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PubMed1995

Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing.

Recent studies on the induction of erythropoietin gene expression by hypoxia have indicated that erythropoietin forms part of a widely operative system of gene regulation by oxygen. Similar responses to hypoxia, cobaltous ions and desferrioxamine have indicated that the action of these agents is closely connected with the mechanism of oxygen sensing. To consider further the mechanisms underlying these responses, the effect of iodonium compounds was tested on five genes which show oxygen-regulated expression; erythropoietin, vascular endothelial growth factor (VEGF), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (GLUT-1) and placental growth factor (PLGF). In each case, the response to hypoxia was specifically inhibited by low doses of diphenylene iodonium (Ph1I+). This occurred irrespective of whether the hypoxic response was induction of gene expression (erythropoietin, vascular endothelial growth factor, lactate dehydrogenase-A, glucose transporter-1) or inhibition of gene expression (PLGF). In contrast, the induction of gene expression by cobaltous ions or desferrioxamine was not inhibited by Ph2I+. The differential action of Ph2I+ on the response to hypoxia versus the response to cobaltous ions or desferrioxamine must reflect a difference in the mechanism of action of these stimuli, which will require accommodation in any model of the oxygen-sensing mechanism. Based on the known properties of Ph2I+, the implication of these findings is that the mechanism of oxygen sensing most probably involves the operation of a flavoprotein oxidoreductase.

Base Sequence↗
PubMed2004

In vitro activities of iodonium salts against oral and dental anaerobes.

The comparative in vitro activities of 11 iodonium salt compounds, 0.12% chlorhexidine, and four antimicrobial agents against 322 anaerobic and fastidious potential dental and periodontal bacterial pathogens were studied. Iodonium salts 3, 4, 5, 9, and 10 had in vitro activities comparable to that of chlorhexidine against most isolates. These compounds may be suitable for incorporation into an oral mouthwash.

Anti-Infective Agents↗
PubMed1993

Membrane transport of tetraphenylphosphonium and its homologues through the planar phospholipid bilayer: concentration dependence and mutually competitive inhibition in membrane passive transport.

The concentration dependence and mutually competitive inhibition in the membrane transport of the lipophilic cations tetraphenylphosphonium (TPP+) and its homologues were studied by use of a planar phospholipid bilayer. The current-voltage characteristics were analyzed by use of a model developed by Ketterer et al. (J. Membr. Biol. 1971, 5, 225-245). The conductance at 0 mV [G(0)], which represents the transport ability, increased with an increase in concentration, and the G(0) values revealed saturation at higher concentrations. The relationship between G(0) and concentration was well fitted to a Michealis-Menten-type equation with a saturable component (maximum conductance [G(0)max] and concentration yielding one-half of G(0)max[Km]). The G(0)max and Km values were calculated to be 4.1 x 10(-8)-242 x 10(-8) S/cm2 and 13-489 microM, respectively, depending on the lipophilic cations used. The G(0) values normalized by the corresponding G(0)max values were plotted against the concentrations normalized by the corresponding Km values. Normalized curves for TPP+ homologues were all superimposed on a single curve. The mechanism for the saturation of G(0) values may involve the adsorption of TPP+ homologues to a planar phospholipid bilayer, because the Km values calculated in the present study were comparable to those for adsorption to phosphatidylcholine liposomes. We further determined the mutual inhibition of membrane transport by these homologues. Tetraphenylmethylphosphonium at 1 mM, a concentration threefold higher than the Km value for this compound, reduced the G(0) for other TPP+ homologues by 38-55%, whereas reduction by 10 mM tetraethylammonium was minimal. The type of inhibition was classified as mutually competitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion↗
PubMed1994

Antagonism by lipophilic quaternary ions of the K+ channel opener, levcromakalim, in vascular smooth muscle.

1. The aim of this study was to characterize the interaction between the K+ channel opener levcromakalim (LKM) and several quaternary ions, in vascular smooth muscle, in vitro. Segments of isolated, thoracic aorta of the rat were suspended in organ baths filled with Krebs solution at 37 degrees C. Cumulative concentration-response curves to LKM were obtained in the absence and in the presence of increasing concentrations of quaternary ions using a number of agents to pre-constrict the vessel. The ions tested were tetraphenylphosphonium (chloride TPP-Cl and bromide TPP-Br salts), tetrapentylammonium (TPeA), tetraethylammonium (TEA), tetraphenylarsonium (TPAs) and tetraphenylboron (TPB). 2. For the compounds which antagonized the vasorelaxation responses of LKM, 'apparent pKB' values were estimated on the basis of a single concentration of antagonist. These were then used to obtain the following order of potency: TPP-Br (7.22 +/- 0.25) = TPAs (7.12 +/- 0.04) = TPP-Cl (7.11 +/- 0.15) > TPeA (6.23 +/- 0.20). TEA and TPB were both found to be inactive at the maximum concentrations used. 3. The interaction between the cationic TPP and anionic TPB was also investigated. The shift in the LKM concentration-response curve constructed in the presence of both of these compounds was compared to that when each agent was present separately. We found that TPB, at concentrations greater than 1 microM, reversed the blockade of the LKM-mediated relaxation induced by TPP (3 microM). 4. Similar experiments were undertaken combining TPB with either alinidine or glibenclamide (both functional antagonists of K+ channel openers). It was found that TPB (10 microM) partially reversed the antagonism induced by alinidine (30 and 100 microM) but had no effect on the action of glibenclamide(3 microM).5. These studies show that lipophilic cations such as TPP and TPAs are potent antagonists of levcromakalim-mediated vasorelaxation responses in the rat thoracic aorta. The mechanism by which these compounds cause their antagonism is not known. However, given the lipophilicity of these compounds, it is possible they may act at a number of sites including the KATP channel itself or possibly via some other intracellular mechanism.

Animals↗
PubMed2005

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium.

Diphenyleneiodonium (DPI) inhibits activity of flavoenzymes like NADPH oxidase, the major source of superoxide anion in cardiovascular system, but affects also other oxidoreductases. Contradictory data have been published concerning the effect of diphenyleneiodonium on the production of reactive oxygen species in cells, both inhibitory and stimulatory action of DPI being reported. We have examined the effect of DPI on the cellular production of reactive oxygen and nitrogen species (ROS/RNS) and on the proliferation and apoptosis of human vascular endothelial cells. We found increased oxidation of ROS-sensitive probes (dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate) when DPI (20 microM-100 microM) was present in the treated cells. However, oxidation of the fluorogenic probes was inhibited if DPI (20 microM-100 microM) was removed from the reaction medium after cell preincubation. These results suggest an artifactual oxidation of the fluorogenic probes by DPI or its metabolites. A similar pattern of influence of DPI on the production of NO (measured with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) was observed. Modulation of generation of reactive oxygen and nitrogen species in DPI-treated cells influenced the nitration of tyrosine residues of cellular proteins, estimated by Western blotting. Decreased level of nitration generally paralleled the lowered production of ROS. A decreased 3-(4,5-dimethylthiazolyl)-3-3(4-sulphophenyl) tetrazolium (MTT) reducing activity of cells for was observed immediately after 1h treatment of human endothelial cells with DPI (1 microM-100 microM), in spite of lack of changes in cell viability estimated by other methods. These results point to a next limitation of MTT in estimation of viability of cells treated with oxidoreductase inhibitors. DPI inhibited the proliferation of HUVECs as well as immortalized cell line HUVEC-ST, as assessed by acid phosphatase activity test and measurement of total nucleic acid content. Proapoptotic action of DPI was observed 12 h after incubation with this compound.

Acid Phosphatase↗
PubMed1999

The qacG gene on plasmid pST94 confers resistance to quaternary ammonium compounds in staphylococci isolated from the food industry.

The 2.3 kb resistance plasmid pST94 revealed a new gene (qacG) encoding resistance to benzalkonium chloride (BC), a commonly used quaternary ammonium disinfectant, and the intercalating dye ethidium bromide (Eb) in staphylococci isolated from the food industry. The 107 amino acid QacG protein showing 69.2% identity to the staphylococcal multi-drug resistance protein Smr is a new member of the small multi-drug resistance (SMR) protein family. QacG conferred resistance via proton dependent efflux. An additional ORF on pST94 encoded a protein with extensive similarity to replication proteins of other Gram-positive bacteria. Gene constructs containing the qacG and smr gene region combined with the smr or qacG promoter, respectively, indicated that QacG is more efficient than Smr and that qacG has a weaker promoter. Resistant qacG-containing cells could be adapted to withstand higher concentrations of BC. Adapted qacG-containing cells showed increased resistance mainly to BC. In contrast, adaptation of sensitive cells showed cross-resistance development to a range of compounds. Induction of proton-dependent efflux was observed for BC-adapted staphylococci cells not containing qacG. The ability of sublethal concentrations of BC to develop cross-resistance and induce efflux mechanisms could be of practical significance; it should be considered before use of any new disinfectant and in the design of better disinfection procedures.

Anti-Infective Agents, Local↗
PubMed2005

Increased superoxide production in hypertensive patients with diabetes mellitus: role of nitric oxide synthase.

BACKGROUND: Hypertension and diabetes are important independent risk factors for increased oxidative stress and increased cardiovascular risk. The combination of hypertension and diabetes results in a dramatic increase in cardiovascular risk. Enhanced oxidative stress in hypertension and diabetes is linked to decreased nitric oxide (NO) bioavailability because of its interaction with vascular superoxide (O(2)(*-)), derived predominantly from NAD(P)H-dependent oxidases. When uncoupled from essential cofactors, NO synthase III (NOS III) can also produce O(2)(*-). We studied platelet superoxide production in patients with hypertension alone and in patients with coexistent diabetes mellitus, investigating the contribution of NOS III uncoupling to platelet superoxide production. METHODS AND RESULTS: Gel-filtered platelets were obtained and were stimulated with Phorbol 12-myristate 13-acetate, and O(2)(*-) production was detected using lucigenin-enhanced chemiluminescence. Superoxide production was significantly higher in patients with diabetes and hypertension (6.4 +/- 1.6 pmol/min/10(8) platelets) than in patients with hypertension (1.6 +/- 0.6 pmol/min/10(8) platelets) (P < .04). After incorporation of N(omega)-nitro-l-arginine methyl ester (L-NAME, 1 mmol/L), O(2)(*-) detection increased in 40% of patients with diabetes and hypertension and in 87% of patients with hypertension. This expected response results from L-NAME inhibition of NO production preventing NO scavenging of O(2)(*-). A reduction in O(2)(*-) production in response to L-NAME occurred in the remaining patients and indicates O(2)(*-) production by the uncoupled NOS III enzyme. CONCLUSIONS: This study provides first published evidence that NOS III can reside in the uncoupled state in patients with hypertension and, to a greater extent, in patients with coexisting hypertension and diabetes, and that it contributes significantly to increased superoxide production in these disease states.

Adult↗
PubMed2005

Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery.

In addition to endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium-derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H2O2) in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh). ACh (10(-6), 10(-5), and 10(-4) M) induced a transient contraction of rat renal artery with intact endothelium in a concentration-related manner, but not in the artery with endothelium removed. In phenylephrine-precontracted renal arteries, ACh induced an endothelium-dependent relaxation response at lower concentrations (10(-8)-10(-6) M), and a relaxation followed by a contraction at higher concentrations (10(-5) M). Inhibition of nitric oxide synthase by N(omega)-nitro-L-arginine (10(-4) M) enhanced the EDC to ACh. Catalase (1000 U ml(-1)) reduced the EDC to ACh. H2O2 (10(-6), 10(-5), and 10(-4) M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium-independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh-induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17-octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H2 receptors (SQ 29548) and thromboxane A2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H2O2. In isolated endothelial cells, ACh (10(-5) M) induced a transient H2O2 production detected with a fluorescence dye sensitive to H2O2 (2',7'-dichlorofluorescein diacetate). The peak concentration of H2O2 was 5.1 x 10(-4) M at 3 min and was prevented by catalase. Taken together, these results show that ACh triggers H2O2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H2O2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H2O2 is most likely the EDCF in rat renal artery in response to ACh stimulation.

Acetylcholine↗
PubMed2005

Cytochrome P4502E1 primes macrophages to increase TNF-alpha production in response to lipopolysaccharide.

Kupffer cells become activated in response to elevated levels of LPS during ethanol feeding, but the role of ethanol in the molecular processes of activation remains unclear. Because cytochrome P4502E1 (CYP2E1) is upregulated in Kupffer cells after ethanol, we hypothesized that this effect primes Kupffer cells, sensitizing them to increase TNF-alpha production in response to LPS. However, cultured Kupffer cells rapidly lose their CYP2E1. This difficulty was overcome by transfecting CYP2E1 to RAW 264.7 macrophages. Macrophages with stable increased CYP2E1 expression (E2) displayed increased levels of CD14/Toll-like receptor 4, NADPH oxidase and H2O2, accompanied by activation of ERK1/2, p38, and NF-kappaB. These increases primed E2 cells, sensitizing them to LPS stimuli, with amplification of LPS signaling, resulting in increased TNF-alpha production. Diphenyleneiodonium, a NADPH oxidase inhibitor, and diallyl sulfide, a CYP2E1 inhibitor, decreased approximately equally H2O2 levels in E2 cells, suggesting that NADPH oxidase and CYP2E1 contribute equally to H2O2 generation. Because CYP2E1 expression also enhanced the levels of the membrane localized NADPH oxidase subunits p47phox and p67phox, thereby contributing to the oxidase activation, it may augment H2O2 generation via this mechanism. H2O2, derived in part from NADPH and CYP2E1, activated ERK1/2 and p38. ERK1/2 stimulated TNF-alpha production via activation of NF-kappaB, whereas p38 promoted TNF-alpha production by stabilizing TNF-alpha mRNA. Oxidant generation after CYP2E1 overexpression appears to be central to macrophage priming and their sensitization to LPS. Accordingly, CYP2E1 priming could explain the sensitization of Kupffer cells to LPS activation by ethanol, a critical early step in alcoholic liver disease.

Allyl Compounds↗
PubMed1993

Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.

Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.

Animals↗
PubMed2000

Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity.

Available evidence for oxidative stress after angioplasty is indirect or ambiguous. We sought to characterize the pattern, time course, and possible sources of free radical generation early after arterial balloon injury. Ex vivo injury performed in arterial rings in buffer with lucigenin yielded a massive oxygen-dependent peak of luminescence that decayed exponentially and was proportional to the degree of injury. Signals for injured vs. control arteries were 207. 1 +/- 17.9 (n = 13) vs 4.1 +/- 0.7 (n = 22) cpm x 10(3)/mg/min (p <. 001). Data obtained with 0.25 mmol/l lucigenin were validated with 0. 005-0.05 mmol/l lucigenin or the novel superoxide-sensitive probe coelenterazine (5 micromol/l). Gentle removal of endothelium prior to injury scarcely affected the amount of luminescence. Lucigenin signals were amplified 5- to 20-fold by exogenous NAD(P)H, and were >85% inhibited by diphenyliodonium (DPI, a flavoenzyme inhibitor). Antagonists of several other potential free radical sources, including xanthine oxidase, nitric oxide synthase, and mitochondrial electron transport, were without effect. Overdistension of intact rabbit iliac arteries in vivo (n = 7) induced 72% fall in intracellular reduced glutathione and 68% increase in oxidized glutathione, so that GSH/GSSG ratio changed from 7.93 +/- 2.14 to 0. 81 +/- 0.16 (p <.005). There was also 28.7% loss of the glutathione pool. Further studies were performed with electron paramagnetic resonance spectroscopy. Rabbit aortas submitted to ex vivo overdistension in the presence of the spin trap DEPMPO (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide, 100 mmol/l, n = 5) showed formation of radical adduct spectra, abolished by DPI or superoxide dismutase. Computer simulation indicated a mixture of hydroxyl and carbon-centered radical adducts, likely due to decay of superoxide adduct. Electrical mobility shift assays for NF-kappaB activation were performed in nuclear protein extracts from intact or previously injured rabbit aortas. Balloon injury induced early NF-kappaB activation, which was decreased by DPI. In conclusion, our data show unambiguously that arterial injury induces an immediate profound vascular oxidative stress. Such redox imbalance is likely accounted for by activation of vessel wall NAD(P)H oxidoreductase(s), generating radical species potentially involved in tissue repair.

Acridines↗
PubMed1976

Inhibitors of tRNA methyltransferases. S-Adenosylsulfonium salts.

Three new compounds have been synthesized and tested as in vitro inhibitors of normal and tumor tRNA methyltransferases. These compounds are 5'-methylethyl(5'adenosyl) sulfonium chloride (MEAS), 5'-methylpropyl-(5'adenosyl)sulfonium chloride (MPAS), and 5'-ethylpropyl(5'-adenosyl)sulfonium chloride (EPAS) They were prepared by reacting an alkyl iodide with the appropriate alkyladenosyl thioether. Inhibition assays revealed all three compounds to be inhibitors of normal and tumor tRNA methyltransferases. The propyl compounds were slightly better inhibitors of the tumor tRNA methyl transferases. MPAS, EPAS, and MEAS had KI's of 58.5, 61.7, and 24.5, respectively, for the normal tRNA methyltransferases and 15.3, 13.8, and 44.3, respectively, for the tumor tRNA methyltransferases.

Animals↗
PubMed2000

Stimulation of NADPH oxidase by oxidized low-density lipoprotein induces proliferation of human vascular endothelial cells.

Oxidized low-density lipoprotein (OxLDL) exerts proliferation and apoptosis in vascular cells, depending on its concentration and the duration of exposure. Recent studies indicate that [O(2)](-) is involved in cell cycle regulation and that OxLDL stimulates endothelial cells to produce [O(2)](-). This study examined the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a potential source for [O(2)](-) in the proliferation-inducing activity of OxLDL in cultured human umbilical vein endothelial cells (HUVEC). Human LDL was oxidized by Cu(++), and proliferation of HUVEC was detected by 3H-thymidine incorporation. OxLDL (5 microg/ml) caused an increase in proliferation of HUVEC of 250 to 300%. OxLDL-induced proliferation was blocked by addition of the antioxidants superoxide dismutase and catalase, suggesting that enhanced [O(2)](-) formation was involved. Diphenylene iodonium (DPI, 1 microM), an inhibitor of NADPH oxidase, also prevented OxLDL-induced proliferation of HUVEC, indicating that NADPH oxidase was the source for enhanced [O(2)](-) formation. The OxLDL effect was mimicked by lysophosphatidylcholine (LPC, 10 microM), a compound formed during oxidation of LDL. LPC-induced proliferation was also prevented by coincubation with DPI. Treatment of HUVEC with [O(2)](-) generated by the xanthine/xanthine oxidase reaction resulted in proliferation as did treatment with OxLDL. As expected, this stimulation could not be blocked by DPI. With the use of the cytochrome c-assay, it was demonstrated that OxLDL and LPC enhanced [O(2)](-) formation in HUVEC (by factor 3.2 and by factor 3.5, respectively). Supporting the assumption that NADPH oxidase was the enzyme responsible for [O(2)](-) formation, cells transfected with antisense oligonucleotides for NADPH oxidase showed a significantly reduced [O(2)](-) formation after stimulation with OxLDL and LPC. OxLDL and its compound LPC induce proliferation of HUVEC through activation of NADPH oxidase. The active NADPH oxidase generates [O(2)](-), which mediates the proliferative effects.

Catalase↗
PubMed1993

Calcium antagonists and Bay K8644 promote depolarization of the rat heart mitochondrial membrane potential. Further evidence for a role in alteration of oxidative metabolism.

Studies were carried out using a tetraphenylphosphonium (TPP+)-selective electrode to monitor the effect of selected calcium (Ca2+) antagonists and the dihydropyridine Ca2+ agonist Bay K8644 on membrane potential (psi) associated with isolated rat heart mitochondria. Verapamil and diltiazem (10-500 microM), standard Ca2+ antagonists, produced a depolarization of both liver and heart mitochondria at concentrations > 150 microM. In contrast, nitrendipine (10-200 microM), a dihydropyridine compound, produced a concentration-related inhibition of psi in mitochondria from both sources, effects which were statistically significant at concentrations > 50 microM. Cinnarizine (10-100 microM) and bepridil (10-100 microM) also produced inhibition of heart psi, these effects being particularly noted in the presence of bepridil, where depolarization of the membrane was statistically significant with only 10 microM drug. The results indicate the complexity of action of these drugs at the mitochondrial level. In general, drug actions on psi appear to be correlated with previously reported effects on Ca2+ transportation rather than oxidative phosphorylation associated with rat heart mitochondria. The findings also illustrate that the mitochondrial actions of cardiovascular compounds may be of relevance in situ, particularly during ischaemia/reperfusion when mitochondria become loaded with Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1997

CD18/ICAM-1-dependent oxidative NF-kappaB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells.

Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in reponse to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-kappaB activation occurs in Kupffer cells and activated NF-kappaB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-kappaB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-kappaB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-kappaB activation, and NO production. Therefore, this study suggests that CD18/ICAM-1-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-kappaB, which may lead to the increased production of NO in Kupffer cells.

Animals↗
PubMed2006

Microtubule disassembly and inhibition of mitosis by a novel synthetic pharmacophore.

Microtubule drugs, which block cell cycle progression through mitosis, have seen widespread use in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. A new microtubule pharmacophore, diphenyleneiodonium, causing disassembly of the microtubule cytoskeleton is described here. Although this synthetic compound does not affect the assembly state of purified microtubules, it profoundly suppresses microtubule assembly in vivo, causes paclitaxel-stabilized microtubules to cluster around the centrosomes, and selectively disassembles dynamic microtubules. Similar to other microtubule drugs, this new pharmacophore blocks mitotic spindle assembly and mitotic cell division.

Animals↗
PubMed1987

Methotrexate efflux in L1210 cells. Kinetic and specificity properties of the efflux system sensitive to bromosulfophthalein and its possible identity with a system which mediates the efflux of 3',5'-cyclic AMP.

Methotrexate exits L1210 mouse leukemia cells via multiple routes that include a unidirectional efflux component which is sensitive to bromosulfophthalein. This efflux component has been characterized in the present study after eliminating the contribution from the other efflux routes by treatment of the cells with an active ester of methotrexate and by reducing the assay pH to 6.2. The remaining efflux at pH 6.2 was greater than 90% sensitive to bromosulfophthalein. This route was also inhibited by probenecid, prostaglandin A1, diamide, 1-methyl-3-isobutylxanthine, various metabolic inhibitors, and by transfer of the cells to a buffer containing high concentrations of KCl. The inhibition by prostaglandin A1 was exceptionally potent and reached 50% at a concentration of 0.5 microM. An enhancement in efflux occurred upon the addition of glucose or by transfer of the cells to a non-saline buffer. When parameters relating to cellular energetics were measured, a reduction in ATP level was associated with the inhibition of efflux by probenecid, carbonylcyanide m-chlorophenylhydrazone, valinomycin, and antimycin A, whereas the increase in efflux by glucose was accompanied by an increase in intracellular ATP. Changes in ATP, however, were not associated with the inhibition by various other compounds or additions or with the enhancement in efflux by the non-anionic buffer. When the relative sensitivity of methotrexate efflux to bromosulfophthalein, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and lactic anhydride was compared with other anion transport systems, differences in specificity indicated that methotrexate was not exiting the cells via the bicarbonate/chloride exchange carrier, the lactate/H+ co-transport system, or a system which mediates the efflux of phthalate. However, a correlation was apparent between the sensitivity of methotrexate efflux to inhibition by prostaglandin A1, probenecid, and certain metabolic inhibitors and the ability of these same compounds to inhibit the unidirectional efflux of 3',5'-cyclic AMP in other cell lines, suggesting that methotrexate may share a common efflux route with cyclic nucleotides.

Adenosine Triphosphate↗