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PubMed2006

Novel frequenin-modulated Ca2+-signaling membrane guanylate cyclase (ROS-GC) transduction pathway in bovine hippocampus.

Frequenin is a member of the neuronal Ca(2+) sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca(2+)-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca(2+) myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca(2+) myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca(2+)-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca(2+)-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.

Amino Acid Sequence↗

PubMed1989

Internalization and degradation of insulin by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells.

Chinese hamster ovary cell lines expressing either the wild-type human insulin receptor or a hybrid molecule in which the tyrosine kinase domain of the insulin receptor is replaced with that of the oncogene, v-ros were examined for their ability to internalize and degrade insulin. Cells expressing the hybrid receptor were found to internalize and degrade insulin at approximately half the rate of cells expressing the native insulin receptor. Moreover, insulin was incapable of inducing the internalization of the cell-surface hybrid molecule. In contrast, the constitutive rate of receptor internalization was found to be the same for the hybrid and wild-type receptors. These results obtained were similar to those with cells expressing either wild-type or mutant receptors lacking kinase activity. In conclusion, the substitution of the specificity of tyrosine kinase of the insulin receptor with that of the v-ros oncogene product results in defective internalization and degradation of insulin, and loss of ligand-induced receptor internalization.

Animals↗

PubMed1990

Lead impairs the production of osteocalcin by rat osteosarcoma (ROS 17/2.8) cells.

The serum level of osteocalcin, a bone-specific protein produced by osteoblasts and an index of bone formation, is decreased in lead-intoxicated children. To elucidate the effect of lead on the hormonal regulation of osteocalcin production, ROS 17/2.8 cells were treated with 0, 5, 10, or 25 microM lead acetate for 24 hr, followed by an additional 24-hr lead treatment with or without 100 pg 1,25-dihydroxyvitamin D3/ml medium. At the end of this period a radioimmunoassay was conducted to determine the amount of osteocalcin present in the cells and secreted into the medium. 1,25-Dihydroxyvitamin D3 increased osteocalcin secretion in control cultures, but this increase was prevented by lead in a concentration-dependent manner. Osteocalcin secretion by cultures treated with 10 or 25 microM lead was even lower than in cultures not stimulated with 1,25-dihydroxyvitamin D3. Intracellular levels of osteocalcin were slightly elevated with 1,25-dihydroxyvitamin D3, and there was no lead effect on cellular levels. These data indicate that lead attenuates basal and 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin in ROS 17/2.8 cells. Because osteocalcin appears to play a central role in bone mineralization, altered osteocalcin production may be a key event in the skeletal toxicity of lead.

Animals↗

PubMed1989

Calcium modulation of the parathyroid hormone-sensitive adenylate cyclase in ROS 17/2.8 cells: effects of N-(6-aminohexyl-5-Cl-naphthalene sulfonamide) (W-7) and trifluoperazine (TFP).

The calcium modulation of the cyclic 3',5'-adenosine monophosphate (cAMP) response to parathyroid hormone (PTH) was studied in a clonal osteosarcoma cell line ROS 17/2.8. CaCl2 was found to stimulate the PTH-sensitive cAMP response of intact cells. At the maximal concentration of 1 mM CaCl2, the maximum response to PTH was increased, but the ED50 for PTH and the time course of maximal cAMP production were not affected. Verapamil blunted, while the cation ionophore A23187 enhanced, the stimulatory effect of CaCl2. Trifluoperazine (TFP) and N-(6-aminohexyl-5-Cl-naphthalene sulfonamide) (W-7) inhibited the stimulatory effect of CaCl2. In membranes prepared in the presence of 0.1 mM CaCl2, a biphasic effect of CaCl2 was demonstrated: stimulation at concentrations of 60-100 microM, and an inhibition above 200 microM, when adenylate cyclase was assayed in the presence of 200 microM EGTA. Addition of exogenous calmodulin to membranes prepared in the presence of EGTA did not have any effect on the PTH-sensitive adenylate cyclase activity, suggesting that endogenous calmodulin was not effectively stripped from the membranes by EGTA treatment. It is concluded that Ca2+ has both a stimulatory and an inhibitory role in modulating PTH-sensitive adenylate cyclase in ROS 17/2.8 cells by as yet unknown mechanisms, and that the involvement of endogenous calmodulin is implicated.

Adenylyl Cyclases↗

PubMed1991

Continuously active sodium channels in osteoblastic ROS 17/2.8 cells.

Cell-attached patch clamp experiments revealed 13-20 pS Na(+)-conducting channels active at normal resting potentials (-28 +/- 1 mV; +/- SEM; 7 cells) in the rat osteosarcoma cell line, ROS 17/2.8. These channels were not blocked by tetrodotoxin, Cd2+, verapamil, or nifedipine. Replacing all cations in the patch pipette except Ca2+ with tetraethylammonium (TEA+) abolishes channel activity; but adding TEA+ to a pipette solution containing only Na+ does not. Depolarization was not necessary to activate these channels, and the open times were much longer than the millisecond open times characteristic of Na+ channels in excitable cells. Current-voltage curves reconstructed from mean single channel currents and mean channel open times resemble L-type Ca2+ current-voltage curves obtained from whole-cell experiments, with current peaks shifted to resting or more hyperpolarized potentials. The voltage sensitivity of these channels has implications on membrane potential stability and on the hyperpolarizing membrane potential spiking activity exhibited by ROS 17/2.8 cells.

Animals↗

PubMed1986

Disposition kinetics of adriamycin, adriamycinol and their 7-deoxyaglycones in AKR mice bearing a sub-cutaneously growing ridgway osteogenic sarcoma (ROS).

Disposition kinetics of Adriamycin (ADR), adriamycinol (AOL) and their 7-deoxyaglycones (ADR-DONE and AOL-DONE) have been studied in AKR mice bearing a s.c. growing ROS tumour after i.v. administration of 10 mg/kg. ADR and its metabolites were extracted from tissues by two different methods, separated and identified by HPLC. Tissue 7-deoxyaglycones were isolated, purified and then identified by HPLC, TLC and mass spectrometry. Kinetic profiles of ADR showed rapid equilibration of the drug with well perfused tissues but a slower and complex equilibration of the drug with the ROS tumour. Serum and tissue profiles of AOL were similar to the parent drug. From the kinetic profiles of the 7-deoxyaglycones it appeared that in the tissues their formation was rapid, with ADR-DONE always appearing first. Maximum concentrations of ADR-DONE were reached in the liver and heart only 10 min after drug administration. Estimated half lives of ADR-DONE were in liver, 1.1 hr and in heart, 2.8 hr and for AOL-DONE in liver, 5.4 hr, in heart, 5.1 hr and in serum, 4.1 hr.

Animals↗

PubMed1990

Effect of 5-azacytidine on metallothionein inducibility and sensitivity to lethality of cadmium in rat osteosarcoma (ROS 17/2.8) cells.

ROS 17/2.8 cells, a cloned rat osteoblastic osteosarcoma cell line, were found to be extremely sensitive to the lethal effects of cadmium and to synthesize little, if any, metallothionein in response to cadmium exposure. Culture of cells for 24 h in the presence of 1 microM 5-azacytidine, a cytidine analog, increased the inducibility of metallothionein by cadmium and significantly reduced (P less than 0.001) cytotoxicity. Anion exchange chromatographic analysis of cadmium binding to low molecular mass cytotoxicity. Anion exchange chromatographic analysis of cadmium binding to low molecular mass cytosolic proteins showed that cells treated with cadmium and 5-azacytidine expressed at least 2 isoforms of metallothionein. One isoform of metallothionein with a low affinity for cadmium was constitutively expressed by these cells. The association of poor inducibility of metallothionein by cadmium with extreme sensitivity of cells to cadmium emphasizes the role of this protein in the cellular response to this toxic metal. The modulation of metallothionein inducibility and sensitivity to cadmium by 5-azacytidine treatment suggest that metallothionein gene structure and regulation are altered in ROS 17/2.8 cells.

Animals↗

PubMed1994

Transforming growth factor-beta1 regulates steady-state PTH/PTHrP receptor mRNA levels and PTHrP binding in ROS 17/2.8 osteosarcoma cells.

The effect of transforming growth factor beta1 (TGF-beta1) on the expression of mRNA for the parathyroid hormone receptor and binding of iodinated parathyroid hormone-related protein in ROS 17/2.8 osteosarcoma cells was evaluated. TGF-beta1 stimulated a 2-7-fold increase in steady state mRNA levels for the parathyroid hormone receptor at a maximal dose of 5 ng/ml, with increased levels of expression at 6 h of TGF-beta1-incubation, and peak levels at 8-24 h. Receptor binding studies revealed a significant increase in PTHrP-specific binding with TGF-beta1 doses as low as 0.5 ng/ml and a 55% increase in numbers of receptors with no alteration in binding affinity with 5.0 ng/ml TGF-beta1. Time course studies indicated that receptor binding was increased at 24 h with peak levels reached at 48 h of treatment. PTH-stimulated cAMP levels were significantly increased in ROS 17/2.8 cells treated with TGF-beta1 (0.5 ng/ml) for 48 h. These data indicate that TGF-beta1 upregulates steady-state mRNA, ligand binding and PTH/PTHrP receptor signaling in rat osteosarcoma cells. The effects of TGF-beta1 on bone may be attributed in part to regulation of the PTH/PTHrP receptor at the molecular level.

Animals↗

PubMed1992

A mouse c-ros genomic clone: identification of a highly conserved 22-amino acid segment in the juxta-membrane domain.

The mouse c-ros protooncogene genomic sequences have been cloned; an analysis of the partial genomic clone revealed a high conservation of the exons encoding the juxta-membrane (JM) and the 5' most protein tyrosine kinase domains. We have identified a segment of 22 amino acids conserved between the human and mouse JM domains; this segment may have a critical role in the function of the c-ros-encoded protein tyrosine kinase receptor.

Amino Acid Sequence↗

PubMed1991

DNA damage by smoke: protection by turmeric and other inhibitors of ROS.

Twigs-dry leaves smoke condensate (TDS), as a source of clastogenic ROS and carcinogenic PAH, was investigated for its in vitro DNA-damaging effect in calf thymus DNA and human peripheral lymphocytes. An aqueous turmeric component--Aq.T--with an established antioxidant activity, was tested as a DNA protectant. TDS induced 13-fold damage to calf thymus DNA as judged by the emergence of a DNA damage specific, fluorescent product (em: 405 nm). Aq.T at 800 ng/microL extended 69% protection to calf thymus DNA and was comparable to the other protectants such as curcumin, BHA, vitamin E, SOD, and CAT. In human peripheral lymphocytes, TDS induced extensive DNA damage in comparison with the tumor promoter TPA, as judged by FADU. Aq.T at 300 ng/microL extended 90% protection to human lymphocyte DNA against TDS-induced damage, and was more effective than the other protectants--DABCO, D-mannitol, sodium benzoate, vitamin E (ROS quenchers), SOD, CAT (antioxidant enzymes), tannic acid, flufenamic acid, BHA, BHT, n-PG, curcumin and quercetin (antioxidants). Aq.T offered 65% protection to human lymphocyte DNA against TPA-induced damage and was comparable to SOD. The above results indicate that TDS induces substantial DNA damage in calf thymus DNA and human lymphocytes and Aq.T is an efficient protectant.

Animals↗

PubMed2006

Ethanol impairs CCK-8-evoked amylase secretion through Ca2+-mediated ROS generation in mouse pancreatic acinar cells.

In the present study, we have investigated the effect of ethanol on amylase release in response to cholecystokinin octapeptide (CCK-8). We have also studied the effect of ethanol on cytosolic free Ca(2+) concentration ([Ca(2+)](c)) and reactive oxygen species (ROS) production by loading of cells with fura-2 and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3nM of 19.39+/-2.73% of the total content of amylase. Treatment of pancreatic acini with ethanol did not induce any significant effect on amylase release at a wide range of concentrations (1-50mM). In contrast, incubation of cells with 50mM ethanol clearly reduced amylase release stimulated by CCK-8. The inhibitory effect of ethanol on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulfhydryl reducing agent. Ethanol induced an increase in [Ca(2+)](c) resulting in a level higher than the prestimulation level both in the presence and in the absence of extracellular Ca(2+). Additionally, ethanol led to an increase in fluorescence of CM-H(2)DCFDA, reflecting an increase in oxidation. A decrease in oxidation was observed in the absence of extracellular Ca(2+) and in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid. Similarly, when the cells were challenged in the presence of the intracellular Ca(2+) chelator 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and in the absence of extracellular Ca(2+), the responses to ethanol were reduced, although not completely inhibited. Taken together, our results suggest that ethanol induces generation of ROS by a Ca(2+)-dependent mechanism and reduces CCK-8-evoked amylase secretion in exocrine pancreatic cells.

Amylases↗

PubMed2006

Cytotoxic mechanisms of Zn2+ and Cd2+ involve Na+/H+ exchanger (NHE) activation by ROS.

The signaling mechanism induced by cadmium (Cd) and zinc (Zn) in gill cells of Mytilus galloprovincialis was investigated. Both metals cause an increase in *O2- production, with Cd to be more potent (216 +/- 15%) than Zn (150 +/- 9.5%), in relation to control value (100%). The metals effect was reversed after incubation with the amiloride analogue, EIPA, a selective Na+/H+ exchanger (NHE) inhibitor as well as in the presence of calphostin C, a protein kinase C (PKC) inhibitor. The heavy metals effect on *O2- production was mediated via the interaction of metal ions with alpha1- and beta-adrenergic receptors, as shown after incubation with their respective agonists and antagonists. In addition, both metals caused an increase in intracellular pH (pHi) of gill cells. EIPA together with either metal significantly reduced the effect of each metal treatment on pHi. Incubation of gill cells with the oxidants rotenone, antimycin A and pyruvate caused a significant increase in pHi (delta pHi 0.830, 0.272 and 0.610, respectively), while in the presence of the anti-oxidant N-acetyl cysteine (NAC) a decrease in pHi (delta pHi -0.090) was measured, indicating that change in reactive oxygen species (ROS) production by heavy metals affects NHE activity. When rosiglitazone was incubated together with either heavy metal a decrease in O2- production was observed. Our results show a key role of NHE in the signal transduction pathway induced by Zn and Cd in gill cells, with the involvement of ROS, PKC, adrenergic and PPAR-gamma receptors. In addition, differences between the two metals concerning NHE activation, O2- production and interaction with adrenergic receptors were observed.

Acetylcysteine↗

PubMed2004

Heme deficiency causes apoptosis but does not increase ROS generation in HeLa cells.

Mitochondria provide cellular energy supply via respiration and are the major sites for the generation of reactive oxygen species (ROS). Mitochondria also play a fundamental role in apoptosis. Heme is a key factor in mitochondrial function. Defective heme synthesis or altered heme metabolism is associated with numerous diseases. Here we investigated the molecular mechanism by which heme promotes HeLa cell growth and survival. We found that heme deficiency-induced apoptosis involves the release of cytochrome c and the activation of caspase 3. However, heme deficiency-induced apoptosis appears to occur by a unique mechanism distinct from those known to mediate mitochondrial-dependent apoptosis. Specifically, our data show that heme deficiency causes apoptosis in a pathway that is independent of ROS generation and the collapse of mitochondrial membrane potential. These results provide insights into how defective heme synthesis or altered heme metabolism causes diseases and how heme may control cell growth and cell death.

Apoptosis↗

PubMed2004

Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1).

Chronic inflammation through foam cells and macrophages is important in atherosclerosis development, and can be considered as therapeutic targets. Cyclooxygenase and NADPH-oxidase were expressed within atherosclerotic lesions. Reactive oxygen species produced by NADPH oxidase were found to trigger the cyclooxygenase-2 expression. The effects of preferential COX-2 inhibitors on ROS produced by Chlamydia-primed human monocytes (THP-1 cells) were evaluated by fluorescence, chemiluminescence, oxymetry, and EPR spin trapping. Fluorescence assays showed an increased production of ROS with Chlamydia versus cells primed by 10(-8)M PMA. COX-2 inhibitors inhibited in a dose-dependent manner the luminol-enhanced CL while ibuprofen and diclofenac increased the chemiluminescence response. By EPR spin trapping, COX-2 inhibitors, ibuprofen, and diclofenac, exhibited a dose-dependent inhibiting effect (10 and 100muM) on the EPR signal appearance. Our cell model combining EPR, chemiluminescence, and oxymetry appeared relevant to study the modulating effects of preferential COX-2 inhibitors on the cell oxidant activity and chronic inflammatory diseases.

Arteriosclerosis↗

PubMed2005

Age-dependent increase of etheno-DNA-adducts in liver and brain of ROS overproducing OXYS rats.

Reactive oxygen species (ROS) and lipid peroxidation (LPO) play a role in aging and degenerative diseases. To correlate oxidative stress and LPO-derived DNA damage, we determined etheno-DNA-adducts in liver and brain from ROS overproducing OXYS rats in comparison with age-matched Wistar rats. Liver DNA samples from 3- and 15-month-old OXYS and Wistar rats were analyzed for 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) by immunoaffinity/32P-postlabelling. While epsilondA and epsilondC levels were not different in young rats, adduct levels were significantly higher in old OXYS rats when compared to old Wistar or young OXYS rats. Frozen rat brain sections were analyzed for epsilondA by immunostaining of nuclei. Brains from old OXYS rats accumulated epsilondA more frequently than age-matched Wistar rats. Our results demonstrate increased LPO-induced DNA damage in organs of OXYS rats which correlates with their known shorter life-span and elevated frequency of chronic degenerative diseases.

Aging↗

PubMed2006

beta cell cytoprotective strategies: establishing the relative roles for iNOS and ROS.

Cytokine-induced beta cell destruction may be mediated by the generation of nitric oxide and/or reactive oxygen species. The relative importance of NO and ROS in cytokine-induced beta cell pathophysiology remains unclear. This investigation evaluates and contrasts the cytoprotective potential of antioxidant gene transfer, versus NF-kappaB inhibition, using a degradation-resistant mutant of IkappaBalpha. NF-kappaB inhibition conferred significant protection against cytokine-induced damage whereas antioxidant overexpression failed to provide protection. Conferred cytoprotection was associated with a suppression of iNOS activation and nitrite accumulation. Our data implicates iNOS, as opposed to ROS, as the pivotal player in cytokine-induced beta cell damage. From a therapeutic standpoint, strategies aimed at targeting the activation of iNOS may harbor therapeutic potential in preserving beta cell survival in the face of proinflammatory cytokine exposure.

Animals↗

PubMed2004

Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells.

Leptomycin B (LMB), which is originally isolated from Streptomyces, possesses anti-tumor properties in vivo and in vitro. Though it was previously reported that LMB induces cell cycle arrest and p53-mediated apoptosis in certain cancer cells, however, the mechanism by which LMB induces apoptosis remains poorly understood. Here, we investigated the mechanisms of apoptosis induced by LMB in U937 cells. Treatment with LMB concentration-dependently induced cytotoxicity and apoptosis in U937 cells that correlated temporally with activation of caspases and down-regulation of Mcl-1 and XIAP. LMB did not change the expressions of Bcl-2 or Bax. A broad spectrum caspase inhibitor, z-VAD-fmk, blocked caspase-3 activation and elevated the survival in LMB-treated U937 cells, suggesting that caspase-3 activation is critical for LMB-induced apoptosis. Interestingly, Bcl-2 overexpression that blocked cytochrome c release by LMB effectively attenuated the apoptotic response to LMB, suggesting that LMB-induced apoptosis is mediated through the mitochondrial pathway. Antioxidants or antioxidant enzymes had no effects on LMB-induced apoptosis. Data of flow cytometry analysis using 2',7'-dichlorofluorescein-diacetate further revealed no reactive oxygen species (ROS) generation by LMB, indicating that apoptosis induced by LMB is ROS-independent. However, the apoptotic response to LMB was not shown in U937 cells pretreated with the sulfhydryl group-containing antioxidant N-acetylcysteine (NAC). Further analysis suggested that NAC directly binds LMB and abolishes the apoptotic effects of LMB. Collectively, these findings suggest that LMB potently induces apoptosis in U937 cells, and LMB-induced apoptosis in U937 cells is related with cytochrome c release, activation of caspases, and selective down-regulation of Mcl-1 and XIAP.

Antibiotics, Antineoplastic↗

PubMed2005

ROS: really involved in oxygen sensing.

The role of reactive oxygen species (ROS) in the cellular response to cellular oxygen sensing has been controversial. Three papers in this issue of Cell Metabolism (Brunelle et al., Guzy et al., 2005; Mansfield et al., 2005) used genetic tools to establish that ROS produced by mitochondria are required for the normal induction of HIF (hypoxia-inducible factor), which is a master regulator of oxygen-sensitive gene expression, by low oxygen.

Animals↗