Vincenzo Mollace

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.

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.

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.

Defective autophagy in Parkinson's disease: Role of oxidative stress

Parkinson’s disease (PD) is a paradigmatic example of neurodegenerative disorder with a critical role of oxidative stress in its etiopathogenesis. Genetic susceptibility factors of PD, such as mutations in Parkin, PTEN-induced kinase 1, and DJ-1 as well as the exposure to pesticides and heavy metals, both contribute to altered redox balance and degeneration of dopaminergic neurons in the substantia nigra. Dysregulation of autophagy, a lysosomal-driven process of self degradation of cellular organelles and protein aggregates, is also implicated in PD and PD-related mutations, and environmental toxins deregulate autophagy. However, experimental evidence suggests a complex and ambiguous role of autophagy in PD since either impaired or abnormally upregulated autophagic flux has been shown to cause neuronal loss. Finally, it is generally believed that oxidative stress is a strong proautophagic stimulus. However, some evidence coming from neurobiology as well as from other fields indicate an inhibitory role of reactive oxygen species and reactive nitrogen species on the autophagic machinery. This review examines the scientific evidence supporting different concepts on how autophagy is dysregulated in PD and attempts to reconcile apparently contradictory views on the role of oxidative stress in autophagy regulation. The complex relationship between autophagy and oxidative stress is also considered in the context of the ongoing search for a novel PD therapy.

Spinal ceramide modulates the development of morphine antinociceptive tolerance via peroxynitrite-mediated nitroxidative stress and neuroimmune activation.

The effective treatment of pain is typically limited by a decrease in the pain-relieving action of morphine that follows its chronic administration (tolerance). Therefore, restoring opioid efficacy is of great clinical importance. In a murine model of opioid antinociceptive tolerance, repeated administration of morphine significantly stimulated the enzymatic activities of spinal cord serine palmitoyltransferase, ceramide synthase, and acid sphingomyelinase (enzymes involved in the de novo and sphingomyelinase pathways of ceramide biosynthesis, respectively) and led to peroxynitrite-derive nitroxidative stress and neuroimmune activation [activation of spinal glial cells and increase formation of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6]. Inhibition of ceramide biosynthesis with various pharmacological inhibitors significantly attenuated the increase in spinal ceramide production, nitroxidative stress, and neuroimmune activation. These events culminated in a significant inhibition of the development of morphine antinociceptive tolerance at doses devoid of behavioral side effects. Our findings implicate ceramide as a key upstream signaling molecule in the development of morphine antinociceptive tolerance and provide the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells

The role of the L‐arginine‐nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) was investigated. Incubation of T 67 astroglial cell line with IL‐β (10 ng ml−1) and TNF‐α (500 u ml−1) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. Nω‐nitro‐L‐arginine methyl ester (L‐NAME; 20–300 μM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine‐treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL‐1β and TNF‐α was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 μM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 μM). The inhibition elicited by L‐NAME on PGE2‐release by cytokine‐treated astroglial cells was reversed by adding AA (40 μM), showing that the effect of NO on cytokine‐dependent PGE2 release occurred at the cyclo‐oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine‐treated astroglial cells was inhibited by indomethacin (10 μM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 μM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX‐2) was involved. On the other hand, pretreating astroglial cells with methylene blue (MB; 10 μM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine‐treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites. The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL‐1β and TNF‐α is due to enhanced COX‐2 activity via activation of the L‐arginine‐NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV‐infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV‐infected M/M on an astrocytic cell‐line lacking CD4‐receptor expression. Exposure to supernatants of HIV‐infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV‐DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV‐infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces <10% apoptosis, and gp120 was totally ineffective. Treatment of HIV‐infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death.