Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3β pathway

Mitochondrial perturbation and oxidative stress are key factors in neuronal vulnerability in several neurodegenerative diseases or during brain ischemia. Here we have investigated the protective mechanism of action of guanosine, the guanine nucleoside, in a human neuroblastoma cell line, SH-SY5Y, subjected to mitochondrial oxidative stress. Blockade of mitochondrial complexes I and V with rotenone plus oligomycin (Rot/oligo) caused a significant decrease in cell viability and an increase in ROS production. Guanosine that the protective effect of guanosine incubated concomitantly with Rot/oligo abolished Rot/oligo-induced cell death and ROS production in a concentration dependent manner; maximum protection was achieved at the concentration of 1mM. The cytoprotective effect afforded by guanosine was abolished by adenosine A(1) or A(2A) receptor antagonists (DPCPX or ZM241385, respectively), or by a large (big) conductance Ca(2+)-activated K(+) channel (BK) blocker (charybdotoxin). Evaluation of signaling pathways showed that the protective effect of guanosine was not abolished by a MEK inhibitor (PD98059), by a p38(MAPK) inhibitor (SB203580), or by a PKC inhibitor (cheleritrine). However, when blocking the PI3K/Akt pathway with LY294002, the neuroprotective effect of guanosine was abolished. Guanosine increased Akt and p-Ser-9-GSK-3β phosphorylation confirming this pathway plays a key role in guanosine's neuroprotective effect. Guanosine induced the antioxidant enzyme heme oxygenase-1 (HO-1) expression. The protective effects of guanosine were prevented by heme oxygenase-1 inhibitor, SnPP. Moreover, bilirubin, an antioxidant and physiologic product of HO-1, is protective against mitochondrial oxidative stress. In conclusion, our results show that guanosine can afford protection against mitochondrial oxidative stress by a signaling pathway that implicates PI3K/Akt/GSK-3β proteins and induction of the antioxidant enzyme HO-1.     

Ethyl pyruvate-mediated Nrf2 activation and hemeoxygenase 1 induction in astrocytes confer protective effects via autocrine and paracrine mechanisms

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule under various pathological conditions, such as, during cerebral ischemia and sepsis in animal models. Here, the authors investigated the novel molecular mechanism underlying the anti-oxidative effect of EP in primary astrocyte cultures, particularly with respect to nuclear factor E2-related factor 2 (Nrf2) activation and hemeoxygenase 1 (HO-1) induction. EP was found to induce Nrf2 translocation and the inductions of various genes downstream of Nrf2 and these resulted in the amelioration of the oxidative damage of H(2)O(2). Furthermore, EP dose-dependently suppressed H(2)O(2)-induced astrocyte cell death (12h preincubation with 5mM EP increased cell survival after 1h exposure to 100 μM H(2)O(2) from 32.6±0.7% to 63±1.8%). HO-1 was markedly induced (4.9-fold) in EP-treated primary astrocyte cultures and Nrf2 was found to translocate from the cytosol to the nucleus and bind to the antioxidant response element (ARE) located on HO-1 promoter after EP treatment. siRNA-mediated HO-1 or Nrf2 knockdown and zinc protoporphyrin (ZnPP)-mediated inhibition of HO-1 activity showed that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of EP, which was found to involve the ERK and Akt signaling pathways. Furthermore, EP-conditioned astrocyte culture media was found to have neuroprotective effects on primary neuronal cultures exposed to oxidative or excitotoxic stress, and this seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and glutathione (GSH), which accumulated in EP-treated astrocyte culture media. Interestingly, we also found that in addition to HO-1, EP-induced Nrf2 activation increased the expressions of various anti-oxidant genes, including GST, NQO1, and GCLM. The study shows that EP-mediated Nrf2 activation and HO-1 induction in astrocytes act via autocrine and paracrine mechanisms to confer protective effects.     

Adenosine receptors as a new target for resveratrol-mediated glioprotection

Resveratrol, a natural polyphenolic compound, has been studied as a neuroprotective molecule. Our group has demonstrated that such effect is closely associated with modulation of glial functionality, but the underlying mechanisms are not fully understood. Because astrocytes actively participate in the brain inflammatory response, and activation of adenosine receptors can attenuate inflammatory processes, the aim of this study was to investigate the role of adenosine receptors as a mechanism for resveratrol glioprotection, particularly regarding to neuroinflammation. Therefore, primary astrocyte cultures were co-incubated with resveratrol and selective antagonists of A₁, A_2A, and A₃ adenosine receptors, as well as with caffeine (a non-selective adenosine receptor antagonist), and then challenged with bacterial inflammogen lipopolysaccharide (LPS). Caffeine and selective adenosine receptor antagonists abolished the anti-inflammatory effect of resveratrol. In accordance with these effects, resveratrol prevented LPS-induced decrease in mRNA levels of adenosine receptors. Resveratrol could also prevent the activation of pro-inflammatory signaling pathways, such as nuclear factor κB (NFκB) and p38 mitogen-activated protein kinase (p38 MAPK) in a mechanism dependent on adenosine receptors. Conversely, trophic factors and protective signaling pathways, including sirtuin 1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and phosphoinositide 3-kinase (PI3K)/Akt were positively modulated by resveratrol in both LPS-stimulated and unstimulated astrocytes, but adenosine receptor antagonism did not abrogate all effects of resveratrol. To our knowledge, our data provide the first evidence that adenosine receptors are involved in the anti-inflammatory activity of resveratrol in astrocytes, thus exerting an important role for resveratrol-mediated glioprotection.     

Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3,5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). -Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.     

Guanine Nucleotide-Binding Protein Regulation of Microsomal Phospholipase D Activity of Canine Cerebral Cortex

The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline and 1-palmitoyl-2-[9,10(n)-3H]palmitoyl-sn-glycerol-3-phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X-100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 mM, but zinc was greatly inhibitory, causing a loss of greater than 90% activity at the 4 mM concentration. Non-hydrolyzable guanine nucleotide analogues such as guanosine 5'-(3-O-thio)triphosphate and guanyl-5'-yl-(beta, gamma-methylene)diphosphonate but not guanosine 5'-(2-thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5'-(2-thio)diphosphate was capable of partially blocking guanosine 5'-(3-O-thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was able to cause a two- to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet-activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide-binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.     

Gliopreventive effects of guanosine against glucose deprivation in vitro

Guanosine, a guanine-based purine, is recognized as an extracellular signaling molecule that is released from astrocytes and confers neuroprotective effects in several in vivo and in vitro studies. Astrocytes regulate glucose metabolism, glutamate transport, and defense mechanism against oxidative stress. C6 astroglial cells are widely used as an astrocyte-like cell line to study the astrocytic function and signaling pathways. Our previous studies showed that guanosine modulates the glutamate uptake activity, thus avoiding glutamatergic excitotoxicity and protecting neural cells. The goal of this study was to determine the gliopreventive effects of guanosine against glucose deprivation in vitro in cultured C6 cells. Glucose deprivation induced cytotoxicity, an increase in reactive oxygen and nitrogen species (ROS/RNS) levels and lipid peroxidation as well as affected the metabolism of glutamate, which may impair important astrocytic functions. Guanosine prevented glucose deprivation-induced toxicity in C6 cells by modulating oxidative and nitrosative stress and glial responses, such as the glutamate uptake, the glutamine synthetase activity, and the glutathione levels. Glucose deprivation decreased the level of EAAC1, the main glutamate transporter present in C6 cells. Guanosine also prevented this effect, most likely through PKC, PI3K, p38 MAPK, and ERK signaling pathways. Taken together, these results show that guanosine may represent an important mechanism for protection of glial cells against glucose deprivation. Additionally, this study contributes to a more thorough understanding of the glial- and redox-related protective properties of guanosine in astroglial cells.     

Sulforaphane Suppresses Hepatitis C Virus Replication by Up-Regulating Heme Oxygenase-1 Expression through PI3K/Nrf2 Pathway

Hepatitis C virus (HCV) infection-induced oxidative stress is a major risk factor for the development of HCV-associated liver disease. Sulforaphane (SFN) is an antioxidant phytocompound that acts against cellular oxidative stress and tumorigenesis. However, there is little known about its anti-viral activity. In this study, we demonstrated that SFN significantly suppressed HCV protein and RNA levels in HCV replicon cells and infectious system, with an IC₅₀ value of 5.7 ± 0.2 μM. Moreover, combination of SFN with anti-viral drugs displayed synergistic effects in the suppression of HCV replication. In addition, we found nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 induction in response to SFN and determined the signaling pathways involved in this process, including inhibition of NS3 protease activity and induction of IFN response. In contrast, the anti-viral activities were attenuated by knockdown of HO-1 with specific inhibitor (SnPP) and shRNA, suggesting that anti-HCV activity of SFN is dependent on HO-1 expression. Otherwise, SFN stimulated the phosphorylation of phosphoinositide 3-kinase (PI3K) leading Nrf2-mediated HO-1 expression against HCV replication. Overall, our results indicated that HO-1 is essential in SFN-mediated anti-HCV activity and provide new insights in the molecular mechanism of SFN in HCV replication.     

Effects of guanosine on insulin secretion and adenylyl and guanylyl cyclase activities of isolated rat islets of Langerhans

The effect of guanosine on insulin secretion adenylyl and guanylyl cyclase activities of isolated rat islets of Langerhans was investigated. Guanosine (1–100 μM) inhibited glucose, tolbutamide, theophylline and prostaglandin E₂-stimulated insulin secretion although it failed to affect glucagon stimulated secretion. Prostaglandin E₂-stimulated adenylyl cyclase of islets was inhibited by guanosine although guanosine had no effect on basal, fluoride, glucagon or GTP-stimulated activity. Guanosine markedly decreased basal guanylyl cyclase activity of islets.These results suggest that guanosine may affect insulin release by inhibiting adenylyl and guanylyl cyclase activities in the ß-cell thereby decreasing the intracellular concentrations of cyclic nucleotides.This effect may be important in modulating the secretory response of the islets to a variety of hormonal agents.     

Possible Role of Heme Oxygenase-1 and Prostaglandins in the Pathogenesis of Cerebral Malaria: Heme Oxygenase-1 Induction by Prostaglandin D2 and Metabolite by a Human Astrocyte Cell Line

Astrocytes are the most abundant cells in the central nervous system that play roles in maintaining the blood-brain-barrier and in neural injury, including cerebral malaria, a severe complication of Plasmodium falciparum infection. Prostaglandin (PG) D₂ is abundantly produced in the brain and regulates the sleep response. Moreover, PGD₂ is a potential factor derived from P. falciparum within erythrocytes. Heme oxygenase-1 (HO-1) is catalyzing enzyme in heme breakdown process to release iron, carbon monoxide, and biliverdin/bilirubin, and may influence iron supply to the P. falciparum parasites. Here, we showed that treatment of a human astrocyte cell line, CCF-STTG1, with PGD₂ significantly increased the expression levels of HO-1 mRNA by RT-PCR. Western blot analysis showed that PGD₂ treatment increased the level of HO-1 protein, in a dose- and time-dependent manner. Thus, PGD₂ may be involved in the pathogenesis of cerebral malaria by inducing HO-1 expression in malaria patients.     

Guanosine prevents nitroxidative stress and recovers mitochondrial membrane potential disruption in hippocampal slices subjected to oxygen/glucose deprivation

Guanosine, the endogenous guanine nucleoside, prevents cellular death induced by ischemic events and is a promising neuroprotective agent. During an ischemic event, nitric oxide has been reported to either cause or prevent cell death. Our aim was to evaluate the neuroprotective effects of guanosine against oxidative damage in hippocampal slices subjected to an in vitro ischemia model, the oxygen/glucose deprivation (OGD) protocol. We also assessed the participation of nitric oxide synthase (NOS) enzymes activity on the neuroprotection promoted by guanosine. Here, we showed that guanosine prevented the increase in ROS, nitric oxide, and peroxynitrite production induced by OGD. Moreover, guanosine prevented the loss of mitochondrial membrane potential in hippocampal slices subjected to OGD. Guanosine did not present an antioxidant effect per se. The protective effects of guanosine were mimicked by inhibition of neuronal NOS, but not of inducible NOS. The neuroprotective effect of guanosine may involve activation of cellular mechanisms that prevent the increase in nitric oxide production, possibly via neuronal NOS.     

Guanosine metabolism in Neurospora crassa

Two aspects of guanosine metabolism in Neurospora have been investigated. (a) The inability of adenine mutants (blocked prior to IMP synthesis) to use guanosine as a nutritional supplement; and (b) the inhibitory effect of guanosine on the utilization of hypoxanthine as a purine source for growth by these mutants. Studies on the utilization of guanosine indicated that the proportion of adenine derived from guanosine may be limiting for the growth of adenine mutants. In wild type, adenine is produced through the biosynthetic pathway when grown in the presence of guanosine. The amount of adenine produced through the de novo biosynthesis in wild type increases with increasing concentrations of guanosine in the medium. However, the total purine synthesis does not increase. Guanosine inhibits the uptake of hypoxanthine severely. In addition, guanosine and its nucleotide derivatives also inhibit the hypoxanthine phosphoribosyltransferase activity, at the same time stimulating the adenine phosphoribosyltransferase activity. Guanosine's effects on the uptake of hypoxanthine and its conversion to the nucleotide form may be the reasons why guanosine inhibits the utilization of hypoxanthine but not adenine by these mutants.     

Role of heme oxygenase-1 in the regulation of manganese superoxide dismutase gene expression in oxidatively-challenged astroglia

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme that reduces superoxide anion to hydrogen peroxide in cell mitochondria. MnSOD is overexpressed in normal aging brain and in various central nervous system disorders; however, the mechanisms mediating the upregulation of MnSOD under these conditions remain poorly understood. We previously reported that cysteamine (CSH) and other pro-oxidants rapidly induce the heme oxygenase-1 (HO-1) gene in cultured rat astroglia followed by late upregulation of MnSOD in these cells. In the present study, we demonstrate that antecedent upregulation of HO-1 is necessary and sufficient for subsequent induction of the MnSOD gene in neonatal rat astroglia challenged with CSH or dopamine, and in astroglial cultures transiently transfected with full-length human HO-1 cDNA. Treatment with potent antioxidants attenuates MnSOD expression in HO-1-transfected astroglia, strongly suggesting that intracellular oxidative stress signals MnSOD gene induction in these cells. Activation of this HO-1-MnSOD axis may play an important role in the pathogenesis of Alzheimer disease, Parkinson disease and other free radical-related neurodegenerative disorders. In these conditions, compensatory upregulation of MnSOD may protect mitochondria from oxidative damage accruing from heme-derived free iron and carbon monoxide liberated by the activity of HO-1.     

GDNF modulates HO-1 expression in substantia nigra postnatal cell cultures

Heme oxygenase-1 (HO-1) has been strongly highlighted because of its induction in many cell types by toxic stimuli, including oxidative stress. The intense HO-1 immunostaining in the substantia nigra of Parkinson disease (PD) patients suggests its involvement in the pathogenesis of this neurodegenerative disease. In this work we investigated HO-1 expression in rat substantia nigra postnatal cell cultures under conditions mimicking dopamine toxicity and its modulation by glial cell line-derived neurotrophic factor (GDNF), a potent neuroprotective factor for dopaminergic neurons. In neuron–glia cultures, we found that H₂O₂, a product of dopamine metabolism, or l-3,4-dihydroxyphenylalanine (l-DOPA), the dopamine precursor used in the therapy of PD, induced a fast up-regulation of HO-1 mRNA and protein levels, followed by a secondary down-regulation. H₂O₂ and l-DOPA also increased HO-1 expression in astrocyte cultures, but with a delayed time course in H₂O₂-treated cultures. HO-1 expression was decreased in neuron–glia cultures under conditions under which GDNF up-regulation was observed. Because exogenously applied GDNF prevented HO-1 up-regulation in cultures treated with H₂O₂ or l-DOPA, and antibody neutralization of GDNF prevented the secondary HO-1 down-regulation observed in neuron–glia cultures, we propose that GDNF negatively modulates HO-1 expression induced by oxidative stress. To our knowledge, this is the first report showing the modulation of HO-1 expression by GDNF.