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Glutamate-stimulated ROS production in neuronal cultures: interactions with lead and the cholinergic system.

Oxidative stress may be an important factor in several pathological brain conditions. A contributing factor in many such conditions is excessive glutamate release, and subsequent glutamatergic neuronal stimulation, that causes increased production of reactive oxygen species (ROS), oxidative stress, excitotoxicity and neuronal damage. Glutamate release is also associated with illnesses such as Alzheimer's disease, stroke, and brain injury. Glutamate may interact with an environmental toxin, lead, and this interaction may result in neuronal damage. Glutamate-induced ROS production is greatly amplified by lead in cultured neuronal cells. Alterations in protein kinase C (PKC) activity seem to be important both for glutamate-induced ROS production, and for the amplification of glutamate-induced ROS production by lead. It is possible that the neurotoxic effects of lead are amplified through glutamate-induced neuronal excitation. Cholinergic stimulation can also trigger ROS production in neuronal cells. PKC seems to play a key-role also in cholinergic-induced ROS production superoxide anion being the primary reactive oxygen species. There seems to be a close relationship between the responses of cholinergic muscarinic and glutamatergic receptors because glutamate receptor antagonists inhibit cholinergic-induced activation of human neuroblastoma cells. Glutamatergic neuronal stimulation may be a common final pathway in several brain conditions in which oxidative stress and ensuing excitotoxicity plays a role.

Animals↗

PubMed1990

Monoclonal antibodies to ROS 17/2.8 cells recognize antigens, some of which are restricted to osteoblasts and chondrocytes.

We have raised a panel of 15 monoclonal antibodies (MAbs) recognizing cell surface antigens of the rat osteoblast-like cell line ROS 17/2.8. The MAbs were selected on the basis of preferential binding to ROS 17/2.8 cells compared to ROS 25/1 cells. Immunohistochemical studies of antigen localization on cryostat sections of rat calvaria, long bone, and soft tissues demonstrated that five of these MAbs, UBIM 1, 2, 3, 12, and 17, recognize antigens that are restricted to normal rat osteoblasts and chondrocytes. The antigens appear to be localized to the cell surface of the osteoblast, with no apparent staining of bone matrix in either undecalcified or decalcified sections. In vitro, these MAbs recognize cell surface antigens present on two additional cell lines, ROS 24/1 and Rat 2 cells, and on the adherent cell population cultured from rat long bone marrow. Of these MAbs, three (UBIM 1, 2, and 3) recognize high-molecular-weight antigens of Mr 200,000-225,000. This study has also identified cell surface antigens of ROS 17/2.8 cells that are not expressed by osteoblasts in vivo. MAbs UBIM 9 and 21 bind to marrow cells in long bone sections, to the 7-day-old nonadherent cell population from cultured marrow, and to lymphoid tissue in sections of spleen. Another four MAbs (UBIM 10, 11, 14, and 22) bind to a variety of cells and tissues both in vitro and in vivo. Studies of the interactions of this panel of MAbs with osteogenic tissues and cell lines may have an important impact on the understanding of osteoblast physiology.

Animals↗

PubMed1999

Different types of ROS-scavenging enzymes are expressed during cutaneous wound repair.

Injury to the skin initiates a series of events including inflammation, new tissue formation, and matrix remodeling. During the early inflammatory phase, polymorphonuclear leukocytes and macrophages infiltrate the wounded tissue. Once activated, they produce large amounts of reactive oxygen species (ROS) as part of their defense mechanism. Although this process is beneficial, increased levels of ROS can inhibit cell migration and proliferation and can even cause severe tissue damage. Therefore, cells must develop strategies for the detoxification of these molecules. To gain insight into the mechanisms which underlie this process, we analyzed the temporal and spatial expression pattern of various ROS-scavenging enzymes during the healing process of full-thickness excisional wounds in mice. Here we demonstrate a strong mRNA expression of two types of superoxide dismutase (SOD), as well as of catalase, and the selenoenzymes glutathione peroxidase (SeGPx) and phospholipid hydroperoxide glutathione peroxidase in normal and wounded skin. Most importantly, mRNA levels of the SODs and of SeGPx increased strongly after skin injury. In situ hybridization and immunofluorescence studies revealed the presence of these transcripts at multiple places in the wound, whereby particularly high expression levels were detected in the hyperproliferative epithelium and the hair follicles at the wound edge. These data suggest an important role of ROS-scavenging enzymes in the detoxification of ROS during cutaneous wound repair.

Animals↗

PubMed2004

Inhibitory phlorotannins from the edible brown alga Ecklonia stolonifera on total reactive oxygen species (ROS) generation.

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human degenerative diseases such as cancer, aging, arteriosclerosis, and rheumatism. Much attention has been focused on the development of safe and effective antioxidants. To discover sources of antioxidative activity in marine algae, extracts from 17 kinds of seaweed were screened for their inhibitory effect on total ROS generation in kidney homogenate using 2',7'-dichlorofluorescein diacetate (DCFH-DA). ROS inhibition was seen in three species: Ulva pertusa, Symphyocladia latiuscula, and Ecklonia stolonifera. At a final concentration of 25 microg/mL, U. pertusa inhibited 85.65+/-20.28% of total ROS generation, S. latiscula caused 50.63+/-0.09% inhibitory, and the Ecklonia species was 44.30+/-7.33% inhibition. E. stolonifera Okamura (Laminariaceae), which belongs to the brown algae, has been further investigated because it is commonly used as a foodstuff in Korea. Five compounds, phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5), isolated from the ethyl acetate soluble fraction of the methanolic extract of E. stolonifera inhibited total ROS generation.

Animals↗

PubMed1999

Tracing of benzidine-reactive substances in ROS, RPE and choroid after light-induced peroxidation.

BACKGROUND: A new method for the ultrastructural localization of lipid peroxides as benzidine-reactive substances (BRS) was recently developed in our laboratory. The aim of the present study was to localize BRS in the eye after intense light exposure. The light protocol was chosen to either hamper disc shedding or to induce a shedding peak. METHODS: Long-Evans rats were either kept under constant irradiation to enhance lipid peroxidation or under physiological light conditions. The light-induced peroxidation was carried out either by constant irradiation for 24 h or by constant irradiation for 20 h followed by a dark period of 4 h. The eye cups were fixed by glutaraldehyde, incubated with or without tetramethylbenzidine and embedded for electron microscopy. RESULTS: After constant irradiation for 24 h smooth nonlamellar BRS appear exclusively intracellularly over the complete rod outer segments (ROS). After the initiation of disc shedding smooth BRS are localized in the extracellular space of the ROS and extracellularly in the basal labyrinth of the retinal pigment epithelium (RPE). Fine-lamellar BRS emerge in vacuoles of the RPE, in the basal labyrinth and in the lumen of choroidal capillaries. CONCLUSION: Light conditions that trigger the disc shedding possibly activate a mechanism to extrude peroxidative damaged material over the complete ROS into the extracellular space to diminish peroxidative damage inside the ROS. Indigestible residual material from the ROS degradation in the phagosomes consists of membranous lipids associated with peroxidative damaged proteins. The residual material seems to be transported through Bruch's membrane into the choriocapillaris.

Animals↗

PubMed2006

Induction of early apoptosis and ROS-generation activity in human gingival fibroblasts (HGF) and human submandibular gland carcinoma (HSG) cells treated with curcumin.

OBJECTIVE: Curcumin [1] is well known to possess apoptosis-inducing activity in some cancer cells, but little is known about its activity in normal cells of oral origin, such as HGF. The aim of the present study was to clarify the relationship between early apoptosis in HGF and the induction of reactive oxygen species (ROS) generation by curcumin. DESIGN: We treated HGF and HSG cells with curcumin [1] and the related compounds biseugenol [2], eugenol [3], alpha-diisoeugenol [4], and isoeugenol [5] and measured cell survival (MTT method), ROS generation (DCFH-DA staining), and induction of early apoptosis. Early apoptosis was detected by monitoring loss of mitochondrial membrane potential (DeltaPsi(m)) by JC-1 staining and externalization of phosphatidylserine (PS) on the cell surface by annexin V-FITC/PI staining combined with flow cytometry. RESULTS: The cytotoxic activities of curcumin [1] and [4] were similar and were nearly 10- to 100-fold stronger than those of the other compounds. Only curcumin was able to induce ROS generation and early apoptosis. Loss of DeltaPsi(m), PS externalization and ROS generation were significantly more pronounced in HGF cells than in HSG cells at curcumin concentrations lower than about 15microM, and were inhibited by the addition of the antioxidants N-acetyl-l-cysteine and glutathione. CONCLUSION: The potent PS externalization and loss of DeltaPsi(m) in curcumin-treated HGF cells appears to be mediated by ROS generation.

Antineoplastic Agents↗

PubMed2005

Identification of novel Nox4 splice variants with impact on ROS levels in A549 cells.

NAD(P)H oxidases (Nox) generate reactive oxygen species (ROS) that function in host defense and cellular signaling. While analyzing the expression of Nox4 at the protein and the mRNA levels, we identified four novel Nox4 splice-variants Nox4B, Nox4C, Nox4D, and Nox4E, which are expressed in human lung A549 cell line and lung tissues. One Nox4 isoform lacks the first NAD(P)H binding site (Nox4B) while another lacks all FADH and NAD(P)H binding sites (Nox4C). Cells over-expressing NoxB or Nox4C exhibited a decrease in ROS levels. Thus, these isoforms have dominant negative characteristics for ROS generation. Two other splice-variants (Nox4D, Nox4E) lack the transmembrane domains, suggesting these as non-membrane associated isoforms. Nox4D contains all FADH and NAD(P)H binding domains and shows the same rate of ROS generation as Nox4 prototype. Taken together, we suggest that Nox4 exists as several isoforms that may have different functions in ROS-related cell signaling.

Alternative Splicing↗

PubMed2006

Inhibition of receptor internalization attenuates the TNFalpha-induced ROS generation in non-phagocytic cells.

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)alpha, although the events through which TNFalpha induces ROS generation are not well characterized. Here, we report that TNFalpha-induced ROS production was blocked by pretreatment with internalization inhibitor monodansyl cadaverine (MDC). Similarly, a transient expression of a GTP-binding and hydrolysis-defective dynamin mutant (dynamin(K44A)) that had been shown to be defective in internalization significantly attenuated the TNFalpha-induced intracellular ROS production. Importantly, the inhibition of receptor internalization suppressed TNFalpha signaling to mitogen-activated protein kinases (MAPKs) stimulation. Together, our results suggest that receptor internalization is somehow necessary for the TNFalpha-induced ROS generation and subsequent intracellular downstream signaling in non-phagocytes.

Cadaverine↗

PubMed2005

Synergistic activation of JNK/SAPK induced by TNF-alpha and IFN-gamma: apoptosis of pancreatic beta-cells via the p53 and ROS pathway.

IFN-gamma and TNF-alpha are major proinflammatory cytokines implicated in islet beta-cell destruction, which results in type-1 diabetes; however, the underlying mechanism is not clear. Using pancreatic beta-cell line MIN6N8 cells, co-treatment with TNF-alpha and IFN-gamma, but neither cytokine alone, synergistically induced apoptosis, correlated with the activation of the JNK/SAPK, which resulted in the production of reactive oxidative species (ROS) and loss of mitochondrial transmembrane potential (delta psi m). Additionally, cells transfected with wild-type JNK1 became more susceptible to apoptosis induced by TNF-alpha/IFN-gamma through ROS production and loss of delta psi m, while cascading apoptotic events were prevented in dominant-negative JNK1-transfected or JNK inhibitor SP600125-treated cells. As the antioxidant, N-acetyl-cysteine, failed to completely suppress apoptosis induced by TNF-alpha/IFN-gamma, an additional pathway was considered to be involved. The level of p53 was significantly increased through synergistic activation of JNK by TNF-alpha/IFN-gamma. Furthermore, the synergistic effect of TNF-alpha/IFN-gamma on apoptosis and ROS production was further potentiated by the overexpression of wild-type p53, but not with mutant p53. This synergistic activation of JNK/SAPK by TNF-alpha/IFN-gamma was also induced in insulin-expressing pancreatic islet cells, and increased ROS production and p53 level, which was significantly inhibited by SP600125. Collectively, these data demonstrate that TNF-alpha/IFN-gamma synergistically activates JNK/SAPK, playing an important role in promoting apoptosis of pancreatic beta-cell via activation of p53 pathway together with ROS.

Animals↗

PubMed2006

Two components in pathogenic mechanism of mitochondrial ATPase deficiency: energy deprivation and ROS production.

Isolated defects of mitochondrial ATPase due to diminished biosynthesis of the enzyme represent new class of severe mitochondrial diseases of nuclear origin. The primary cause of decreased cellular content of ATPase appears to be a problem in assembly of the F1 catalytic part of the enzyme. With the aim to elucidate how the low ATPase content affects mitochondrial energy provision and ROS production, we have investigated fibroblasts from patients with ATPase decrease to 10-30%. Measurements of cellular respiration showed pronounced decrease in ATPase capacity for basal respiration, mitochondrial ATP synthesis was decreased to 26-33%. Cytofluorometric analysis using TMRM revealed altered discharge of mitochondrial membrane potential (DeltaPsim) in patient cells, which was 20 mV increased at state 3-ADP. Analysis of ROS production by CM-H2DCFDA demonstrated 2-fold increase in ROS production in patient cells compared to controls. ROS production rate was sensitive to uncoupler (FCCP) and thus apparently related to increased DeltaPsim. Our studies clearly demonstrate that low ATPase content and decreased mitochondrial ATP production lead to high values of DeltaPsim and are associated with activation of ROS generation by the mitochondrial respiratory chain. In conclusion, both the energetic deprivation and increased oxidative stress are important components of the pathogenic mechanism of ATPase disorders.

Adenosine Triphosphatases↗

PubMed2004

Primary cirrhotic hepatocytes resist TGFbeta-induced apoptosis through a ROS-dependent mechanism.

BACKGROUND/AIMS: The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. METHODS: Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents. RESULTS: Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment. CONCLUSIONS: These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.

Animals↗

PubMed2005

Differential profiles of copper-induced ROS generation in human neuroblastoma and astrocytoma cells.

To determine neuronal and glial responses to copper (Cu) elevation in the CNS, human neuroblastoma and astrocytoma cells were used to compare their responses to Cu in terms of reactive oxygen species (ROS) generation and expression of enzymes responsible for anti-oxidation. Astrocytoma cells, not neuroblastoma cells, were responsive to Cu and Cu elevation was associated with ROS generation. Intracellular Cu levels as determined by inductively coupled plasma-mass spectrometry (ICP-MS), and expression levels of copper-transporting ATPase (ATP7A) and human copper transporter 1 (hCtr1) as detected by quantitative reverse transcription-polymerase chain reaction (RT-PCR), were comparable in both cell lines. Differences in Cu-induced ROS between two cell lines paralleled superoxide dismutase (SOD)-catalase expression as detected by Western blot analysis. Copper,zinc-SOD (Cu,Zn-SOD) and catalase protein levels were upregulated by Cu in neuroblastoma cells while Cu,Zn-SOD was down-regulated by Cu and catalase level was not changed in astrocytoma cells. Manganese-SOD (Mn-SOD) was not responsive to Cu in either cell line. Furthermore, 78-kDa glucose-regulated protein aggregation and upregulation were observed in Cu-treated astrocytoma cells, but not neuroblastoma cells. These data suggest that neurons use the SOD-catalase system to scavenge Cu-induced ROS while glia rely on the endoplasmic reticulum stress response to compensate for the reduction of ROS scavenging capacity.

Adenosine Triphosphatases↗

PubMed2005

Enhanced ROS production and antioxidant defenses in cybrids harbouring mutations in mtDNA.

It has been suggested that mutations in mitochondrial DNA (mtDNA) can produce an increase in reactive oxygen species (ROS) and that this can play a major role in the pathogenic mechanisms of mitochondrial encephalomyopathies. Many studies exist using electron transport chain (ETC) inhibitors, however there are only a few studies that examine ROS production associated with mutations in the mtDNA. To investigate this issue, we have studied ROS production, antioxidant defences and oxidative damage to lipids and proteins in transmitochondrial cybrids carrying different mtDNA mutations. Here, we report that two different mutant cell lines carrying mutations in their mitochondrial tRNA genes (A3243G in tRNA LeuUUR and A8344G in tRNA Lys) showed an increased ROS production with a parallel increase in the antioxidant enzyme activities, which may protect cells from oxidative damage in our experimental conditions (no overt oxidative damage to lipids and proteins has been observed). In contrast, cytochrome c oxidase (COX) mutant cybrids (carrying the stop-codon mutation G6930A in the COXI gene) showed neither an increase in ROS production nor elevation of antioxidant enzyme activities or oxidative damage. These results suggest that the specific location of mutations in mtDNA has a strong influence on the phenotype of the antioxidant response. Therefore, this issue should be carefully considered when antioxidant therapies are investigated in patients with mitochondrial disorders.

Adaptation, Physiological↗

PubMed2005

Benzoquinone activates the ERK/MAPK signaling pathway via ROS production in HL-60 cells.

Benzene (BZ) is a class I carcinogen and its oxidation to reactive intermediates is a prerequisite of hematoxicity and myelotoxicity. The generated metabolites include hydroquinone, which is further oxidized to the highly reactive 1,4-benzoquinone (BQ) in bone marrow. Therefore, we explored the mechanisms underlying BQ-induced HL-60 cell proliferation by studying the role of BQ-induced reactive oxygen species (ROS) in the activation of the ERK-MAPK signaling pathway. BQ treatment (0.01-30 microM) showed that doses below 10 microM did not significantly reduce viability. ROS production after 3 microM BQ treatment increased threefold; however, catalase addition reduced ROS generation to basal levels. FACS analysis showed that BQ induced a fivefold increase in the proportion of cells in S-phase. We also observed a high proportion of Bromodeoxyuridine (BrdU) stained cells, indicating a higher DNA synthesis rate. BQ also produced rapid and prolonged phosphorylation of ERK1/2 proteins. Simultaneous treatment with catalase or PD98059, a potent MEK protein inhibitor, reduced cell recruitment into the S-phase and also abolished the ERK1/2 protein phosphorylation induced by BQ, suggesting that MEK/ERK is an important pathway involved in BQ-induced ROS mediated proliferation. The prolonged activation of ERK1/2 contributes to explain the increased S-phase cell recruitment and to understand the leukemogenic processes associated with exposure to benzene metabolites. Thus, the possible mechanism by which BQ induce HL-60 cells to enter the cell cycle and proliferate is linked to ROS production and its growth promoting effects by specific activation of regulating genes known to be activated by redox mechanisms.

Benzoquinones↗

PubMed2006

The role of ROS in microcystin-LR-induced hepatocyte apoptosis and liver injury in mice.

Microcystin-LR (MC-LR) produced by cyanobacteria in diverse water systems is a potent specific hepatotoxin and has been documented to induce hepatocyte apoptosis and liver injury; however, the mechanisms have not been fully elucidated. In the present study, we investigated whether MC-LR stimulated ROS generation in the liver of mice and the role of ROS in the pathogenesis of MC-LR-induced liver injury in vivo. MC-LR treatment (60 microg/kg of body weight) for 12h prompted large amount of ROS generation in mice liver, upregulated the expression of Bax and Bid, caused the mitochondrial membrane potential (MMP) loss and hepatocyte apoptosis as well as liver injury. While pretreatment with antioxidants, oral administration of vitamin C (250mg/kg of body weight, dissolved in double distill water) and vitamin E (200mg/kg of body weight, dissolved in corn oil) per day for 3 days continually, significantly reduced the generation of ROS and effectively inhibited the MC-LR-induced hepatocyte apoptosis and liver injury, suggesting that ROS played a critical role in MC-LR-induced hepatocyte apoptosis and liver injury. The protective effect of vitamin C and E also suggested the potential interest in the clinical treatment of MC-LR-induced liver injury and hepatotoxicity.

Alanine Transaminase↗

PubMed2005

Rhodostomin inhibits thrombin-enhanced adhesion of ROS 17/2.8 cells through the blockade of alphavbeta3 integrin.

Osteosarcoma is a very malignant bone tumor which has a high metastatic potential and usually lead to poor prognosis. The adhesion of tumor cells to the endothelium or extracellular matrix (ECM) is an essential step in the metastatic cascade. We investigated the effect of thrombin on the adhesion activity of the osteosarcoma cell line, ROS 17/2.8. Incubation with the low concentrations of thrombin (0.01-5 U/ml, 5 min to 24 h) elevated the adhesion activity of ROS 17/2.8 to both human umbilical vein endothelial cells (HUVEC) and extracellular matrix, with the peak effect at the concentration of 0.5 U/ml for 30 min at 37 degrees C. The ROS 17/2.8 cells responded to thrombin by a peak effect of increased adhesion to HUVEC (5.5 folds vs. control) and fibronectin (4.8 folds) after thrombin pretreatment (0.5 U/ml, 30 min, 37 degrees C). Pretreatment with monoclonal antibodies against beta3 integrins, including anti-alphavbeta3, 10E5 and 7E3, effectively antagonized the thrombin-enhanced cell adhesion activity, whereas anti-alpha3beta1 and anti-alpha5beta1 did not antagonize the enhanced cell adhesion. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, and synthetic peptide RGDS also blocked the thrombin-enhanced ROS 17/2.8 cell adhesion. This study demonstrated that thrombin enhanced the cell adhesion of ROS 17/2.8 cells to HUVEC or ECM through an upregulation of beta3 integrins, and rhodostomin was a strong inhibitor on thrombin-enhanced cell adhesion, either to HUVEC or fibronectin substratum.

Animals↗

PubMed2006

Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death.

Vertebrate limb development is a well-studied model of apoptosis; however, little is known about the intracellular molecules involved in activating the cell death machinery. We have shown that high levels of reactive oxygen species (ROS) are present in the interdigital 'necrotic' tissue of mouse autopod, and that antioxidants can reduce cell death. Here, we determined the expression pattern of several antioxidant enzymes in order to establish their role in defining the areas with high ROS levels. We found that the genes encoding the superoxide dismutases and catalase are expressed in autopod, but they are downregulated in the interdigital regions at the time ROS levels increased and cell death was first detected. The possible role of superoxide and/or peroxide in activating cell death is supported by the protective effect of a superoxide dismutase/catalase mimetic. Interestingly, we found that peroxidase activity and glutathione peroxidase-4 gene (Gpx4) expression were restricted to the non-apoptotic tissue (e.g., digits) of the developing autopod. Induction of cell death with retinoic acid caused an increase in ROS and decrease in peroxidase activity. Even more inhibition of glutathione peroxidase activity leads to cell death in the digits, suggesting that a decrease in antioxidant activity, likely due to Gpx4, caused an increase in ROS levels, thus triggering apoptosis.

Animals↗

PubMed2004

Lysophosphatidylcholine-induced modulation of Ca(2+)-activated K(+)channels contributes to ROS-dependent proliferation of cultured human endothelial cells.

Proliferation of endothelial cells plays a crucial role in the process of atherosclerotic plaque destabilization. The major component of oxidized low-density lipoprotein lysophosphatidylcholine (LPC) has been shown to promote endothelial proliferation by increasing the production of reactive oxygen species (ROS). Since K(+) channels are known to control the cell cycle, we investigated the role of Ca(2+)-activated K(+) channels (BK(Ca)) in the regulation of LPC-induced endothelial proliferation and ROS generation. A significant increase of cell growth induced by LPC (20 micromol/l; cell counts (CCs): +87%, thymidin incorporation: +89%; n = 12, P < 0.01) was observed, which was inhibited by the BK(Ca) inhibitor iberiotoxin (IBX; 100 nmol/l), by the NAD(P)H-oxidase inhibitor diphenyleneiodonium (5 micromol/l) and by transfection with antisense (AS) oligonucleotides against NAD(P)H oxidase, whereas N(G)-monomethyl-l-arginine (l-NMMA) further increased LPC-induced cell growth. Using the patch-clamp technique a significant increase of BK(Ca) open-state probability (control: 0.004 +/- 0.002; LPC: 0.104 +/- 0.035; n = 21, P < 0.05) by LPC was observed. Using dichlorofluorescein fluorescence microscopy a significant increase of ROS induced by LPC was reported, that was blocked by IBX and Ca(2+) antagonists. Intracellular Ca(2+) measurements revealed a capacitative Ca(2+) influx caused by LPC. Bioactivity of nitric oxide (NO) was measured using a [(3)H]-cGMP radioimmunoassay. LPC significantly decreased acetylcholine-induced NO synthesis. LPC significantly increased cGMP levels in endothelial cells transfected with AS, which was blocked by IBX. In conclusion, our results demonstrate that LPC activates BK(Ca) thereby increasing ROS production which induces endothelial proliferation. In addition LPC-induced BK(Ca)-activation contributes to increased cGMP levels, if ROS production is prevented by AS.

Calcium↗