry oxygen from the lungs to the tissues via the circulation. However, hemoglobin also acts as a redox regulator and as a scavenger of the gaseous mediators nitric oxide (NO) and hydrogen sulfide (H2S). Here we show that upregulation of hemoglobin (α, β and δ variants of globin proteins) occurs in human peripheral blood mononuclear cells (PBMCs) in critical illness (patients with severe third-degree burn injury and patients with sepsis). The increase in intracellular hemoglobin concentration is a result of a combination of enhanced protein expression and uptake from the extra-cellular space via a CD163-dependent mechanism. Intracellular hemoglobin preferentially localizes to the mitochondria, where it interacts with complex I and, on the one hand, increases mitochondrial respiratory rate and mitochondrial membrane potential, and on the other hand, protects from H2O2-induced cytotoxicity and mitochondrial DNA damage. Both burn injury and sepsis were associated with increased plasma levels of H2S. Incubation of mononuclear cells with H2S induced hemoglobin mRNA upregulation in PBMCs in vitro. Intracellular hemoglobin upregulation conferred a protective effect against cell dysfunction elicited by H2S. Hemoglobin uptake also was associated with a protection from, and induced the upregulation of, HIF-1α and Nrf2 mRNA. In conclusion, PBMCs in critical illness upregulate their intracellular hemoglobin levels by a combination of active synthesis and uptake from the extracellular medium. We propose that this process serves as a defense mechanism protecting the cell against cytotoxic concentrations of H2S and other gaseous transmitters, oxidants and free radicals produced in critically ill patients.

Prostaglandins, NSAIDs, and Gastric Mucosal Protection: Why Doesn't the Stomach Digest Itself?

Except in rare cases, the stomach can withstand exposure to highly concentrated hydrochloric acid, refluxed bile salts, alcohol, and foodstuffs with a wide range of temperatures and osmolarity. This is attributed to a number of physiological responses by the mucosal lining to potentially harmful luminal agents, and to an ability to rapidly repair damage when it does occur. Since the discovery in 1971 that prostaglandin synthesis could be blocked by aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), there has been great interest in the contribution of prostaglandins to gastric mucosal defense. Prostaglandins modulate virtually every aspect of mucosal defense, and the importance of this contribution is evident by the increased susceptibility of the stomach to injury following ingestion of an NSAID. With chronic ingestion of these drugs, the development of ulcers in the stomach is a significant clinical concern. Research over the past two decades has helped to identify some of the key events triggered by NSAIDs that contribute to ulcer formation and/or impair ulcer healing. Recent research has also highlighted the fact that the protective functions of prostaglandins in the stomach can be carried out by other mediators, in particular the gaseous mediators nitric oxide and hydrogen sulfide. Better understanding of the mechanisms through which the stomach is able to resist injury in the presence of luminal irritants is helping to drive the development of safer anti-inflammatory drugs, and therapies to accelerate and improve the quality of ulcer healing.

Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide

The gaseous mediators hydrogen sulphide (H 2S) and nitric oxide ( NO) are synthesised in the body from l-cysteine and l-arginine, respectively. In the cardiovascular system, NO is an important regulator of vascular tone and its over- or under-production has been linked to a variety of diseases. The physiological significance of H 2S is not yet clear but, like NO, it exhibits vasodilator activity and may play a part in septic and haemorrhagic shock, hypertension, regulation of cardiac contractility, and in inflammation. To date, there have been no reports of a chemical interaction between H 2S and NO. Here we show that incubation of the H 2S donor, sodium hydrosulphide, with a range of NO donors and NO gas in vitro leads to the formation of a nitrosothiol molecule as determined by a combination of techniques; electron paramagnetic resonance, amperometry, and measurement of nitrite. We further show that this nitrosothiol did not induce cGMP accumulation in cultured RAW264.7 cells unless NO was released with Cu 2+. Finally, using liver homogenates from LPS treated rats we present evidence for the endogenous formation of this nitrosothiol. These findings provide the first evidence for the formation of a novel nitrosothiol generated by reaction between H 2S and NO. We propose that generation of this nitrosothiol in the body may regulate the physiological effects of both NO and H 2S.

Inhibition of Caspase-1-Like Activity by Ac-Tyr-Val-Ala-Asp-Chloromethyl Ketone Induces Long-Lasting Neuroprotection in Cerebral Ischemia through Apoptosis Reduction and Decrease of Proinflammatory Cytokines

Broad spectrum caspase inhibitors have been found to reduce neurodegeneration caused by cerebral ischemia. We studied whether blockade of group I caspases, mainly caspase-1, using the inhibitor Ac-YVAD.cmk reduced infarct volume and produced prolonged neuroprotection. Ac-YVAD.cmk (300 ng/rat) was injected intracerebroventricularly 10 min after permanent middle cerebral artery occlusion in the rat. Drug treatment induced a significant reduction of infarct volume not only 24 hr after ischemia (total damage, percentage of hemisphere volume: control, 41.1 +/- 2.3%; treated, 26.5 +/- 2.1%; p < 0.05) but also 6 d later (total damage: control, 30.6 +/- 2.2%; treated, 23.0 +/- 2.2%; p < 0.05). Ac-YVAD. cmk treatment resulted in a reduction not only of caspase-1 (control, 100 +/- 20.3%; treated, 3.4 +/- 10.4%; p < 0.01) but also of caspase-3 (control, 100 +/- 30.3%; treated, 13.2 +/- 9.5%; p < 0.05) activity at 24 hr and led to a parallel decrease of apoptosis as measured by nucleosome quantitation (control, 100 +/- 11.8%; treated, 47 +/- 5.9%; p < 0.05). Six days after treatment no differences in these parameters could be detected between control and treated animals. Likewise, brain levels of the proinflammatory cytokines IL-1beta and TNF-alpha were reduced at 24 hr (39.5 +/- 23.7 and 51.9 +/- 10.3% of control, respectively) but not at 6 d. Other cytokines, IL-10, MCP-1, MIP-2, and the gaseous mediator nitric oxide, were not modified by the treatment. These findings indicate that blockade of caspase-1-like activity induces a long-lasting neuroprotective effect that, in our experimental conditions, takes place in the early stages of damage progression. Finally, this effect is achieved by interfering with both apoptotic and inflammatory mechanisms.

Role of nitric oxide in the inhibition of BMP-2-mediated stimulation of proteoglycan synthesis in articular cartilage

Objective Bone morphogenetic protein-2 (BMP-2)-mediated stimulation of articular cartilage proteoglycan (PG) synthesis is suppressed in arthritic murine knee joints and by interleukin-1 (IL-1). The goal of this study was to investigate whether the gaseous mediator nitric oxide (NO) plays a crucial role in the inhibition of BMP-2 effects by IL-1.Methods Bone morphogenetic protein-2 alone or in combination with IL-1 was injected into the right knee joint of wild-type and NOS2 deficient C57Bl/6×129/Sv mice. Proteoglycan synthesis was measured ex vivo by incorporation of35S-sulfate on day 1, 2 and 3 after injection. To study the role of NO in the inhibition BMP-2-mediated stimulation of PG synthesis in arthritic joints, BMP-2 was injected intra-articularly in the joints of wild-type and NOS2 deficient mice with zymosan-induced arthritis. To check for NOS2 deficiency, NO production was measured in conditioned medium after challenge of patellae with surrounding tissue with IL-1.Results BMP-2 potently stimulated proteoglycan synthesis in articular cartilage of normal knees (up to 4-fold) but not in arthritic knees. Co-injection of BMP-2 with tumor necrosis factor α had no effect on BMP-2-mediated stimulation of PG synthesis but co-injection with IL-1 α resulted in a nearly total inhibition of BMP-2-mediated stimulation. In contrast, in NOS2 deficient mice IL-1 had no effect on BMP-2-mediated stimulation of PG synthesis. However, injection of BMP-2 into arthritic knee joints of NOS2 knock out mice did not result in significant stimulation of PG synthesis.Conclusions In this study we show that NO plays a role in the inhibition of BMP-2-mediated stimulation of PG synthesis by IL-1. However, NO, or at least NOS2, plays no dominant role in the inhibition of BMP-2 effects in arthritic knee joints.