Neuroendocrine cells of nasal mucosa are a cellular source of brain-derived neurotrophic factor.

There is growing evidence for extensive interaction between sensory neurons, immune and mucosal epithelial cells during airway inflammation and hyperreactivity. This neuro-immune cross-talk (neurogenic inflammation) involves different groups of mediators, which include the neurotrophin family (nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 and -4). Neurotrophins modulate airway inflammation by enhancing sensory nerve excitability and production of neuropeptides, and by interaction with different immune cell types. In the present study, it was questioned whether airway epithelial cells express BDNF, and if proinflammatory cytokines (tumour necrosis factor-alpha, interleukin-1beta and interferon-gamma) and a glucocorticoid (budesonide) affect this expression. Primary cultures of nasal epithelial cells were used. It was found that BDNF was stored in chromogranin A-containing secretory granules of specialised epithelial cells, i.e. neuroendocrine cells, and was secreted in a polarised manner. Apical secretion appears to be constitutive, whereas basolateral secretion is markedly enhanced upon stimulation with cytokines. This enhanced basolateral secretion was not due to enhanced synthesis and was not affected by inhibitors of the processing enzymes, such as furin and the metalloproteinases involved in the maturation of BDNF, but was considerably diminished by budesonide. Therefore, airway mucosa might contribute to neurogenic inflammation through increased secretion of brain-derived neurotrophic factor by neuroendocrine cells under inflammatory conditions.

BDNF and DPP-IV in polyps and middle turbinates epithelial cells.

HYPOTHESIS: Neuropeptides released from sensory nerves may contribute to airway inflammation, particularly if their metabolism is impaired through defective inactivation and/or increased production. In the airways, neuropeptides are subjected to degradation by enzymes such as dipeptidyl peptidase (DPP-IV), and are upregulated by neurotrophins such as brain derived neurotrophic factor (BDNF). We therefore assessed in primary human nasal epithelial cells the expression of DPP-IV and BDNF under basal and inflammatory conditions. METHODS: Human epithelial cells were isolated from nasal polyps and middle turbinates, and grown on collagen-coated polycarbonate filters with an air liquid-interface. After three weeks of culture, constitutive cellular expression of DPP-IV and BDNF was assessed by measuring its activity and by ELISA, respectively. To mimick in vivo inflammatory conditions, cells were exposed apically and basolaterally to 50 ng/ml of TNFalpha, IL-1beta, and IFN-gamma for two days. DPP-IV activity and BDNF protein expression were measured in cell lysates and in the apical and basolateral media. RESULTS: Constitutive DPP-IV activity was similar in polyps and turbinates cells. In contrast, polyps epithelial cells expressed higher amounts of BDNF compared to turbinates derived cells. On the other hand, TNFalpha, IL-1beta, and IFN-gamma did not affect DPP-IV activity but significantly increased the cellular expression and the basolateral secretion of BDNF. CONCLUSIONS: Our data show for the first time that primary human airway epithelial cells produced DPP-IV and BDNF under basal conditions. Furthermore, the production and secretion of BDNF were markedly increased in response to pro-inflammatory cytokines, confirming the involvement of BDNF in airway inflammation.

Nasal polyps and middle turbinates epithelial cells sensitivity to amphotericin B.

HYPOTHESIS: Intranasal application of the antimycotic agent amphotericin B (AmphoB) has been proposed as an effective treatment of chronic rhinosinusitis (CRS) with polyps. AmphoB is a sterol-binding agent known to modify cell membrane structure. The cytotoxic effects of AmphoB were studied on primary human nasal epithelial cells in vitro. METHODS: Human epithelial cells were isolated from nasal polyps and middle turbinates of patients suffering from CRS, and grown on collagen-coated polycarbonate filters with an air liquid-interface. After 15 days of culture, cells were exposed apically to 50 microM AmphoB during 4 h daily for 5 days. Some cells were treated during 4 weeks. The bioelectric properties of cells were then studied in Ussing chambers. Integrity of the cell monolayers was assessed by measurement of the transepithelial resistance (R) and immunofluorescent localization of the tight junction protein occludin. RESULTS: Disruption of the epithelial monolayer integrity was observed in all of the nasal polyps cell cultures, as demonstrated by a 60% drop in R. Immunofluorescence microscopy showed significant loss in cell number and disruption in the distribution of occludin. Turbinate cell cultures elicited no change in R and expression of occludin after AmphoB treatment. However, the transepithelial potential, the basal short-circuit current and the amiloride-sensitive current were reduced by 70%. CONCLUSIONS: AmphoB was cytotoxic for nasal polyp epithelial cells with disruption of the epithelium integrity and loss of tight junctions. In contrast, integrity of turbinate epithelial cells was conserved despite alterations in transepithelial ion transport. These observations may explain the beneficial effect of intranasal application of AmphoB on CRS observed in clinical trials.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

BACKGROUND: It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. METHODS: HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. RESULTS: Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. CONCLUSION: Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases.

Effects of first generation E1E3-deleted and second generation E1E3E4-deleted/modified adenovirus vectors on human endothelial cell death.

Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying beta-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFFR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.

Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells.

The effects of a transient exposure to hydrogen peroxide (10 min at 200 microM H(2)O(2)) on pancreatic beta cell signal transduction and insulin secretion have been evaluated. In rat islets, insulin secretion evoked by glucose (16.7 mM) or by the mitochondrial substrate methyl succinate (5 mM) was markedly blunted following exposure to H(2)O(2). In contrast, the secretory response induced by plasma membrane depolarization (20 mM KCl) was not significantly affected. Similar results were obtained in insulinoma INS-1 cells using glucose (12.8 mM) as secretagogue. After H(2)O(2) treatment, glucose no longer depolarized the membrane potential (DeltaPsi) of INS-1 cells or increased cytosolic Ca(2+). Both DeltaPsi and Ca(2+) responses were still observed with 30 mM KCl despite an elevated baseline of cytosolic Ca(2+) appearing approximately 10 min after exposure to H(2)O(2). The mitochondrial DeltaPsi of INS-1 cells was depolarized by H(2)O(2) abolishing the hyperpolarizing action of glucose. These DeltaPsi changes correlated with altered mitochondrial morphology; the latter was not preserved by the overexpression of the antiapoptotic protein Bcl-2. Mitochondrial Ca(2+) was increased following exposure to H(2)O(2) up to the micromolar range. No further augmentation occurred after glucose addition, which normally raises this parameter. Nevertheless, KCl was still efficient in enhancing mitochondrial Ca(2+). Cytosolic ATP was markedly reduced by H(2)O(2) treatment, probably explaining the decreased endoplasmic reticulum Ca(2+). Taken together, these data point to the mitochondria as primary targets for H(2)O(2) damage, which will eventually interrupt the transduction of signals normally coupling glucose metabolism to insulin secretion.

Reactive oxygen metabolites increase mitochondrial calcium in endothelial cells: implication of the Ca2+/Na+ exchanger.

In endothelial cells, a bolus of hydrogen peroxide (H2O2) or oxygen metabolites generated by hypoxanthine-xanthine oxidase (HX-XO) increased the mitochondrial calcium concentration [Ca2+]m. Both agents caused a biphasic increase in [Ca2+]m which was preceded by a rise in cytosolic free calcium concentration [Ca2+]c (18 and 6 seconds for H2O2 and HX-XO, respectively). The peak and plateau elevations of [Ca2+] were consistently higher in the mitochondrial matrix than in the cytosol. In Ca2+-free/EGTA medium, the plateau phase of elevated [Ca2+] evoked by H2O2 due to capacitative Ca2+ influx was abolished in the cytosol, but was maintained in the mitochondria. In contrast to H2O2 and HX-XO, ATP which binds the P2Y purinoceptors induced an increase in [Ca2+]m that was similar to that of [Ca2+]c. When cells were first stimulated with inositol 1,4, 5-trisphosphate-generating agonists or the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), subsequent addition of H2O2 did not affect [Ca2+]c, but still caused an elevation of [Ca2+]m. Moreover, the specific inhibitor of the mitochondrial Ca2+/Na+ exchanger, 7-chloro-3,5-dihydro-5-phenyl-1H-4.1-benzothiazepine-2-on (CGP37157), did not potentiate the effects of H2O2 and HX-XO on [Ca2+]m, while causing a marked increase in the peak [Ca2+]m and a significant attenuation of the rate of [Ca2+]m efflux upon addition of histamine or CPA. In permeabilized cells, H2O2 mimicked the effects of CGP37157 causing an increase in the basal level of matrix free Ca2+ and decreased efflux. Dissipation of the electrochemical proton gradient by carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and blocade of the Ca2+ uptake by ruthenium red prevented [Ca2+]m increases evoked by H2O2. These results demonstrate that the H2O2-induced elevation in [Ca2+]m results from a transfer of Ca2+ secondary to increased [Ca2+]c, and an inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria.

Clusterin gene expression mediates resistance to apoptotic cell death induced by heat shock and oxidative stress.

Clusterin is a widely expressed, well conserved, secreted glycoprotein, which is highly induced in tissues regressing as a consequence of apoptotic cell death in vivo. It has recently been shown that clusterin expression is only confined to surviving cells following the induction of apoptosis in vitro, suggesting that it is involved in cell survival rather than death. In the hypothesis that clusterin may be implicated in cellular responses to stress, clusterin gene expression was analyzed in the A431 human epidermoid cancer cell line following heat shock and oxidative stress. Our results show that both a transient heat shock (20 min at 42 degrees C) and various oxidative stresses, including hydrogen peroxide, superoxide anion, hyperoxia and UVA exposure, induce a strong increase in clusterin mRNA levels as assessed by northern blot. Nuclear run-on analysis suggests that transcriptional activation is involved in inducing clusterin mRNA in response to heat shock. Using pulse-chase analysis of control and heat shocked cells, it is shown that clusterin mRNA is translated and secreted, thus resulting in increased extracellular levels of the protein following heat shock. To investigate the function of clusterin in response to these stresses, clusterin anti-sense transfectants that stably express virtually no clusterin at the mRNA and protein level were generated in A431 cells. These anti-sense transfectants are shown to be highly sensitive to apoptotic cell death induced by heat shock or oxidative stress compared with wild-type A431 cells or control transfectants. Taken together, our results show that clusterin gene expression is induced in response to heat shock and oxidative stress in human A431 cells, and confers cellular protection against heat shock and oxidative stress.

Hydrogen peroxide-induced DNA damage is independent of nuclear calcium but dependent on redox-active ions.

In cells undergoing oxidative stress, DNA damage may result from attack by .OH radicals produced by the Fenton reaction, and/or by nucleases activated by nuclear calcium. In the present study, the participation of these two mechanisms was investigated in HeLa cells. Nuclear-targeted aequorin was used for selectively monitoring Ca2+ concentrations within the nuclei ([Ca2+]n), in conjunction with the cell-permeant calcium chelator bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), the lipid-soluble broad-spectrum metal chelator with low affinity for Ca2+ and Mg2+ N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and the high-affinity iron/copper chelator 1, 10-phenanthroline (PHE). In Ca2+-containing medium, H2O2 induced extensive DNA strand breaks and an increase in [Ca2+]n that was almost identical to that observed in the cytosol ([Ca2+]c). In cells bathed in Ca2+-free/EGTA medium, in which the increases in [Ca2+]n and [Ca2+]c due to H2O2 were significantly reduced, similar levels of DNA fragmentation also occurred. In cells preloaded with BAPTA/AM or TPEN, the small increase of [Ca2+]n normally elicited by H2O2 in Ca2+-free medium was completely buffered, and DNA damage was largely prevented. On the other hand, pretreatment with PHE did not affect the calcium response in the nuclei, but completely prevented DNA strand breakage induced by H2O2. Re-addition of 100 microM CuSO4 and 100 microM FeSO4 to TPEN- and PHE-treated cells prior to H2O2 challenge reversed the effect of TPEN and PHE, whereas 1 mM was necessary to negate the effect of BAPTA/AM. The levels of DNA strand breakage observed, however, did not correlate with the amounts of 8-hydroxy 2'-deoxyguanosine (8-OHdG): H2O2 did not produce 8-OHdG, whereas PHE alone slightly increased 8-OHdG levels. CuSO4 and FeSO4 enhanced the effects of PHE, particularly in the presence of H2O2. Exposure of cells to a mixture of CuSO4/FeSO4 also resulted in a significant increase in 8-OHdG levels, which was prevented in cells preloaded with BAPTA/AM. Similar results were obtained in a cell-free system using isolated calf thymus DNA exposed to CuSO4/FeSO4, regardless of whether H2O2 was present or not. These results suggest that BAPTA/AM prevents H2O2-induced DNA damage by acting as an iron/copper chelator. These data also indicate that caution must be exercised in using Ca2+ chelating agents as evidence for a role in cellular Ca2+ levels in experimental conditions in which transition-metal-ion-mediated oxidant production is also occurring.

Modulation of the DNA binding activity of transcription factors CREP, NFkappaB and HSF by H2O2 and TNF alpha. Differences between in vivo and in vitro effects.

Human endothelial cells exposed to H2O2 showed reduced CREP DNA binding activity, enhanced HSF activation, and no induction of NFkappaB binding activity. Interestingly, H2O2 was able to induce NFkappaB subunit p65 translocation in the nucleus. In contrast, cells exposed to TNF alpha showed enhanced CREP binding activity, activation of NFkappaB and no induction of HSE-HSF complex. Addition of H2O2, diamide and iodoacetic acid to the binding reaction mixture markedly reduced the DNA binding ability of the three transcription factors. Thus free sulfhydryls were important in DNA binding activity of CREP, NFkappaB and HSF, and the lack of induction of NFkappaB by H2O2 in intact cells was likely caused by oxidation on a thiol, and not by a deficiency in the activation pathway.

Hyperoxia, unlike phorbol ester, induces glutathione peroxidase through a protein kinase C-independent mechanism.

Human selenium-dependent glutathione peroxidase (GP) is implicated as a mechanism of resistance against oxygen free radicals. The 5' flanking sequence upstream from the coding region of GP contained an oxygen-responsive element termed ORE1 that is responsive to hypoxia, as well as several copies of the activator protein-1 (AP-1)- and AP-1-like-binding sites. In this study, we sought to define the molecular events that lead to GP gene transcription in response to hyperoxia in human umbilical-vein endothelial cells, and asked whether such induction is mimicked and sustained by activation of protein kinase C (PKC) by phorbol esters. Treatment of cells with 100 nM phorbol 12,13-dibutyrate (PdBu) induced a delayed (24-48 h) but significant (2-fold) increase in steady-state GP mRNA levels. Steady-state GP mRNA levels also rose after exposure to 95% O2, again after considerable delay (48-72 h). For both PdBu and oxygen, induction was transcriptionally regulated, as demonstrated by nuclear run-on experiments. The simulations by PdBu and oxygen were additive. In contrast with PdBu, hyperoxia did not stimulate translocation of PKC from the cytosol to the particulate fraction, although the specific activity of both cytosolic and particulate-associated PKC was increased 2-fold in cells exposed to 95% O2 for 5 days. In addition, gel mobility-shift assays using double-stranded tumour-promoting-agent-responsive element (TRE) and nuclear extracts derived from phorbol- and oxygen-treated cells revealed that PdBu, but not hyperoxia, increased AP-1 DNA-binding activity. On the other hand, the up-regulation of GP expression by oxygen could not be accounted for by the ORE1 core sequence, since no specific protein-DNA binding activity could be detected using nuclear extracts from hyperoxic cells and ORE1. Taken together, these results suggest that there may be different molecular mechanisms controlling GP expression. After exposure to PdBu, GP undergoes transcriptional activation via a process that can be readily explained by a classic AP-1 interaction with the TRE sites in the GP promoter. During hyperoxia, GP also undergoes transcriptional activity, but via a process that appears to involve neither TRE nor ORE1.

Reactive oxygen intermediates induce regulated secretion of von Willebrand factor from cultured human vascular endothelial cells.

Exocytosis from Weibel-Palade bodies, the secretory granules of vascular endothelial cells, causes the rapid release of von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis, and cell surface expression of P-selectin, a membrane protein involved in neutrophil binding. Thus, exocytosis may represent a link between hemostasis and inflammation. We investigated the effect of reactive oxygen intermediates (ROIs) on vWF secretion. Incubation of cultured endothelial cells with xanthine oxidase (XO), which generates superoxide anions (O2-), induces a potent, rapid secretory response. However, vWF release was not observed in response to H2O2. Extracellular, subendothelial vWF deposits typically seen after exocytosis from Weibel-Palade bodies were observed after exposure to XO. XO caused a rapid, sustained increase in intracellular free calcium concentration ([Ca2+]i). vWF secretion was markedly inhibited by BAPTA-AM, a cell-permeant calcium chelator. Removal of extracellular calcium did not inhibit vWF release, although the sustained phase of the [Ca2+]i increase was suppressed. These results suggest that XO-induced vWF release is mediated by the initial increase in [Ca2+]i which is caused by calcium mobilization from intracellular stores rather than by calcium influx. Exocytosis from Weibel-Palade bodies may contribute to the pathogenic effect of ROIs in atherosclerosis and inflammation.

Differential expression and regulation of hsp70 and hsp90 by phorbol esters and heat shock.

Human peripheral blood monocytes (PBM) produce superoxide anions (O2-.) by a process involving electron transfer from NADPH to O2, catalyzed by the respiratory burst enzyme NADPH oxidase. We have previously shown that phagocytosis, while activating NADPH oxidase, induced in PBM the synthesis of heat shock (HS) proteins (HSP). The present study was undertaken to establish whether this increase in HSP expression was related to O2-. and/or to classical second messengers such as protein kinase C (PKC). Thus, the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) were compared with those of heat shock on the expression, in PBM, of the major HSP, hsp70 and hsp90, using biometabolic labeling, Western and Northern blotting, and gel mobility shift assays. PMA induced the accumulation of mRNA and an increased expression of hsp90 and, to a lesser extent, hsp/hsc70 (hsc is the cognate, constitutive form). This induction was also observed in PBM from patients with chronic granulomatous disease, a genetic defect in NADPH oxidase, and was abolished by the PKC inhibitors staurosporine and H-7. PMA did not cause activation of the HS factor, and the PMA-induced overexpression expression of HSP was not blocked by the transcriptional inhibitor actinomycin D. HSP-specific mRNA stability was increased after PMA exposure as compared with heat shock. These results suggest that O2-. is not involved in the PMA-mediated induction of hsp70 and hsp90 and that, in contrast to HS, PMA increases the expression of HSP as a result of PKC-induced mRNA stabilization rather than of transcriptional activation of HS genes.

Differential regulation of glutathione peroxidase by selenomethionine and hyperoxia in endothelial cells.

We have studied the effect of selenomethionine (SeMet) and hyperoxia on the expression of glutathione peroxidase (GP) in human umbilical vein endothelial cells. Incubation of HUVEC with 1 x 10(-6) M SeMet for 24 h and 48 h caused a 65% and 86% increase in GP activity respectively. The same treatment did not result in significant changes in GP gene transcription and mRNA levels. Pactamycin, a specific inhibitor of the initiation step of translation, prevented the rise in GP activity induced by SeMet and caused an increase in GP mRNA in both cells grown in normal and SeMet-supplemented medium. Interestingly, SeMet supplementation stimulated the recruitment of GP mRNA from an untranslatable pool on to polyribosomes, so that the concentration of GP mRNA in polyribosomal translatable pools was 50% higher in cells grown in SeMet-supplemented medium than in cells grown in normal medium. On the other hand, cells exposed to 95% O2 for 3 days in normal medium showed a 60%, 394% and 81% increase in GP gene transcription rate, mRNA levels and activity respectively. Hyperoxia also stabilized GP mRNA. Hyperoxic cells grown in SeMet-supplemented medium did not show any change in GP gene transcription and mRNA levels, but expressed an 81% and 100% increase in GP activity and amount of GP mRNA associated with polyribosomes respectively, when compared with hyperoxic cells maintained in normal medium. Thus, GP appeared to be regulated post-transcriptionally, most probably co-translationally, in response to selenium availability, and transcriptionally and post-transcriptionally in response to oxygen.

Effects of hypoxanthine-xanthine oxidase on Ca2+ stores and protein synthesis in human endothelial cells.

We have investigated the effects of reactive O2 metabolites generated by the hypoxanthine-xanthine oxidase (HX-XO) system on intracellular Ca2+ and its relation with protein synthesis in human umbilical vein endothelial cells (HUVECs). Spectrofluorometry with fura 2 showed that the oxidative stress induced a rapid transient rise in cytosolic [Ca2+], followed by a sustained elevation above the baseline value. In the presence of La3+, which blocks Ca2+ influx from the extracellular medium, a transient [Ca2+] increase was still observed, but the sustained rise was suppressed. The HX-XO-related [Ca2+] changes were completely prevented by pretreatment with thapsigargin, which depletes intracellular Ca2+ stores. Hence, the effects of HX-XO on Ca2+ homeostasis were due to mobilization of Ca2+ from the intracellular stores with subsequent influx of extracellular Ca2+. HX-XO mobilized more of sequestered Ca2+ than did thrombin, a receptor agonist that depletes only a part of the intracellular Ca2+ stores (the hormone-sensitive stores). To determine the relevance of the HX-XO-related depletion of Ca2+ stores for cell function, we investigated the role of Ca2+ mobilization in the regulation of protein synthesis. Overall protein synthesis in HUVECs was markedly reduced by thapsigargin, which depletes both hormone-sensitive and -insensitive stores, but was not substantially affected by thrombin. Manipulation of the refilling of the Ca2+ stores via the availability of extracellular Ca2+ significantly influenced the thapsigargin-related and the HX-XO-related inhibition of overall protein synthesis. A corresponding effect of extracellular [Ca2+] was seen in polyribosome distribution profiles, which reflected an inhibition of translation initiation in both treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of inhibition of catalase and superoxide dismutase activity on antioxidant enzyme mRNA levels.

We examined the effects of inhibition of Cu,Zn superoxide dismutase (Cu,Zn SOD) and catalase (Cat) activities on the steady-state mRNA levels of the three major antioxidant enzymes [Cu,Zn SOD, Cat, and glutathione peroxidase (GP)] in human umbilical vein endothelial cells under normoxia and hyperoxia. Inhibition of Cat activity by aminotriazole was not associated with alteration of the other antioxidant enzymes or with potentiation of cell injury. On the other hand, inhibition of Cu,Zn SOD activity by N-N'-diethyl-dithiocarbamate (DDC) was associated with an increase in Cu,Zn SOD mRNA level and a decrease in Cat and GP mRNA level. The combination of DDC and hyperoxia treatments was associated with an additive effect on Cu,Zn SOD message. We propose that these coordinate mRNA changes might be an adaptation to the oxidative environment. This proposal supports the concept that the intracellular O2 metabolites themselves could be the signals that trigger the antioxidant enzymes gene expression.

Variable glutathione levels and expression of antioxidant enzymes in human endothelial cells.

We have investigated the relationship between intracellular glutathione levels and the inducibility of the mRNAs encoding the major antioxidant enzymes Cu,Zn superoxide dismutase (Cu,Zn SOD), catalase (CAT), glutathione peroxidase (GP), and the stress protein heme oxygenase (HO) following exposure of human umbilical vein endothelial cells (HUVEC) to either hypoxanthine-xanthine oxidase or 95% O2. Treatment of HUVEC with 2 and 200 microM buthionine sulfoximine (BSO) for 16 h reduced total glutathione (GSH) levels by 51 and 95%, respectively, whereas treatment with 100 microM diethylmaleate (DEM) for 24 h increased the cellular GSH content by 58%. None of these treatments affected the responsiveness of HUVEC to a subsequent oxidant challenge, in terms of antioxidant enzymes activities and mRNA levels. On the contrary, HO mRNA was significantly induced by both BSO and DEM, as well as by hyperoxia, albeit to a different extent. We conclude that intracellular redox changes do not appear to regulate the expression of the mRNAs encoding Cu,Zn SOD, CAT, and GP. Furthermore, factors other than endogenous thiols may play a role in the control of HO mRNA expression.

Response of human endothelial cell antioxidant enzymes to hyperoxia.

To explore the level of regulation of the expression of the major antioxidant enzymes in response to hyperoxia, we exposed human umbilical vein endothelial cells to 95% O2 for 3 and 5 days and measured (1) the steady-state mRNA levels, (2) the activities, and (3) the immunoreactive content of CuZn and Mn superoxide dismutases (SOD), catalase (CAT), and glutathione peroxidase (GP). We found that a 3-day exposure to 95% O2 caused (1) an increase in CuZnSOD mRNA (by 41%), CAT mRNA (by 26%), and GP mRNA (by 173%); (2) an increase in CuZnSOD activity (by 30%), a decrease in CAT activity (by 37%), and an increase in GP activity (by 60%); and (3) an increase in CuZnSOD immunodetectable protein (by 26%) and a loss in CAT immunoreactive protein (by 27%). After a 5-day exposure to 95% O2, there was (1) a 93% increase in CuZnSOD mRNA, a 71% increase in CAT mRNA, and a 127% increase in GP mRNA; (2) a 56% increase in CuZnSOD activity, a 70% decrease in CAT activity, and an 89% increase in GP activity; and (3) a 35% increase in CuZnSOD immunoreactive protein and a 55% loss in CAT immunoreactive protein. There was no change in the steady-state MnSOD mRNA level after 3 days in 95% O2, but a 100% increase was observed on day 5 of oxygen exposure. MnSOD activity was unchanged in cells exposed to hyperoxia for 3 and 5 days. These data suggest that, in human umbilical vein endothelial cells, the regulation of antioxidant enzymes expression in response to O2 is complex and exerted at different levels.

Differential protective effects of O-phenanthroline and catalase on H2O2-induced DNA damage and inhibition of protein synthesis in endothelial cells.

The respective roles of H2O2 and .OH radicals was assessed from the protective effects of catalase and the iron chelator o-phenanthroline on 1) the inhibition of protein synthesis, and 2) DNA damage and the related events (activation of the DNA repairing enzyme poly(ADP)ribose polymerase with the associated depletion of NAD and ATP stores) in cultured endothelial cells exposed to the enzyme reaction hypoxanthine-xanthine oxidase (HX-XO) or pure H2O2. Catalase added in the extracellular phase completely prevented all of these oxidant-induced changes. O-phenanthroline afforded a complete protective effect against DNA strand breakage and the associated activation of the enzyme poly(ADP)ribose polymerase. By contrast, iron chelation was only partially effective in maintaining the cellular NAD and ATP contents, as well as the protein synthetic activity. In addition, the ATP depletion following oxidant injury was much more profound than NAD depletion. These results indicate that: 1) .OH radical was most likely the ultimate O2 species responsible for DNA damage and activation of poly(ADP)ribose polymerase; 2) both H2O2 and .OH radicals were involved in the other cytotoxic effects (inhibition of protein synthesis and reduction of NAD and ATP stores); and 3) NAD and ATP depletion did not result solely from activation of poly(ADP)ribose polymerase, but other mechanisms are likely to be involved. These observations are also compatible with the existence of a compartmentalized intracellular iron pool.

Differential expression of hsp70 stress proteins in human endothelial cells exposed to heat shock and hydrogen peroxide.

The potential role of oxidative stress conditions in the induction of heat shock proteins was studied in human umbilical vein endothelial cells. We compared the effects of temperature (43 to 45 degrees C), exposure to hydrogen peroxide (H2O2) and oxygen metabolites generated by the enzyme system hypoxanthine-xanthine oxidase (O2- plus H2O2), as well as exposure to 95% O2, on the expression of the major 70-kD heat shock proteins (hsp70). Northern blot analysis indicated that: (1) heat shock induced a rapid and marked increase in hsp70 mRNA levels that reached a maximum during recovery from a 30-min exposure to 45 degrees C; (2) treatment with a 5-mM H2O2 bolus or 50 mU/ml xanthine oxidase also increased hsp70 mRNA levels but to a lesser extent than heat shock (about 10 and 25 times less, respectively); (3) no change was detected after a 5-day exposure to 95% O2. Nuclear run on transcription data and kinetics of mRNA decay in the presence of actinomycin D indicated that the observed increase in hsp70 mRNA levels in both heat-shocked and H2O2-treated cells was mainly due to a transcriptional induction. The kinetics of hsp70 synthesis correlated with the accumulation of hsp70 mRNA. Two-dimensional gel electrophoresis and immunologic analysis of these heat shock proteins revealed a series of at least five distinct hsp70 isoforms induced in heat-shocked cells, whereas only a specific subset of these proteins, mainly one acidic isoform, was induced in very low amounts in response to H2O2 treatment. These results clearly indicate that the endothelial cell responses to oxidative stress and heat shock differ in both qualitative and quantitative terms in respect to hsp70 induction. They also suggest that the intensity of this response to oxidative stress conditions may vary depending on the nature of the oxidative challenge.