Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study investigated the role of nitric oxide (NO)/cGMP signal transduction in the M(3) muscarinic acetylcholine receptor (mAChR)-stimulated increase in aquaporin-5 (AQP5) levels in the apical plasma membrane (APM) of rat parotid glands. Pretreatment of rat parotid tissue with the NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium inhibited both acetylcholine (ACh)- and pilocarpine-induced increases in AQP5 in the APM. NO donors [3-morpholinosydnonimine (SIN-1) and (S)-nitroso-N-acetylpenicillamine (SNAP)] mimicked the effects of mAChR agonists. A selective protein kinase G inhibitor [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg-3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823)] and an NO synthase inhibitor (N(6)-imminoethyl-L-lysine) blocked SIN-1- and SNAP-induced increases in AQP5 in the APM. A calmodulin kinase II inhibitor [(8)-5-isoquinolinesulfonic acid, 4-[2-(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (KN-62)] decreased the pilocarpine-induced increase of AQP5 in the APM. Using diaminofluorescinein-2 diacetate, enhanced NO synthase activity was detected in isolated parotid acinar cells after ACh-treatment. Treatment with dibutyryl cGMP, but not dibutyryl cAMP, induced an increase in AQP5 levels in the APM. BAPTA-AM inhibited the cGMP-induced increase in AQP5 in the APM. Pretreatment of the tissues with a myosin light chain kinase inhibitor [(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9)] inhibited a mAChR-stimulated increase in AQP5 levels in the APM. Although there was a significant ACh-induced increase in AQP5 in the APM in the absence of extracellular Ca(2+), the maximal effect of ACh on the AQP5 levels in the APM occurred in the presence of extracellular Ca(2+). These results suggest that NO/cGMP signal transduction has a crucial role in Ca(2+) homeostasis in the mAChR-stimulated increase in AQP5 levels in the APM of rat parotid glands.
Mol Pharmacol 2002 Jun
PMID:The muscarinic acetylcholine receptor-stimulated increase in aquaporin-5 levels in the apical plasma membrane in rat parotid acinar cells is coupled with activation of nitric oxide/cGMP signal transduction. 1202 4

Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide-dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH-3T3(IR) cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulin-stimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2+-mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulin-signaling pathways.
Mol Endocrinol 2002 Aug
PMID:Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells. 1214 46

We had shown previously that the novel, marine, anticancer compound dehydrothyrsiferol (DHT) does not modulate P-glycoprotein (P-gp) dependent drug efflux. Many chemotherapeutics with clinical impact are substrates for the structurally distant related membrane transport protein MRP1 (multidrug resistance-associated protein 1). Thus, we were interested in analysing the behaviour of DHT and control compounds in specific drug transport of MRP1 overexpressing cells. We established a fluorescence based drug efflux system for specific, functional detection of interference of a test compound in MRP1 mediated drug extrusion. Briefly, MRP1 overexpressing HL60/Adr cells were incubated to uptake and then efflux fluorescent 5(6)-carboxyfluorescein diacetate (CFDA), rhodamine 123 (Rh123), or 3,3-diethylocarbocyanine iodide (DiOC2), respectively. Changes in cell fluorescence intensity after coincubation with the compound of interest were determined by flow cytometry. MRP1 mediated efflux of CFDA was analysed in the presence of DHT, the known substrates genistein, probenecid, and the specific inhibitor MK-571. To exclude unknown P-gp related interference in drug transport, efflux of the fluorescent P-gp substrate DiOC2 and specific inhibition by cyclosporin A (CsA) were analysed. Cytotoxicity of DHT in resistant HL60/Adr cells was found to be even superior to that in the parental HL60 leukaemia cell line. Consequently, DHT did not interfere in MRP1 mediated drug transport. In contrast to DiOC2, rhodamine 123 was not specifically effluxed by P-gp but also by MRP1. Therefore, we propose the MRP1 specific CFDA efflux model as a screening and/or excluding system for MRP1 substrates. Together with previous data our results suggest DHT to be an interesting candidate for further investigation directed towards a drug development regimen.
Int J Mol Med 2002 Nov
PMID:Dehydrothyrsiferol does not modulate multidrug resistance-associated protein 1 resistance: a functional screening system for MRP1 substrates. 1237

We previously reported the isolation of the novel human DENN gene, which is differentially expressed in normal and neoplastic cells. DENN is identical to MADD (mitogen-activated protein kinase-activating death domain), which interacts with tumor necrosis factor receptor 1 through their death domains. DENN is also homologous to Rab3 GEP, a rat Rab3 GDP/GTP exchange protein. Real-time reverse transcription-polymerase chain reaction analysis showed that DENN expression in cancer cell lines was 26-50 times that in normal cells. The Jurkat human leukemia, PLC/PRF/5 human hepatoma, and NS-1 mouse myeloma cell lines as well as the MRC-5 human fetal lung and Vero monkey kidney cell lines were treated successfully with four separate DENN-targeted antisense oligodeoxynucleotides (ODNs) to abrogate DENN expression. Quantitative assessment of cell viability and apoptosis by flow cytometry via fluorescein diacetate and propidium iodide membrane-integrity tests, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end-labeling, and annexin V assays showed that antisense silencing of DENN resulted in markedly more pronounced cell death in cancer cells compared with nonmalignant cells. Antisense-treated cell lines exhibited extensive loss of DNA content, forming distinct sub-G(1) peaks, while cell proliferation diminished significantly. Ultrastructural features of programmed cell death in cells subjected to antisense ODNs were authenticated by electron microscopy. In contrast, transfection of cell lines with a plasmid construct to achieve DENN overexpression augmented cellular proliferation and could reverse the apoptotic effect of antisense and staurosporine treatment. Our findings suggest that DENN is intimately involved in anti-apoptotic and cell-survival processes.
Mol Carcinog 2002 Nov
PMID:Induction of marked apoptosis in mammalian cancer cell lines by antisense DNA treatment to abolish expression of DENN (differentially expressed in normal and neoplastic cells). 1241 May 63

Previously, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h, respectively, after irradiation of 5 Gy. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c-release was confirmed. In order to elucidate the mechanism which acts prior to the mitochondrial membrane potential changes, we examined in the previous study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we examined in the present study reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation.
Int J Mol Med 2003 Feb
PMID:Radiation-induced reactive oxygen species formation prior to oxidative DNA damage in human peripheral T cells. 1252 68

Ion movements are among the early signals that could play important roles in cancer cell proliferation and metastasis. In this work, we investigated the role of K+ channels in adhesion and proliferation of H35 hepatocarcinoma cells. A variety of K+ channel blockers were used in order to differentiate the critical subtype(s) of K+ channels involved. 4-Aminopyridine, an inhibitor of voltage-gated K+ channels, significantly reduced the attachment of H35 cells to primary rat endothelial layer as determined by CFSE (5-(6-)-carboxyfluorescein diacetate succinimidyl ester) fluorescence assay. 4-Aminopyridine also inhibited the proliferation of H35 cells as measured by [3H]-thymidine incorporation. Non-selective K+ channel blockers TPeA and verapamil had similar inhibitory effects on H35 cell adhesion and proliferation. In contrast, iberiotoxin, a selective inhibitor of KCa channels, had no effect on the adhesion and proliferation of H35 cells. Glibenclamide, a potent inhibitor of KATP channels, could inhibit the cell adhesion and proliferation only at a very high concentration (100 micro M) that may block Kv channels. These experiments suggest that Kv channels play an important role in the metastasis and proliferation of hepatocarcinoma cells. Since inhibition of K+ channels would reduce Ca2+ influx in these cells, it is likely that the influence of Kv channels on H35 cell adhesion and proliferation is mediated by a Ca2+-dependent mechanism.
Int J Mol Med 2003 Feb
PMID:Blockage of voltage-gated K+ channels inhibits adhesion and proliferation of hepatocarcinoma cells. 1252 89

Excessive generation of reactive oxygen species (ROS) has been suggested as a causal factor in various neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease [Brain Res. 830 (1999) 10-15; Biochem. J. 310 (1995) 83-90; Free Radic. Biol. Med. 27 (1999) 612-616]. The present work examined the role of ROS in the neurotoxicity of methylmercury (MeHg). ROS formation in primary astrocytic cultures of neonatal rat cerebral cortex was monitored by 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) fluorescence. MeHg, at 10 and 20 microM caused a significant increase in ROS formation (10 microM, P<0.01; 20 microM, P<0.001). Additional studies established the effectiveness of antioxidants/free radical scavengers in attenuating the MeHg-stimulated ROS formation in the following rank-order: (1) Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a non-thiol containing antioxidant, (2) n-propyl gallate (PG), a free radical scavenger, (3) superoxide dismutase (SOD), an antioxidant enzyme that dismutates superoxide anion radical, (4) alpha-phenyl-tert-butyl nitrone (PBN), a lipophilic hydroxyl radical spin trapping agent. A significant inhibition of MeHg-induced ROS generation was also noted in astrocytes preincubated (3 h) with arachidonyl trifluoromethyl ketone (AACOCF(3,) 20 microM, P<0.05), a specific inhibitor of cytosolic phospholipase A(2) (cPLA(2)). Conversely, pretreatment (24 h) with 100 microM buthionine-L-sulfoxamine [BSO, a glutathione (GSH) synthesis inhibitor], significantly increased (P<0.05) ROS formation in MeHg treated astrocytes compared to controls. Combined, these studies invoke ROS as potent mediators of MeHg cytotoxicity and support the hypothesis that excessive ROS generation, at least in part, plays an important role in MeHg-induced neurotoxicity.
Brain Res Mol Brain Res 2003 Jan 31
PMID:Methylmercury-induced reactive oxygen species formation in neonatal cerebral astrocytic cultures is attenuated by antioxidants. 1257 36

Endogenous sources of reactive nitrogen species (RNS) act as second messengers in a variety of cell signaling events, whereas environmental sources of RNS like nitrogen dioxide (NO2) inhibit cell survival and growth through covalent modification of cellular macromolecules. To examine the effects of RNS on cell cycle progression, murine type II alveolar C10 cells arrested in G0 by serum deprivation were exposed to either NO2 or SIN-1, a generator of RNS, during cell cycle re-entry. In serum-stimulated cells, RNS did not prevent the immediate early gene response by AP-1, but rather blocked cyclin D1 gene expression, resulting cell cycle arrest at the boundary between G0 and G1. Dichlorofluorescin diacetate (DCF) fluorescence indicated that RNS induced sustained production of intracellular hydrogen peroxide (H2O2), which normally is produced only transiently in response to serum growth factors. Loading cells with catalase did not diminish the formation of 3-nitrotyrosine on the cell surface, but rather prevented enhanced DCF fluorescence and rescued cyclin D1 expression and S phase entry. These studies indicate environmental RNS interfere with cell cycle re-entry through an H2O2-dependent mechanism that influences expression of cyclin D1 and progression from G0 to the G1 phase of the cell cycle.
Am J Respir Cell Mol Biol 2003 Jun
PMID:Reactive nitrogen species block cell cycle re-entry through sustained production of hydrogen peroxide. 1260 Aug 34

Previously, we examined the formation of reactive oxygen species (ROS) in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In this study, we examined radiation-induced ROS formation in adult articular chondrocytes, which were demonstrated to be highly resistant to apoptosis in our previous study. We found that ROS formation was actually scarcely seen after irradiation of up to 20 Gy in these cells. Therefore, the origin of the great difference of radiosensitivity between T lymphocytes and adult articular chondrocytes is considered to lie in the degree of ROS formation following irradiation, with this difference possibly resulting from the scavenging acuity of these two kinds of normal tissue cells for free radicals including hydroxyl radicals.
Int J Mol Med 2003 Apr
PMID:Comparison of radiation-induced reactive oxygen species formation in adult articular chondrocytes and that in human peripheral T cells: possible implication in radiosensitivity. 1263 97

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are known to play an important role in the proliferation and viability of vascular smooth muscle cells. In this study, we determined the effects of increased superoxide dismutase and catalase activity on fetal pulmonary arterial smooth muscle cell (FPASMC) proliferation and viability using EUK-134, a superoxide dismutase/catalase mimetic. Treatment of FPASMC with EUK-134 or with a combination of superoxide dismutase and catalase enzymes decreased superoxide and hydrogen peroxide levels as detected by the fluorescent dyes dihydroethidium and dichlorodihydrofluorescein diacetate, respectively. EUK-134 (5 microM) attenuated serum-induced FPASMC proliferation, whereas 50 microM EUK-134 decreased the number of viable cells, suggesting cell death. Conversely, combined superoxide dismutase and catalase enzyme activity equivalent to 50 microM EUK-134 prevented proliferation but did not reduce the number of viable FPASMC. The loss of mitochondrial membrane potential after 18 h, an increase in caspase-9 and caspase-3 activity after 24 h, and the subsequent appearance of TdT-mediated dUTP nick end labeling-positive nuclei were detected in FPASMC after treatment with 50 microM EUK-134. This indicates an induction of programmed rather than necrotic cell death and suggests that prolonged removal of ROS is required to stimulate apoptosis. Compounds such as EUK-134 may, therefore, prove more effective than enzymic antioxidants over longer periods, especially when the aim is to decrease the number of smooth muscle cells in diseases resulting from excessive muscularization.
Am J Physiol Lung Cell Mol Physiol 2003 Aug
PMID:Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic. 1266 66


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