Nitric oxide modulation of interleukin-1[beta]-evoked intracellular Ca2+ release in human astrocytoma U-373 MG cells and brain striatal slices.
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Intracellular Ca(2+) mobilization and release into mammal CSF plays a fundamental role in the etiogenesis of fever induced by the proinflammatory cytokine interleukin-1beta (IL-1beta) and other pyrogens. The source and mechanism of IL-1beta-induced intracellular Ca(2+) mobilization was investigated using two experimental models. IL-1beta (10 ng/ml) treatment of rat striatal slices preloaded with (45)Ca(2+) elicited a delayed (30 min) and sustained increase (125-150%) in spontaneous (45)Ca(2+) release that was potentiated by l-arginine (300 microm) and counteracted by N-omega-nitro-l-arginine methyl ester (l-NAME) (1 and 3 mm). The nitric oxide (NO) donors diethylamine/NO complex (sodium salt) (0.3 and 1 mm) and spermine/NO (0.1 and 0.3 mm) mimicked the effect of IL-1beta on Ca(2+) release. IL-1beta stimulated tissue cGMP concentration, and dibutyryl cGMP enhanced Ca(2+) release. The guanyl cyclase inhibitors 1H-[1,2, 4]oxadiazole[4,3-a] quinoxalin-1-one (100 microm) and 6-[phenylamino]-5,8 quinolinedione (50 microm) counteracted Ca(2+) release induced by 2.5 but not 10 ng/ml IL-1beta. Ruthenium red (50 microm) and, to a lesser extent, heparin (3 mg/ml) antagonized IL-1beta-induced Ca(2+) release, and both compounds administered together completely abolished this response. Similar results were obtained in human astrocytoma cells in which IL-1beta elicited a delayed (30 min) increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (402 +/- 71.2% of baseline), which was abolished by 1 mm l-NAME. These data indicate that the NO/cGMP-signaling pathway is part of the intracellular mechanism transducing IL-1beta-evoked Ca(2+) mobilization in glial and striatal cells and that the ryanodine and the inositol-(1,4,5)-trisphosphate-sensitive Ca(2+) stores are involved. (+info)
Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes.
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OBJECTIVE: We have recently shown that tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response. METHODS: CMs were isolated from 10-day-old chick embryos and grown to confluence. After 20 h of serum starvation the cells were treated with TNFalpha and LPS, and/or specific agonists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities and cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of [3H]-thymidine. RESULTS: Treatment of CMs with TNFalpha and LPS increased cell number and [3H]-thymidine incorporation. Addition of TNFalpha and LPS provoked an induction of ODC, with consequent polyamine accumulation, and a more delayed enhancement of NOS activity, which appeared to be independent of the activation of the ODC-polyamine system. TNFalpha and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments with specific inhibitors of ODC and NOS, as well as with inhibitors of sGC and cGMP-dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP-dependent pathways are required for the mitogenic action of TNFalpha and LPS. Moreover, addition of exogenous polyamines to untreated cells raised the cGMP level in a NO-dependent fashion, and enhanced [3H]-thymidine incorporation. The latter effect was inhibited by sGC or PKG inhibitors. Treatment of quiescent cells with NO donors, 8-bromo-cGMP or YC-1, an sGC activator, also promoted DNA synthesis. Furthermore, putrescine and NO donor can additively activate sGC in cell-free extracts. CONCLUSION: TNFalpha and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP. (+info)
NO Inhibits NaCl absorption by rat thick ascending limb through activation of cGMP-stimulated phosphodiesterase.
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In the isolated, perfused rat thick ascending limb (THAL), L-arginine (L-Arg) stimulates endogenous nitric oxide (NO) production, which inhibits NaCl absorption. However, the intracellular cascade responsible for the effects of NO has not been studied. We hypothesized that endogenous NO inhibits THAL NaCl transport by increasing cGMP, which activates protein kinase G (PKG) and cGMP-stimulated phosphodiesterase (PDE II), which, in turn, decreases cAMP levels. THALs from rats were isolated and perfused, and net chloride flux (J(Cl-)) was measured. L-Arg was used to stimulate NO production. Adding L-Arg (0.5 mmol/L) to the bath decreased J(Cl-) from 154.4+/-9.9 to 101.9+/-14.1 pmol. mm(-1). min(-1), a 35.2% decrease (n=6; P<0.05). In the presence of the soluble guanylate cyclase inhibitor LY-83583 (10 micromol/L), adding L-Arg to the bath did not affect THAL J(Cl-) (143.7+/-28.1 versus 136.7+/-22.2 pmol. mm(-1). min(-1); n=6). LY-83583 alone had no effect on J(Cl-). In the presence of the PDE II inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) 50 micromol/L, L-Arg reduced J(Cl-) by only 13% (142.1+/-8.9 versus 122.7+/-11.5 pmol. mm(-1). min(-1); P<0.05; n=6). EHNA alone had no effect on THAL J(Cl-). In the presence of 10(-5) mol/L dibutyryl (db)-cAMP, L-Arg did not significantly reduce J(Cl-) (116.3+/-18.2 versus 102.6+/-15.6 pmol. mm(-1). min(-1); n=6). db-cAMP (10(-5) mol/L) had no effect on THAL J(Cl-). In the presence of the PKG inhibitor KT-5823 (2 micromol/L), L-Arg lowered J(Cl-) from 142.6+/-14.1 to 85.9+/-8.3 pmol. mm(-1). min(-1), a decrease of 35.6% (n=8; P<0.05). We conclude that (1) endogenous NO inhibits THAL J(Cl-) by stimulating soluble guanylate cyclase and increasing cGMP; (2) NO inhibits THAL J(Cl-) by stimulation of PDE II, which, in turn, decreases cAMP levels; and (3) PKG does not mediate NO-induced inhibition of THAL J(Cl-). (+info)
Identification of genes induced by a macrophage activator, S-28463, using gene expression array analysis.
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S-28463 and imiquimod are imidazoquinoline compounds which stimulate microbicidal activity by inducing a local immune response at the site of application. Imiquimod-containing cream is an effective clinical treatment against cervical warts caused by human papillomavirus infection. Imiquimod also induces leishmanicidal activity both in vitro in macrophages and in vivo in a mouse model for cutaneous leishmaniasis. The major target cells of S-28463 and imiquimod are macrophages. To explore the molecular basis in which imidazoquinolines generate macrophage microbicidal activity, a cDNA gene array analysis was undertaken to identify genes induced by S-28463. Out of 588 genes screened in this assay, only 13 genes were significantly induced by S-28463. Remarkably, virtually all of the induced genes are involved in macrophage activation and inflammatory response. This experimental approach defines the mechanism of action of this clinically relevant compound in the induction of microbicidal activity in macrophages and also potentially identifies novel genes associated with microbicidal activity in this cell type. (+info)
Phase I clinical and pharmacogenetic study of weekly TAS-103 in patients with advanced cancer.
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PURPOSE: TAS-103 is an inhibitor of both topoisomerase I and II enzymes with broad antitumor activity. It is metabolized to TAS-103-glucuronide (TAS-103-G) predominantly by uridine diphosphate glucuronosyltransferase isoform 1A1 (UGT1A1). We conducted a phase I study to determine the maximum-tolerated dose (MTD) and dose-limiting toxicity (DLT) of TAS-103 when administered on a weekly schedule to patients with advanced cancer. In addition, we evaluated the influence of UGT1A1 genotype on the pharmacokinetics and toxicity of TAS-103. PATIENTS AND METHODS: Thirty-two patients were treated with escalating doses (50 to 200 mg/m(2)) of TAS-103, administered intravenously over 1 hour each week for 3 weeks. Pharmacokinetic analysis was performed at the 130-, 160-, and 200-mg/m(2) dose levels. UGT1A1 genotypes were determined using reverse-transcription polymerase chain reaction techniques. RESULTS: DLT (grade 3 neutropenia) was observed in 5 of 12 patients at 160 mg/m(2) and in 3 of 6 patients at 200 mg/m(2). At 160 mg/m(2), there was a significant correlation between areas under the curve (AUCs) for TAS-103 and TAS-103-G (r = 0.76, P <.05) and an apparent relationship between TAS-103 AUC and D 15 absolute neutrophil count (r = -0.63, P <.05, n = 11, one outlier excluded). UGT1A1 genotype did not influence clearance of TAS-103. CONCLUSION: We recommend a dose of 130 to 160 mg/m(2), or 250 to 300 mg administered using the above weekly schedule for phase II studies. Further studies to characterize the pharmacodynamics and pharmacogenetics of TAS-103 are warranted. (+info)
Inhibition of intramacrophage growth of Penicillium marneffei by 4-aminoquinolines.
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The antimicrobial activities of chloroquine (CQ) and several 4-aminoquinoline drugs were tested against Penicillium marneffei, an opportunistic fungus that invades and grows inside macrophages and causes disseminated infection in AIDS patients. Human THP1 and mouse J774 macrophages were infected in vitro with P. marneffei conidia and treated with different doses of drugs for 24 to 48 h followed by cell lysis and the counting of P. marneffei CFU. CQ and amodiaquine exerted a dose-dependent inhibition of fungal growth, whereas quinine and artemisinin were fungistatic and not fungicidal. The antifungal activity of CQ was not due to an impairment of fungal iron acquisition in that it was not reversed by the addition of iron nitrilotriacetate, FeCl3, or iron ammonium citrate. Perl's staining indicated that CQ did not alter the ability of J774 cells to acquire iron from the medium. Most likely, CQ's antifungal activity is due to an increase in the intravacuolar pH and a disruption of pH-dependent metabolic processes. Indeed, we demonstrate that (i) bafilomycin A1 and ammonium chloride, two agents known to alkalinize intracellular vesicles by different mechanisms, were inhibitory as well and (ii) a newly synthesized 4-amino-7-chloroquinoline molecule (compound 9), lacking the terminal amino side chain of CQ that assists in drug accumulation, did not inhibit P. marneffei growth. These results suggest that CQ has a potential for use in prophylaxis of P. marneffei infections in human immunodeficiency virus-infected patients in countries where P. marneffei is endemic. (+info)
In vitro activities of DU-1102, a new trioxaquine derivative, against Plasmodium falciparum isolates.
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The antimalarial trioxaquine derivative DU-1102, synthesized by covalent linkage between aminoquinoline and trioxane moieties, was highly active against Cameroonian isolates (mean 50% inhibitory concentration of 43 nmol/liter) of Plasmodium falciparum. There was no correlation between the responses to DU-1102 and chloroquine and only a low correlation between the responses to DU-1102 and pyrimethamine, suggesting an independent mode of action of the trioxaquine against the parasites. (+info)
Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa.
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Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+ -free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required. (+info)