Carolina Muscoli

Modulation of Prostaglandin Biosynthesis by Nitric Oxide and Nitric Oxide Donors

The biosynthesis and release of nitric oxide (NO) and prostaglandins (PGs) share a number of similarities. Two major forms of nitric-oxide synthase (NOS) and cyclooxygenase (COX) enzymes have been identified to date. Under normal circumstances, the constitutive isoforms of these enzymes (constitutive NOS and COX-1) are found in virtually all organs. Their presence accounts for the regulation of several important physiological effects (e.g. antiplatelet activity, vasodilation, and cytoprotection). On the other hand, in inflammatory setting, the inducible isoforms of these enzymes (inducible NOS and COX-2) are detected in a variety of cells, resulting in the production of large amounts of proinflammatory and cytotoxic NO and PGs. The release of NO and PGs by the inducible isoforms of NOS and COX has been associated with the pathological roles of these mediators in disease states as evidenced by the use of selective inhibitors. An important link between the NOS and COX pathways was made in 1993 by Salvemini and coworkers when they demonstrated that the enhanced release of PGs, which follows inflammatory mechanisms, was nearly entirely driven by NO. Such studies raised the possibility that COX enzymes represent important endogenous “receptor” targets for modulating the multifaceted roles of NO. Since then, numerous papers have been published extending the observation across various cellular systems and animal models of disease. Furthermore, other studies have highlighted the importance of such interaction in physiology as well as in the mechanism of action of drugs such as organic nitrates. More importantly, mechanisticstudies of how NO switches on/off the PG/COX pathway have been undertaken and additional pathways through which NO modulates prostaglandin production unraveled. On the other hand, NO donors conjugated with COX inhibitors have recently found new interest in the understanding of NO/COX reciprocal interaction and potential clinical use. The purpose of this article is to cover the advances which have occurred over the years, and in particular, to summarize experimental data that outline how the discovery that NO modulates prostaglandin production has impacted and extended our understanding of these two systems in physiopathological events.

On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies

The list of pathophysiological conditions associated with the overproduction of superoxide expands every day. Much of the knowledge compiled on the role of this radical in disease has been gathered using the native superoxide dismutase enzyme and, more recently, by the use of superoxide dismutase knockout models or transgenic models that overexpress the various isoforms of the enzyme. Although the native enzyme has shown promising anti‐inflammatory properties in both preclinical and clinical studies, there were drawbacks and issues associated with its use as a therapeutic agent and pharmacological tool. Based on the concept that removal of superoxide modulates the course of inflammation, synthetic, low‐molecular‐weight mimetics of the superoxide dismutase enzymes that could overcome some of the limitations associated with the use of the native enzyme have been designed. In this review, we will discuss the advances made using various superoxide dismutase mimetics that led to the proposal that superoxide (and/or the product of its interaction with nitric oxide, peroxynitrite) is an important mediator of inflammation, and to the conclusion that superoxide dismutase mimetics can be utilized as therapeutic agents in diseases of various etiologies. The importance of the selectivity of such compounds in pharmacological studies will be discussed.

Oxidative stress and neuroAIDS: triggers, modulators and novel antioxidants

Neurological disorders represent one of the most common disturbances accompanying HIV infection. In the past few years, highly antiretroviral active therapy has significantly reduced the incidence of HIV-related diseases. However, neurological dysfunction in AIDS patients still remains an unresolved problem. Oxidative stress, which occurs in brain tissues of patients undergoing HIV infection and is implicated in cell death of both astroglia and neurones, has recently been suggested to play a role in the pathogenesis of neuroAIDS. Thus, a better understanding of the processes that trigger and modulate free radical formation in brain tissues of AIDS patients might help in a successful therapeutic approach to the neuropathogenesis of HIV infection.

Therapeutic manipulation of peroxynitrite attenuates the development of opiate-induced antinociceptive tolerance in mice

Severe pain syndromes reduce quality of life in patients with inflammatory and neoplastic diseases, often because chronic opiate therapy results in reduced analgesic effectiveness, or tolerance, leading to escalating doses and distressing side effects. The mechanisms leading to tolerance are poorly understood. Our studies revealed that development of antinociceptive tolerance to repeated doses of morphine in mice was consistently associated with the appearance of several tyrosine-nitrated proteins in the dorsal horn of the spinal cord, including the mitochondrial isoform of superoxide (O2-) dismutase, the glutamate transporter GLT-1, and the enzyme glutamine synthase. Furthermore, antinociceptive tolerance was associated with increased formation of several proinflammatory cytokines, oxidative DNA damage, and activation of the nuclear factor poly(ADP-ribose) polymerase. Inhibition of NO synthesis or removal of O2- blocked these biochemical changes and inhibited the development of tolerance, pointing to peroxynitrite (ONOO-), the product of the interaction between O2- and NO, as a signaling mediator in this setting. Indeed, coadministration of morphine with the ONOO- decomposition catalyst, Fe(III) 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, blocked protein nitration, attenuated the observed biochemical changes, and prevented the development of tolerance in a dose-dependent manner. Collectively, these data suggest a causal role for ONOO- in pathways culminating in antinociceptive tolerance to opiates. Peroxynitrite (ONOO-) decomposition catalysts may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

Hypolipemic and hypoglycaemic activity of bergamot polyphenols: From animal models to human studies

Bergamot juice produces hypolipemic activity in rats though the mechanism remains unclear. Here we investigated on the effect of bergamot extract (BPF) in diet-induced hyperlipemia in Wistar rats and in 237 patients suffering from hyperlipemia either associated or not with hyperglycaemia. BPF, given orally for 30 days to both rats and patients, reduces total and LDL cholesterol levels (an effect accompanied by elevation of cHDL), triglyceride levels and by a significant decrease in blood glucose. Moreover, BPF inhibited HMG-CoA reductase activity and enhanced reactive vasodilation thus representing an efficient phytotherapeutic approach in combating hyperlipemic and hyperglycaemic disorders.

Targeting the Overproduction of Peroxynitrite for the Prevention and Reversal of Paclitaxel-Induced Neuropathic Pain

Chemotherapy-induced peripheral neuropathy (CIPN) accompanied by chronic neuropathic pain is a major dose-limiting side effect of a large number of antitumoral agents including paclitaxel (Taxol). Thus, CIPN is one of most common causes of dose reduction and discontinuation of what is otherwise a life-saving therapy. Neuropathological changes in spinal cord are linked to CIPN, but the causative mediators and mechanisms remain poorly understood. We report that formation of peroxynitrite (PN) in response to activation of nitric oxide synthases and NADPH oxidase in spinal cord contributes to neuropathological changes through two mechanisms. The first involves modulation of neuroexcitatory and proinflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10 and IL-4) cytokines in favor of the former. The second involves post-translational nitration and modification of glia-derived proteins known to be involved in glutamatergic neurotransmission (astrocyte-restricted glutamate transporters and glutamine synthetase). Targeting PN with PN decomposition catalysts (PNDCs) not only blocked the development of paclitaxel-induced neuropathic pain without interfering with antitumor effects, but also reversed it once established. Herein, we describe our mechanistic study on the role(s) of PN and the prevention of neuropathic pain in rats using known PNDCs (FeTMPyP5+ and MnTE-2-PyP5+). We also demonstrate the prevention of CIPN with our two new orally active PNDCs, SRI6 and SRI110. The improved chemical design of SRI6 and SRI110 also affords selectivity for PN over other reactive oxygen species (such as superoxide). Our findings identify PN as a critical determinant of CIPN, while providing the rationale toward development of superoxide-sparing and “PN-targeted” therapeutics.

Superoxide potentiates NF-κB activation and modulates endotoxin-induced cytokine production in alveolar macrophages

Gram-negative bacterial infection predisposes to the development of shock and acute lung injury with multiple organ dysfunction in the critically ill. Although overexpression of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, IL-6, IL-8, and other mediators is causally implicated in the pathogenesis of shock and lung injury, the underlying mechanisms following cellular exposure to gram-negative endotoxin remain unclear. De novo generation of reactive oxygen species (ROS) by monocytes/macrophages in particular has been proposed as a pivotal regulatory mechanism by which enhanced transactivation of redox-sensitive genes culminates in augmented cytokine expression within the lower respiratory tract. Here we sought to characterize the mechanism of action of a synthetic, nonpeptide, low-molecular-weight, Mn-containing superoxide dismutase mimetic (SODm), M40403, in modulating E. coli lipopolysaccharide serotype 0111:B4 (LPS)-induced cytokine production by cultured rat alveolar macrophages. Intracellular superoxide (O2.−) ion generation was measured using hydroethidine (HE) dye, and the dose-dependent effects of M40403 on TNF-α and IL-6 biosynthesis by ELISAs. Upstream redox-sensitive signaling events involving the pleiotropic transcription factor NF-κB were determined in nuclear extracts by electrophoretic mobility shift assays (EMSAs) and p65 subunit Western blot. The levels of the cytosolic inhibitory protein IκB-α were also assessed by Western analysis. We found that M40403 potently suppressed the production of superoxide, TNF-α, and IL-6 in LPS-stimulated alveolar macrophages, suggesting a key role for superoxide in endotoxin-induced cytokine production in the distal air spaces. In addition, M40403 decreased E. coli LPS-induced activation of NF-κB, and this effect was associated with modest suppression of cytoplasmic IκB-α degradation. Together, these results suggest that removal of superoxide by M40403 inhibits endotoxin-induced production of TNF-α and IL-6 in alveolar macrophages by a mechanism involving suppression of redox-sensitive NF-κB transactivation or signaling.

Superoxide-mediated nitration of spinal manganese superoxide dismutase: a novel pathway in N-methyl-D-aspartate-mediated hyperalgesia

&NA; N‐methyl‐d‐aspartate (NMDA) receptors serve prominent roles in vast physio‐pathological conditions including hyperalgesia (defined as augmented pain intensity in response to painful stimuli) associated with central sensitization. Using M40403 a synthetic low molecular weight superoxide dismutase mimetic that removes superoxide we show for the first time that this radical plays a key role in NMDA‐mediated hyperalgesia. Intrathecal administration of NMDA in rats led to a time‐dependent development of thermal hyperalgesia. Removal of superoxide with M40403 abolished NMDA‐mediated hyperalgesia, while its inactive congener had no effect. Thus NMDA‐mediated hyperalgesic response to heat is mediated through spinal release of superoxide. At time of near‐to‐maximal hyperalgesia, we observed that spinal endogenous manganese superoxide dismutase (MnSOD), the enzyme that normally keeps superoxide under well‐controlled condition was nitrated, as shown by immunoprecipitation. Subsequently and as determined by biochemical analysis, nitration of MnSOD led to its deactivation as shown by the loss of the enzymes ability to dismute and hence remove superoxide. M40403 by preventing MnSOD nitration restored its activity and inhibited the hyperalgesic response to intrathecal NMDA. Thus, superoxide‐mediated nitration and deactivation of spinal MnSOD is a novel pathway of NMDA‐mediated spinal hyperalgesia and hence central sensitization since it helps to maintain high levels of superoxide that in turn maintains nociceptive signaling. The broader implication of our findings is that superoxide may contribute to various forms of pain events that are driven by NMDA‐receptor activation.

The role of oxidative stress in paraquat-induced neurotoxicity in rats: protection by non peptidyl superoxide dismutase mimetic

Herbicides, including paraquat, may produce neurodegenerative effect when given both peripherally and into the brain though the pathophysiological mechanism is still unknown. Microinfusion of paraquat into the Substantia Nigra (50 microg) produced increased motor activity, jumping and circling opposite to the injection site, associated with ECoG desynchronization, high voltage epileptogenic spikes, and with neuropathological effects. These effects were accompanied by increase of malondialdehyde (MDA) levels in the Substantia Nigra, suggesting that paraquat was able to induce oxidative stress when injected directly into the rat brain. Pre-treatment of rats with M40401, a non peptidyl superoxide dismutase (SOD) mimetic given directly into the Substantia Nigra or i.p. prevented both behavioural, electrocorticogram and neuropathological effects and MDA elevation. Taken together, these results demonstrate that paraquat produces brain damage via abnormal formation of oxygen free radicals and that this effect may be counteracted by novel SOD mimetics.

Counter-Regulation of Opioid Analgesia by Glial-Derived Bioactive Sphingolipids

The clinical efficacy of opiates for pain control is severely limited by analgesic tolerance and hyperalgesia. Herein we show that chronic morphine upregulates both the sphingolipid ceramide in spinal astrocytes and microglia, but not neurons, and spinal sphingosine-1-phosphate (S1P), the end-product of ceramide metabolism. Coadministering morphine with intrathecal administration of pharmacological inhibitors of ceramide and S1P blocked formation of spinal S1P and development of hyperalgesia and tolerance in rats. Our results show that spinally formed S1P signals at least in part by (1) modulating glial function because inhibiting S1P formation blocked increased formation of glial-related proinflammatory cytokines, in particular tumor necrosis factor-α, interleukin-1βα, and interleukin-6, which are known modulators of neuronal excitability, and (2) peroxynitrite-mediated posttranslational nitration and inactivation of glial-related enzymes (glutamine synthetase and the glutamate transporter) known to play critical roles in glutamate neurotransmission. Inhibitors of the ceramide metabolic pathway may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.