Leishmaniasis is an infectious disease caused by protozoan parasites of the genusLeishmania. There is no vaccine against human leishmaniasis and the treatment of the disease would benefit from a broader spectrum and a higher efficacy of leishmanicidal compounds. We analyzed the leishmanicidal activity and the mechanism of action of the calcium ionophore, calcimycin.L. majorpromastigotes were coincubated with calcimycin and the viability of the cells was assessed using resazurin assay. Calcimycin displayed dose-dependent effect with IC50= 0.16 μM. Analysis of propidium iodide/LDS-751 stained promastigotes revealed that lower concentrations of calcimycin had cytostatic effect and higher concentrations had cytotoxic effect. To establish the mechanism of action of calcimycin, which is known to stimulate activity of mammalian constitutive nitric oxide synthase (NOS), we coincubatedL. majorpromastigotes with calcimycin and selective NOS inhibitors ARL-17477 or L-NNA. Addition of these inhibitors substantially decreased the toxicity of calcimycin toLeishmaniapromastigotes. In doing so, we demonstrated for the first time that calcimycin has a direct leishmanicidal effect onL. majorpromastigotes. Also, we showed thatLeishmaniaconstitutive Ca2+/calmodulin-dependent nitric oxide synthase is involved in the parasite cell death. These data suggest activation ofLeishmanianitric oxide synthase as a new therapeutic approach.
OBJECTIVES: Elevated plasma uric acid indicates an increased risk of cardiovascular diseases associated with endothelial dysfunction. However, the role of uric acid in the pathogenesis of endothelial dysfunction is still a matter of debate. It is not clear whether uric acid is a real causative risk factor, an inert marker, or even a protective molecule with respect to its antioxidant properties. We have studied the effect of uric acid on intact endothelial cells as well as cells with homocysteine-induced endothelial dysfunction. DESIGN: Bovine aortic endothelial cells were treated with uric acid (100 - 600 muM) and homocysteine (100 muM) or with uric acid only. After 24 hours, the cells were stimulated with 1 mug/ml of calcium ionophore A23187, and nitric oxide (NO) production was measured electrochemically with the use of a NO-sensitive microelectrode. The expression of endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation at Ser1179 was estimated with the use of Western blotting. Interaction between NO and uric acid was measured with a NO electrode. Superoxide generation was measured with the use of the fluorescence dye MitoSox Red. RESULTS: Homocysteine strongly diminished A23187-induced NO release. 100 muM uric acid slightly restored NO production; higher concentrations were ineffective. Interestingly, a dose-dependent decrease of NO release was observed in the cells treated only with uric acid. Uric acid did not scavenge NO and did not change eNOS protein expression or phosphorylation at Ser1179, but dose-dependently increased superoxide production in A23187-stimulated cells. CONCLUSION: In conclusion, uric acid decreased NO bioavailability and enhanced superoxide generation in A23187-stimulated bovine aortic endothelial cells.
- MeSH
- aorta MeSH
- calcimycin farmakologie MeSH
- časové faktory MeSH
- endoteliální buňky účinky léků fyziologie MeSH
- fosforylace MeSH
- homocystein metabolismus farmakologie MeSH
- ionofory farmakologie MeSH
- kinetika MeSH
- kultivované buňky MeSH
- kyselina močová metabolismus farmakologie MeSH
- mikroelektrody MeSH
- oxid dusnatý metabolismus MeSH
- skot MeSH
- superoxidy metabolismus MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: An increased glucose utilization by aldose reductase (ALR-2) has been implicated in the pathogenesis of diabetic vascular complications. In this process, several mechanisms are involved, including the depletion of cofactors required for the action of antioxidant enzymes or endothelial NO synthase. In this study, the effect of a novel ALR-2 inhibitor JMC-2004 on hyperglycemia-induced endothelial dysfunction was studied. METHODS: Bovine aortic endothelial cells (BAEC) were treated with glucose (30 mM), JMC-2004 (0.01mM), or glucose and JMC-2004 for 24 h. The cells were then stimulated with calcium ionophore A23187 after which NO production was measured electrochemically using a porphyrine-coated carbon NO electrode. Nitrite concentrations were determined in the cell supernatants. The peroxyl and hydroxyl radical-scavenging activity of JMC-2004 was measured with luminol-enhanced chemiluminescence. The expression of eNOS was determined by Western blotting. JMC-2004 IC50 for ALR-2 was determined colorimetrically with D-glyceraldehyde as a substrate. RESULTS: Incubating the cells with 30 mM glucose strongly diminished A23187- induced NO production. Treatment with JMC-2004 restored NO production by 40% without affecting eNOS expression. This effect was probably antioxidantindependent, since JMC-2004 did not have any antioxidant capacity. JMC-2004 exerted high selectivity towards ALR-2. CONCLUSIONS: ALR-2 inhibition with JMC-2004 was able to abolish hyperglycemia- induced endothelial dysfunction in bovine aortic endothelial cells.
- MeSH
- aldehydreduktasa antagonisté a inhibitory MeSH
- buněčné linie MeSH
- calcimycin farmakologie MeSH
- cévní endotel patologie účinky léků MeSH
- elektrochemie MeSH
- endoteliální buňky patologie účinky léků MeSH
- fenoly farmakologie MeSH
- glukosa farmakologie MeSH
- glyceraldehyd metabolismus MeSH
- hydroxylový radikál metabolismus MeSH
- hyperglykemie patologie MeSH
- inhibitory enzymů farmakologie MeSH
- luminiscence MeSH
- oxid dusnatý metabolismus MeSH
- peroxidy metabolismus MeSH
- pyrroly farmakologie MeSH
- skot MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca(2+)-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcepsilonRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcepsilonRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.
- MeSH
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- calcimycin farmakologie MeSH
- detergenty farmakologie MeSH
- exocytóza MeSH
- financování organizované MeSH
- fosfatidylseriny chemie MeSH
- fosforylace MeSH
- inhibitory enzymů farmakologie MeSH
- ionofory farmakologie MeSH
- konfokální mikroskopie MeSH
- krysa rodu rattus MeSH
- nádorové buněčné linie MeSH
- proteiny přenášející fosfolipidy metabolismus MeSH
- tyrosin chemie MeSH
- vanadáty farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.
- MeSH
- acetofenony farmakologie MeSH
- benzopyrany farmakologie MeSH
- calcimycin farmakologie MeSH
- indoly farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- ionofory farmakologie MeSH
- izoenzymy antagonisté a inhibitory fyziologie klasifikace MeSH
- karbazoly farmakologie MeSH
- maleimidy farmakologie MeSH
- oocyty fyziologie účinky léků MeSH
- prasata fyziologie MeSH
- proteinkinasa C antagonisté a inhibitory fyziologie klasifikace MeSH
- pyrony farmakologie MeSH
- vápník fyziologie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- kazuistiky MeSH
Induction of penetration gland emptying by cercariae of the bird schistosomes Trichobilharzia szidati and T. regenti employing linoleic acid, linolenic acid, praziquantel and calcium ionophore A23187 showed that both postacetabular and circumacetabular cells released their content at chosen stimulant concentrations. The gland secretions consisted of soluble and insoluble parts. The former one adhering to the ground seemed to have different saccharide composition from the glands of Schistosoma mansoni. It bound labelled saccharides, thus exhibiting lectin-like activity. Protein profiles of the latter one were identical after stimulation by all four stimulants in T. szidati. The soluble secretions contained several proteolytic enzymes; 31 kDa and 33 kDa cysteine proteases were identified in E/S products of T. szidati and T. regenti, respectively. The circumacetabular glands contained a significant amount of calcium. Immunohistochemistry revealed that the origin of E/S products after in vitro stimulation is in both penetration glands and tegumental structures. No crossreactivity was observed between the bird schistosomes and a serum raised against S. mansoni elastase.
- MeSH
- anthelmintika farmakologie MeSH
- calcimycin farmakologie MeSH
- exokrinní žlázy sekrece účinky léků MeSH
- finanční podpora výzkumu jako téma MeSH
- interakce hostitele a parazita genetika MeSH
- kyseliny linolové farmakologie MeSH
- myši inbrední BALB C MeSH
- praziquantel farmakologie MeSH
- proteiny červů chemie metabolismus MeSH
- Schistosomatidae metabolismus účinky léků MeSH
- techniky in vitro MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- MeSH
- calcimycin farmakologie MeSH
- finanční podpora výzkumu jako téma MeSH
- hladké svalstvo metabolismus účinky léků MeSH
- hypoxie metabolismus MeSH
- ionofory farmakologie MeSH
- králíci MeSH
- ucho MeSH
- vazodilatancia MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- MeSH
- azid sodný farmakologie MeSH
- calcimycin farmakologie MeSH
- dospělí MeSH
- katalasa farmakologie MeSH
- lidé MeSH
- N-formylmethionin-leucyl-fenylalanin farmakologie MeSH
- neutrofily fyziologie MeSH
- superoxiddismutasa farmakologie MeSH
- trombocyty fyziologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
