Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intracellular delivery of nonpermeant molecules to cultured cells in situ can be problematic. This work is a comparison between two methods of accomplishing this delivery; liposome-mediated delivery and electroporation. The final goal was to examine the effects of the glutathione S-transferase (GST) inhibitor Basilen Blue (BB) on glyceryl trinitrate biotransformation in porcine kidney epithelial (PK1) cells after intracellular delivery. Initial evaluation used the fluorescent markers carboxyfluorescein and lucifer yellow (LY). This was followed by biochemical analysis of glyceryl trinitrate biotransformation. Liposome-mediated delivery proved ineffective in spite of variations in the lipid composition of the liposomes and the use of an agglutinin and a fusogen. In contrast, electroporation was a very effective method for intracellular delivery of both lucifer yellow and basilen blue to the PK1 cells. The results show that in cells where Basilen Blue was introduced, there was a decrease in both the glyceryl trinitrate (GTN) biotransformation and ratio of glyceryl-1,2-dinitrate (1,2-GDN) to glyceryl-1,3-dinitrate (1,3-GDN) formed. The technique of in situ electroporation shows great promise for the assessment of a variety of nonpermeant molecules of pharmacological interest.
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PMID:Comparison of liposome fusion and electroporation for the intracellular delivery of nonpermeant molecules to adherent cultured cells. 848 59

Nephrotoxic haloalkenes undergo glutathione- and cysteine conjugate beta-lyase-dependent bioactivation, and glutathione S-conjugate formation with haloalkenes as substrates is preferentially catalyzed by the hepatic microsomal glutathione S-transferase (mGST). Porcine kidney-derived LLC-PK1 cells, which are competent to bioactivate glutathione and cysteine S-conjugates of haloalkenes, show low mGST activity. Stable transfection of LLC-PK1 cells with the gene encoding mGST would be expected to increase glutathione S-conjugate formation and, therefore, to increase haloalkene cytotoxicity. Transfection of LLC-PK1 cells with human mGST genes resulted in increased expression of mGST protein in microsomal fractions, in increased glutathione S-conjugate formation with hexachloro-1,3-butadiene and 1-chloro-2,4-dinitrobenzene as the substrates, and in increased cytotoxicity of hexachloro-1,3-butadiene. In addition, transfection with mGST gene also increased the activity of cytosolic glutathione S-transferases.
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PMID:Stable transfection of LLC-PK1 cells with human microsomal glutathione S-transferase gene increases haloalkene glutathione S-conjugate formation and cytotoxicity. 917 97

The level of phosphorylated c-Jun NH2-terminal kinase (JNK) in LLC-PK1 cells treated with CdCl2 increased after 30 min and remained elevated even at 8 hr. And the activity of JNK assayed using glutathione S-transferase-c-Jun as substrate increased dose-dependently. Consistent with the JNK activation, marked increases in the levels of c-Jun and c-Jun phosphorylated on Ser63 and Ser73 were observed in cells treated with CdCl2. The pretreatment with an intracellular Ca2+ chelator, 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), abolished cadmium-induced JNK phosphorylation. However, pretreatment with a cell permeable chelator of heavy metals, N,N,N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), did not. The present results showed that cadmium induces persistent activation of JNK pathway in a renal epithelial cell line, and that intracellular Ca2+ is necessary for the activation.
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PMID:Activation of c-Jun NH2-terminal kinase (JNK/SAPK) in LLC-PK1 cells by cadmium. 979 7

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC-PK1 cells were treated with H2O2 in the absence of serum to induce cell death. Subsequent to exposure to H2O2, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H2O2-treated control cells, it was observed that 0.5 microM of desipramine and 25 mM of NAC exhibited about 90 and 95% of cytoprotection, respectively, against H2O2-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and 3%, respectively, when compared to 71% in H2O2-exposed cells. Cellular glutathione level in 500 microM H2O2-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H2O2-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H2O2-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.
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PMID:Protection of LLC-PK1 cells against hydrogen peroxide-induced cell death by modulation of ceramide level. 1583 19

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).
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PMID:Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex. 1597 99

We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na+/K+-ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na+/K+-ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the 1 subunit of Na+/K+-ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of 1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na+/K+-ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na+/K+-ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na+/K+-ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na+/K+-ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.
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PMID:Binding of Src to Na+/K+-ATPase forms a functional signaling complex. 1626 70

Mutations in the gene encoding polycystin-2 (PC2) result in autosomal dominant polycystic kidney disease and defects in left-right asymmetry during embryogenesis. PC2 is a TRP-type Ca(2+)-permeable non-selective cation channel, which is expressed in kidney and other organs. PC2 is present and functional in microtubule-containing primary cilia of renal epithelial cells. However, no information is yet available as to whether PC2 interacts with microtubules. Here, we assessed the role of microtubular dynamics in regulating PC2 channel function in primary cilia. Isolated ciliary membranes from LLC-PK1 epithelial cells were reconstituted in a lipid bilayer system. The acute addition of the microtubular disrupter colchicine (15 mum) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 mum) increased ciliary PC2 channel activity. The further addition of alpha-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, alpha-tubulin and GTP had no effect on in vitro translated PC2. Using the yeast two-hybrid assay, we found that PC2 interacts with the microtubule-dependent motor kinesin-2 subunit KIF3A, a protein involved in polycystic kidney disease. The interaction occurred through the carboxyl termini domain of both proteins, which was further confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. Co-immunoprecipitation experiments showed that PC2 and KIF3A are in the same complex in native HEK293, Madin-Darby canine kidney cells (MDCK), and LLC-PK1 cells. Immunofluorescent staining also showed substantial PC2 and KIF3A co-localization in primary cilia of renal epithelial cells. The data indicate that microtubular organization regulates PC2 function, which may explain, among others, the regulatory role of PC2 in the sensory function of primary cilia.
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PMID:Polycystin-2 cation channel function is under the control of microtubular structures in primary cilia of renal epithelial cells. 1695 Jul 92

Although glyceryl trinitrate (GTN) has been used in the treatment of angina for many years, details of its conversion to the proximal activator (presumed to be NO or an NO congener) of soluble guanylyl cyclase (sGC) are still unclear. We reported previously that purified microsomal glutathione transferase 1 (MGST1) mediates the denitration of GTN. In the current study, we investigated in intact cells whether this enzyme also converts GTN to species that activate sGC (mechanism-based biotransformation). We utilized LLC-PK1 cells, a cell line with an intact NO/sGC/cGMP system, and generated a stable cell line that overexpressed MGST1. MGST1 in the stably transfected cells was localized to the endoplasmic reticulum, and microsomes from these cells exhibited markedly increased GST activity. Although incubation of these cells with GTN resulted in a 3-4-fold increase in GTN biotransformation, attributed primarily to an increase in formation of the 1,3-glyceryl dinitrate metabolite, GTN-induced cGMP accumulation in cells overexpressing MGST1 was not different than that observed in wild type cells or in cells stably transfected with empty vector. To determine whether overexpression of NADPH cytochrome P450 reductase might act in concert with MGST1 to generate activators of sGC, we assessed GTN-induced cGMP accumulation in MGST1-overexpressing cells that had been transiently transfected with CPR. In this case, GTN-induced cGMP accumulation was also not different than that observed in wild type cells. We conclude that although MGST1 mediates the biotransformation of GTN in intact cells, this biotransformation does not contribute to the formation of activators of sGC.
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PMID:Role of microsomal glutathione transferase 1 in the mechanism-based biotransformation of glyceryl trinitrate in LLC-PK1 cells. 1942 24

Foetal calf serum (FCS) dependent cell viability, cytotoxicity and detoxification were investigated in MDCK and LLC-PK1 cells. FCS was used at 0-10% (v/v). Viability and cytotoxicity were measured by neutral red uptake and by the MTT test. Viability of LLC-PK1 was strongly dependent on the FCS concentration, but that of MDCK cells only to a very limited extent. For both cell lines the cytotoxicity of HgCl(2) was FCS concentration dependent: lower toxicity was observed with 5-10% FCS than with 0-1% FCS. This effect was not observed for paracetamol. The results could not be explained by altered glutathione or glutathione S-transferase. The optimal FCS concentration of 1%, necessary to retain cell viability, had a limited influence on cytotoxicity. FCS concentration must be taken into consideration when cytotoxicity data from different studies are compared.
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PMID:Effects of foetal calf serum on cell viability, cytotoxicity and detoxification in the two kidney-derived cell lines LLC-PK1 and MDCK. 2069 60

Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.
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PMID:An alteration in ELMOD3, an Arl2 GTPase-activating protein, is associated with hearing impairment in humans. 2403 9


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