Lipoic/Capsaicin-Related Amides: Synthesis and Biological Characterization of New TRPV1 Agonists Endowed with Protective Properties against Oxidative Stress.

α-Lipoic acid is a sulfur-containing nutrient endowed with pleiotropic actions and a safe biological profile selected to replace the unsaturated alkyl acid of capsaicin with the aim of obtaining lipoic amides potentially active as a TRPV1 ligand and with significant antioxidant properties. Thus, nine compounds were obtained in good yields following a simple synthetic procedure and tested for their functional TRPV1 activity and radical-scavenger activity. The safe biological profile together with the protective effect against hypoxia damage as well as the in vitro antioxidant properties were also evaluated. Although less potent than capsaicin, almost all lipoic amides were found to be TRPV1 agonists and, specifically, compound 4, the lipoic analogue of capsaicin, proved to be the best ligand in terms of efficacy and potency. EPR experiments and in vitro biological assays suggested the potential protective role against oxidative stress of the tested compounds and their safe biological profile. Compounds 4, 5 and 9 significantly ameliorated the mitochondrial membrane potential caused by hypoxia condition and decreased F2-isoprostanes, known markers of oxidative stress. Thus, the experimental results encourage further investigation of the therapeutic potential of these lipoic amides.

The Shc protein RAI promotes an adaptive cell survival program in hypoxic neuroblastoma cells.

Neuroblastoma (NB) is a highly malignant pediatric solid tumor where a hypoxic signature correlates with unfavorable patient outcome. The hypoxia-inducible factor (HIF)-1α plays an important role in NB progression, contributing to cell proliferation and invasiveness. RAI belongs to the Shc family proteins, it is mainly neuron specific and protects against cerebral ischemia. RAI is also expressed in several NB cell lines, where it promotes cell survival. In this work, hypoxia differently affected cell survival and pro-apoptotic program in two NB cell lines, either expressing RAI (SKNBE) or not (SKNMC). RAI expression appeared to promote NB cell survival and to reduce some pro-apoptotic markers under hypoxia. Accordingly, the RAI silencing in SKNBE cells resulted in a reduction of cell survival and HIF-1α expression. Furthermore, using SKNMC cells stably expressing RAI, we defined a role of RAI in NB cell responses to hypoxia. Of interest, in hypoxic SKNMC cells expressing RAI HIF-1α protein levels were higher than in control cells. This was associated with a) an increased cell survival; b) an increased expression of anti-apoptotic markers; c) a pro-autophagic and not pro-apoptotic phenotype; and d) an increased metabolic activity. We may conclude that RAI plays an important role in hypoxic signaling in NB cells and the interplay between RAI and HIF-1α may be relevant in the protection of NB cells against hypoxia. Our results may contribute to a further understanding the physiology of NB cells and the molecular mechanisms involved in their survival, with important implications in NB progression.

Different Adaptive Responses to Hypoxia in Normal and Multiple Myeloma Endothelial Cells.

BACKGROUND/AIMS: Hypoxia is a powerful stimulator of angiogenesis under physiological as well as pathological conditions. Normal endothelial cells (EC), such as human umbilical vein EC (HUVEC), are relatively affected by hypoxic insult in terms of cell survival. In contrast, EC from tumors are particularly resistant to hypoxia-induced cell death. Previous reports have shown that EC in bone marrow from multiple myeloma (MM) patients had a hypoxic phenotype, even under normoxic conditions. The aim of this study was to evaluate whether HUVEC and MMEC adapt differently to hypoxia. METHODS: Cell proliferation was assessed by the CyQUANT assay. Cdc25A, p21, Bax, Bcl-xl, BNIP3, glucose transporter (GLUT)-1, monocarboxylate transporter (MCT)-4 and carbonic anhydrase (CA)IX mRNA expression was determined by qRT-PCR. HIF-1α, BNIP3, Beclin-1, LC3B, livin, Bax, Bcl-xl, p21, p62 and ß-actin protein expression was analyzed by western blot. Apoptosis was determined by TUNEL assay. Silencing of BNIP3 was achieved by stealth RNA system technology. RESULTS: While HUVEC survival was reduced after prolonged hypoxic exposure, MMEC were completely unaffected. This difference was also significant in terms of livin, cdc25A and p21 expression. Hypoxia induced apoptosis and inhibited autophagy in HUVEC, but not in MMEC, where hypoxic treatment resulted in a more sustained adaptive response. In fact, MMEC showed a more significant increase in the expression of genes regulated transcriptionally by hypoxia-inducible factor (HIF)-1α. Interestingly, they showed higher expression of BNIP3 than did HUVEC, indicating a more pronounced autophagic (and pro-survival) phenotype. The potential role of BNIP3 in EC survival was confirmed by BNIP3 siRNA experiments in HUVEC, where BNIP3 inhibition resulted in reduced cell survival and increased apoptosis. CONCLUSION: These findings provide further information on how hypoxia may affect EC survival and could be important for a better understanding of EC physiology under normal and pathological conditions, such as in multiple myeloma.

Neuroprotective Effects of Castanea sativa Mill. Bark Extract in Human Neuroblastoma Cells Subjected to Oxidative Stress.

One of the major features of neurodegenerative disease is the selective vulnerability of different neuronal populations that are affected in a progressive and often stereotyped manner. Despite the susceptible neuronal population varies between diseases, oxidative stress is implicated as the major pathogenic process in all of them. Natural Extract of Castanea sativa Mill. bark (ENC), recently characterized in its phenolic composition, acts as antioxidant and cardioprotective agent. Its neuroprotettive properties, however, have never been investigated. The aim of this study was to assess neuroprotection of ENC in in vitro models of oxidative-stress-mediate injury. Human neuroblastoma SH-SY5Y cells treated with glutamate (50 mM for 24 h) or hydrogen peroxide (25 µM for 1 h followed by 24 with medium) were used. The results showed that the addition of ENC (1-50 µg/ml) to cell medium before the neuronal damage provided neuroprotection in both experimental models used, while its addition after the injury was ineffective. In conclusion, the present results suggest that ENC could be a valuable support as dietary supplement, combining beneficial preventive neuroprotettive effects with a high antioxidant activity.

Short-term hypoxia enhances the migratory capability of dendritic cell through HIF-1α and PI3K/Akt pathway.

Hypoxia represents an inadequate oxygen supply to tissues, which can modulate cell functions, primarily through the hypoxia-inducible transcription factor HIF-1α. Dendritic cells (DC) are professional antigen-presenting cells and their migration maybe affected by hypoxia, since the local microenvironment in lymphoid organs, as well as in inflamed and tumor tissues, is characterized by low oxygen tensions. In this study we observed an enhanced migratory capability of human monocyte-derived DC, using in vitro migration assays performed under hypoxic conditions. Such enhancement was independent on either the chemoattractants involved or the maturation level of DC. However, HIF-1α appeared to be crucial for the migration only of immature DC and not for mature DC under hypoxia, as indicated by HIF-1α siRNA approaches. Furthermore, we observed that while Akt phosphorylation was enhanced in both immature and mature DC exposed to hypoxia, other signaling pathways, such as p38 and p42/p44 MAPK, were differently affected during hypoxic treatment. More interestingly, aspecific and specific inhibition of PI3K/Akt indicated that such pathway was relevant for the migration of both immature and matured DC under hypoxia, even when DC were transfected with HIF-1α siRNA. Our results indicate that, besides HIF-1α, several other pathways, including PI3K/Akt, may be involved in the response to hypoxia of immature and, more specifically, of mature DC to sustain their trafficking and functions within hypoxic microenvironments.

SQSTM1/p62 inhibition impairs pro-survival signaling in hypoxic human dendritic cells.

The sequestosome 1 (SQSTM1)/p62 is an adaptor protein which plays multiple roles in several cell functions, including cell survival and autophagy. Dendritic cells (DCs) are the most prominent antigen presenting cells and during their lifespan they are exposed to different oxygen tensions, including hypoxia. By using a siRNA approach we found out that p62 was implicated in the maintenance of Erk1/2 phosphorylation and preservation of hypoxic DC survival, as well as in the reduction of AMPK activation. Thus, p62 expression in DCs in hypoxic microenvironments, such as in the lymphoid organs, may extend their lifespan to ensure their functions.

A novel CB2 agonist, COR167, potently protects rat brain cortical slices against OGD and reperfusion injury.

Cannabinoid CB2 receptor activation has been shown to have many pharmacological but not psychotropic effects. The aim of this study was to investigate the potential protection of brain tissues afforded by the novel substituted 4-quinolone-3-carboxylic acid derivative COR167, a selective CB2 agonist, toward ischemia and reperfusion-induced injury, as well as the mechanism of this potential effect. Rat brain cortical slices subjected to oxygen and glucose deprivation (OGD) followed by re-oxygenation were used. Cell damage was quantified by measuring at the end of the reperfusion phase the release into the artificial cerebrospinal fluid (ACSF) of lactate dehydrogenase (LDH), glutamate, IL-6 and TNF-α and by evaluating in tissue the lipid-peroxides (thiobarbituric acid-reactive substances, TBARS), the free, reduced glutathione content (GSH) and the water gain (TWG), taken as an index of cell swelling. COR167 (10nM or 100 nM), added to ACSF during the entire reperfusion phase, markedly reduced LDH and glutamate release, as well as TWG. Lower (0.1-1 nM) or higher concentrations (1,000 nM) were ineffective, suggesting thereby an hormetic behavior. COR167 at 10nM concentration markedly reverted in tissues TBARS increase and GSH decrease, while reducing IL-6 and TNF-α release into ACSF. COR167 effects on glutamate and LDH release were abrogated by the selective CB2 inverse-agonists COR170 (1 nM) and AM630 (1µM) but not by the CB1 antagonist AM251 (1 µM). COR170 as well as AM630 per se were able to revert TWG. The CB2 receptor agonist COR167 potently protected rat brain cortical slices against OGD and reperfusion injury, partly through CB2 receptors activation.

In vitro inhibition of human and rat platelets by NO donors, nitrosoglutathione, sodium nitroprusside and SIN-1, through activation of cGMP-independent pathways.

Three different NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine hydrochloride (SIN-1) were used in order to investigate mechanisms of platelet inhibition through cGMP-dependent and -independent pathways both in human and rat. To this purpose, we also evaluated to what extent cGMP-independent pathways were related with the entity of NO release from each drug. SNP, GSNO and SIN-1 (100 µM) effects on platelet aggregation, in the presence or absence of a soluble guanylate cyclase inhibitor (ODQ), on fibrinogen receptor (α(IIb)ß(3)) binding to specific antibody (PAC-1), and on the entity of NO release from NO donors in human and rat platelet rich plasma (PRP) were measured. Inhibition of platelet aggregation (induced by ADP) resulted to be greater in human than in rat. GSNO was the most powerful inhibitor (IC(50) values, µM): (a) in human, GSNO=0.52±0.09, SNP=2.83 ± 0.53, SIN-1=2.98 ± 1.06; (b) in rat, GSNO = 28.4 ± 6.9, SNP = 265 ± 73, SIN-1=108 ± 85. GSNO action in both species was mediated by cGMP-independent mechanisms and characterized by the highest NO release in PRP. SIN-1 and SNP displayed mixed mechanisms of inhibition of platelet aggregation (cGMP-dependent and independent), except for SIN-1 in rat (cGMP-dependent), and respectively lower or nearly absent NO delivery. Conversely, all NO-donors prevalently inhibited PAC-1 binding to α(IIb)ß(3) through cGMP-dependent pathways. A modest relationship between NO release from NO donors and cGMP-independent responses was found. Interestingly, the species difference in NO release from GSNO and inhibition by cGMP-independent mechanism was respectively attributed to S-nitrosylation of non-essential and essential protein SH groups.

The effects of hypoxia/reoxygenation on the physiological behaviour of U373-MG astrocytes.

Nerve cells are very susceptible to hypoxia responsive for mitochondrial dysfunctions involved in the subsequent oxidative stress, apoptosis and necrosis. In this paper, we examined the effect of 12 h incubation of U-373 MG astrocytes in hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell proliferation, modifications of NO and ATP production, intracellular Ca(2+) concentration [Ca(2+)](i), membrane potential, desferoxamine-chelatable free iron, esterified F2-isoprostanes levels and the production of phosphorylated ERK. The same parameters were evaluated also after a following re-oxygenation period of 24 h. Immediately after hypoxia the NO concentration increased significantly and returned to values similar to those of controls after the re-oxygenation period. At the same time, ATP levels remained similar to controls and the cell proliferation significantly decreased. This involved a significant increase of [Ca(2+)](i) immediately after hypoxia and the value remained significantly elevated after the following re-oxygenation period. Moreover, after hypoxia, astrocytes were slightly although not significantly depolarized. Indeed iron and F2-isoprostanes levels increased significantly after hypoxia. Finally ERK proteins increased slowly and not significantly after hypoxia and the same trend was observed after the re-oxygenation period. On the whole, our results indicate that 2% O(2) hypoxia induces a moderate oxidative stress, well tolerated by U-373 MG cells, remaining the ATP production, mitochondrial membrane potential and activated ERK proteins, similar to the values of controls.

The physiological behaviour of IMR-32 neuroblastoma cells is affected by a 12-h hypoxia/24-h reoxygenation period.

Nervous system cells are highly dependent on adequate tissue oxygenation and are very susceptible to hypoxia, which causes mitochondrial dysfunctions involved in apoptosis and necrosis. In this paper, we examine the effect of a 12-h incubation of differentiated IMR-32 neuroblastoma cells in a hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell viability, modifications of NO, intracellular Ca(2+) concentration [Ca(2+)](i) and membrane potential, the production of phosphorylated ERK, desferoxamine-chelatable free iron and esterified F2-isoprostane levels. The same parameters were evaluated after a subsequent 24-h re-oxygenation period. The NO concentration increased significantly immediately after hypoxia and returned to values similar to those of controls after the reoxygenation period. At the same time, we observed a significant increase of [Ca(2+)](i) immediately after hypoxia. Phosphorylated ERK proteins increased significantly during the first 2 h of hypoxia, then decreased, and remained practically unmodified after 12 h hypoxia and the following reoxygenation period. Moreover, IMR-32 cell mitochondria were significantly depolarized after hypoxia, while membrane potential returned to normal after the reoxygenation period. Finally, desferoxamine-chelatable free iron and F2-isoprostane levels also increased significantly after hypoxia. Our results indicate that 2% O(2) hypoxia induces variations of NO and [Ca(2+)](i) with subsequent mitochondrial depolarization, and it is responsible for oxidative stress, represented by increased free iron and F2-isoprostane, protein carbonyls and 4 hydroxynonenal protein adducts levels.

Hypoxia Shapes Autophagy in LPS-Activated Dendritic Cells.

During their lifespan, dendritic cells (DCs) are exposed to different pO2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.

Hypoxia affects the physiological behavior of rat cortical synaptosomes.

Nerve cells, especially synaptosomes, are very susceptible to hypoxia and the subsequent oxidative stress. In this paper, we examined the effects of hypoxia (93% N(2):2% O(2):5% CO(2), v/v/v) on rat cortical synaptosomes by evaluating modifications of synaptosomal mitochondrial respiration rate and ATP production, membrane potential, intrasynaptosomal mitochondrial Ca(2+) concentration ([Ca(2+)](i)), and desferoxamine-chelatable free iron and esterified F2-isoprostane levels after different periods of hypoxia and after 30 min of reoxygenation. Oxygen consumption decreased significantly during 120 min of hypoxia and was restored after reoxygenation. At the same time, ATP production decreased and remained significantly lower even after reoxygenation. This involved a depolarization of the synaptosomal mitochondrial membrane, although the [Ca(2+)](i) remained practically unchanged. Indeed, iron and F2-isoprostane levels, representing useful prediction markers for neurodevelopmental outcome, increased significantly after hypoxia, and there was a strong correlation between the two variables. On the whole our results indicate that synaptosomal mitochondria undergo mitoptosis after 2 h of hypoxia.

Ozonation of human blood induces a remarkable upregulation of heme oxygenase-1 and heat stress protein-70.

Heme oxygenase-I (HO-1) has emerged as one of the most protective enzymes and its pleiotropic activities have been demonstrated in a variety of human pathologies. Unpublished observations have shown that HO-1 is induced after the infusion of ozonated blood into the respective donors, and many other experimental observations have demonstrated the efficacy of oxidizing agents. It appeared worthwhile to evaluate whether we could better define the activity of potential inducers such as hydrogen peroxide and ozonated human plasma. Human vascular endothelial cells at confluence were challenged with different concentrations of these inducers and the simultaneous production of nitric oxide (NO); and HO-1 was measured by either measuring nitrite, or bilirubin formation, or/and the immune reactivity of the protein by Western blot using a rabbit antihuman HO-1 and Hsp-70. The results show that production of both NO and HO-1 is fairly dose dependent but is particularly elevated using human plasma after transient exposure to a medium ozone concentration. At this concentration, there is also induction of Hsp-70. The results clarify another positive effect achievable by the use of ozone therapy.

Design and Synthesis of New Transient Receptor Potential Vanilloid Type-1 (TRPV1) Channel Modulators: Identification, Molecular Modeling Analysis, and Pharmacological Characterization of the N-(4-Hydroxy-3-methoxybenzyl)-4-(thiophen-2-yl)butanamide, a Small Molecule Endowed with Agonist TRPV1 Activity and Protective Effects against Oxidative Stress.

4-(Thiophen-2-yl)butanoic acid was identified as a cyclic substitute of the unsaturated alkyl chain of the natural ligand, capsaicin. Accordingly, a new class of amides was synthesized in good yield and high purity and their molecular recognition against the target was investigated by means of docking experiments followed by molecular dynamics simulations, in order to rationalize their geometrical and thermodynamic profiles. The pharmacological properties of these new compounds were expressed as activation (EC50) and desensitization (IC50) potencies. Several compounds were found to activate TRPV1 channels, and in particular, derivatives 1 and 10 behaved as TRPV1 agonists endowed with good efficacy as compared to capsaicin. The most promising compound 1 was also evaluated for its protective role against oxidative stress on keratinocytes and differentiated human neuroblastoma cell lines expressing the TRPV1 receptor as well as for its cytotoxicity and analgesic activity in vivo.

Iron release, superoxide production and binding of autologous IgG to band 3 dimers in newborn and adult erythrocytes exposed to hypoxia and hypoxia-reoxygenation.

Iron is released in a desferrioxamine (DFO)-chelatable form when erythrocytes are challenged by an oxidative stress. The release is increased when an accelerated removal of erythrocytes occurs such as in perinatal period, in which iron release is greater in hypoxic than in non-hypoxic newborns. This suggests that an hypoxic environment at birth promotes iron release. To test this possibility, iron release in a model of hypoxia, hypoxia-reoxygenation and normoxia was studied in newborn and adult erythrocytes. In newborn erythrocytes, hypoxia induced a much greater iron release compared to an equal period of normoxia. In adult erythrocytes, hypoxia also induced a greater iron release as compared to normoxia, but it was much lower than that seen with newborn erythrocytes. Methemoglobin (MetHb) formation roughly paralleled iron release. The phenylhydrazine-promoted superoxide anion (O(2)?(-)) production was greater with normoxic but lower with hypoxic erythrocytes from newborns as compared to that from adults. This discrepancy between iron release and O(2)?(-) production may be explained by the shift towards MetHb in hemoglobin autoxidation. Iron diffusion out of the erythrocytes was much higher with hypoxic erythrocytes from newborns as compared to that from adults. Also the binding of autologous IgG to band 3 dimers (AIgGB) is much greater with hypoxic erythrocytes from newborns as compared to that from adults, suggesting that the level of iron release is related to the extent of band 3 clustering and that hypoxia accelerates removal of erythrocytes from bloodstream in in vivo condition.

NMR metabolomic investigation of astrocytes interacted with Aß42 or its complexes with either copper(II) or zinc(II).

Alzheimer's disease (AD) is the leading cause of senile dementia. One of the main hallmarks of AD is the presence of amyloid plaques in the brain, primarily formed by fibrils of the amyloid-ß (Aß) peptide. Transition metal ions, such as Cu(2+) and Zn(2+) have been found at high concentrations in senile plaques isolated from AD patients and evidence have been reached that (i) Aß aggregation is greatly affected by Cu(2+) and Zn(2+) and (ii) Cu(2+), implicated in the formation of reactive oxygen species, leads to mitochondrial dysfunctions ultimately leading to neuronal cells death. Aß, apart from being toxic to neural cells, induces reactive astrocytosis in cell culture. Astrocytes play many crucial roles to sustain normal brain function by maintaining the cerebral homeostasis, modulating the synaptic transmission, and providing a metabolic support for neuronal growth. Although many studies have shown that Aß fibrils interfere in the main astrocytic functions aimed at supporting the neuronal activity, nothing is known about the effects of Zn(2+)- and Cu(2+)-induced Aß aggregates on astrocyte functions. In this study the effects of treatments with Aß(42), either in absence or in the presence of Cu(2+) and Zn(2+), on astrocyte cell cultures were evaluated by using classical cellular assay and by looking at changes in metabolic profiles in the cellular medium by using nuclear magnetic resonance spectroscopy (NMR). Our results indicate that metal induced Aß aggregation strongly affects the metabolites involved in the neurotransmission activity supporting a deleterious impact of Cu(2+) and Zn(2+) Aß amyloidogenesis on astrocyte functions.

Rottlerin exhibits antiangiogenic effects in vitro.

Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF-7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D-1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα-stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D-1 and endothelin-1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme-1 expression, thus contributing to the drop in endothelin-1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis.

Biochemical modifications induced in human blood by oxygenation-ozonation.

Some biochemical effects determined on human blood after addition of a gas mixture composed of oxygen (approximately 96%) and ozone (approximately 4%) have been evaluated. Ozone was used in a mild concentration ranging between 0.21 and 1.68 mM. Within few minutes after rapid mixing of the equal gas-liquid volumes, the ozone was consumed because by instantaneously reacting with biomolecules, generating reactive oxygen species (particularly hydrogen peroxide) having very short lifetime and lipid oxidation products. The following results are oxygen-ozone dose dependent: (1) The pO(2) values have risen from about 40 up to 400 mmHg. (2) By testing the highest ozone concentration, the total antioxidant capacity of blood decreased within 1 min from 1.35 to 0.91 mM but regained its normal values within 20 min owing to the rapid reduction of oxidized antioxidants operated by erythrocytes. (3) Similarly, intraerythrocytic reduced glutathione after ozonation decreased from the initial value of 5.71 to 4.56 micromol/g Hb. (4) Both hemolysis and methemoglobin showed a negligible increase.

Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell lines.

To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 microm) determined a gradual, moderate elevation in [Ca2+]i (46.8 +/- 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 +/- 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 +/- 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50-300 microm) of DETA/NO negatively regulated cell proliferation via a Ca2+-independent mechanism.

Potentiation of intracellular Ca2+ mobilization by hypoxia-induced NO generation in rat brain striatal slices and human astrocytoma U-373 MG cells and its involvement in tissue damage.

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic-ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low-oxygen and Ca2+-free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia-induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine-NO and inhibited by N-omega-nitro-L-arginine methyl ester (L-NAME) or by the NO scavenger 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A membrane-permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U-373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 +/- 13.1% compared to normoxic controls and this effect was inhibited by L-NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L-NAME. Although heparin plus RR or RY antagonized hypoxia-induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO-Ca2+ signalling might be a potential therapeutic target in hypoxia-induced neuronal degeneration.