Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.

Implication of Egr-1 in trifluoperazine-induced growth inhibition in human U87MG glioma cells

The early growth response gene-1 (Egr-1) is a tumor suppressor which plays an important role in cell growth, differentiation and apoptosis. Egr-1 has been shown to be down-regulated in many types of tumor tissues. Trifluoperazine (TFP), a phenothiazine class of antipsychotics, restored serum-induced Egr-1 expression in several cancer cell lines. We investigated the effect of Egr-1 expression on the TFP-induced inhibition of cell growth. Ectopic expression of Egr-1 enhanced the TFP-induced antiproliferative activity and downregulated cyclin D1 level in U87MG glioma cells. Our results suggest that antipsychotics TFP exhibits antiproliferative activity through up-regulation of Egr-1.

Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.

Bioluminescence assay of adenosine triphosphate in drug susceptibility testing of Mycobacterium tuberculosis.

Twenty three clinical isolates M. tuberculosis and the reference strain, M. tuberculosis H37Rv were tested for their susceptibility to trifluoperazine (TFP) by the standard broth dilution method and the bioluminescence assay. The results showed that in 15 of the 23 isolates, the minimal inhibitory concentration (MIC) was identical in both the methods and in the remaining 8 isolates the difference in the MIC values between the methods, was less than two fold and was not significant. The findings suggest that the measurement of adenosine triphosphate (ATP) by bioluminescence assay can be employed as an alternative method for the rapid screening of clinical isolates for their susceptibility to anti-mycobacterial agents.

Evaluation of the hepatoprotective effect of some drugs in paracetamol-induced liver cell toxicity in the rat

This study aimed to clarify the hepatocellular injury induced by paracetamol and the possible hepatoprotective effect of fructose, cocktail of fructose, cyclosporin A and trifluoperazine, Nigella sativa oil, vitamin C and vitamin E

Terapéutica farmacológica del trastorno delirante / Pharmacologic treatment of delusional disorder

La literatura disponible acerca de la terapéutica del trastorno delirante sugiere que esta entidad no responde bien a los tratamientos neurolépticos habituales. Varios trabajos informan de casos que responden a pimozida, luego de no haber respondido a otros antipsicóticos. No obstante, se carece de estudios controlados. Por este motivo no es posible llegar a conclusiones definitivas al respecto

Trifluoperazine reduces the expression of CD69 in phytohemagglutinin-activated lymphocytes

Trifluoperazine (TFP) is a phenothiazine capable of inhibiting lymphocyte proliferation as well as natural killer cells (NK) and lymphokine-activated killer cells (LAK) cytotoxic activity. CD69 is a surface molecule induced by various mechanisms of cellular activation. In the present work the modulation of CD69 expression by TFP was investigated on PHA-stimulated peripheral blood mononuclear cells and compared to that of CD25 (IL-2 receptor) expression. Determination of surface molecules was performed in an indirect immunofluorescence assay using anti-CD69 or anti-CD25 monoclonal antibodies, and analyzed by flow cytometry. The time course of the expression of these two molecules differed: CD69 expression was already declining at 48 h, whereas CD25 was still increasing at 72 h after stimulation. TFP (10 muM) reduced CD69 expression by 71.8 per cent at 24 h, 68.4 per cent at 48 h and 24 per cent at 72 h following activation. In contrast, the same dose of TFP did not significantly affect CD25 expression at 24 h but showed an inhibitory effect at later times. These results suggest that different activation pathways are involved in the expression of CD25 and CD69.

Multidrug resistance in leukemias: the problem and some approaches to its circumvention

The phenomenon of multidrug resistance (MDR), representing cross-resistance among a number of unrelated chemotherapeutic drugs, is the major cause of chemotherapy failure in many tumors. It has been also detected in leukemias and in these cancers, as well as in many others, resistance can be reversed by a number of substances known as modulators or reversing agents. The capacity of identifying tumors resistant to chemotherapy could orientate the treatment employed. In leukemias, tumor cells are easily obtainable and many techniques have been used to evaluate resistance in these cells. Studying 42 leukemia patients we found a correlation of nearly 60 per cent among surface expression of P-glycoprotein, in vitro resistance reversal by cyclosporin A (CS-A) and extrusion of the rhodamine 123 dye. This latter assay has the advantage of measuring a functional aspect related to resistance (intracellular drug accumulation), being reproducible and affordable by most laboratories. The data generated by this assay were in accordance with those reported by other authors using different methods. To allow for an experimental approach in the study of MDR in leukemias, an in vitro model of a vincristine-induced erythroleukemia resistant cell line was established by us, and was shown to display MDR characteristics: resistance to unrelated drugs, surface expression of P-glycoprotein, extrusion of rhodamine 123 and resistance reversal by trifluoperazine, a reversing agent. Furthermore, this vincristine-resistant line was as sensitive to cell mediated lysis by natural killer (NK) cells as the parental line. Models like this one allow for the in vitro testing of new reversing agents, and when combined to in vitro tests for NK and LAK activity, may select for substances capable of modulating resistance without affecting a potentially useful cell mediated immunotherapy.

Effect of cyclosporin A and trifluoperazine on rat liver mitochondria swelling and lipid peroxidation

The effect of cyclosporin A (CsA) or trifluoperazine (TFP) on lipid peroxidation and mitochondrial swelling was determined using liver mitochondria incubated with 30 microM Ca2+ and 250 microM t-butylhydroperoxide or 5 mM inorganic phosphate (P(i)). Lipid peroxidation was not modified by either 1 microM CsA or 40 microM TFP. These compounds presented a distinct effect on mitochondrial permeability. Under oxidative conditions, CsA only showed a transient protective effect whereas TFP completely inhibited mitochondrial swelling. Conversely, CsA was very efficient when Ca2+ and P(i) were used, a condition under which TFP was unable to prevent the swelling. These data are consistent with our previous results (M.F. Nepomuceno, D.V. Macedo and L. Pereira-da-Silva (1991). Brazilian Journal of Medical and Biological Research, 24: 833-836) showing that lipid peroxidation is one among other different components of the permeabilization process. The data suggest that lipid peroxidation is independent of swelling, occurring later than swelling, presumably when the mitochondrial reductant systems are depleted. The differential effects of CsA and TFP suggest that these compounds can be used as specific probes in the elucidation of the two distinct mechanisms responsible for mitochondrial swelling

Calcium homeostasis in Trypanosoma cruzi

By using the fluorescent Ca2+ indicator fura 2, submicromolar levels of intracellular Ca2+ have been detected in Trypanosoma cruzi different stages. The intracellular transport mechanisms involved in maintaining Ca2+ homeostasis in T. cruzi have been characterized by measuring Ca2+ transport in digitonin-permeabilized cells. Two intracellular calcium transport systems have been detected. Ca2+ uptake by the mitochondria occurs by an electrophoretic mechanism, is inhibited by antimycin A, FCCP, and ruthenium red, and stimulated by respiratory substrates, phosphate and acetate. This pool has a high capacity and low affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.6-0.7 microM. Ca2+ uptake by the endoplasmic reticulum is inhibited by high concentrations of vanadate and anticalmodulin agents, and stimulated by ATP. This pool has a low capacity and a high affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.05-1.0 microM. In addition, calmodulin has been purified from T. cruzi epimastigotes and shown to stimulate the homologous plasma membrane Ca(2+)-ATPase and cyclic-AMP phosphodiesterase. The gene encoding this protein has been cloned and sequenced and shown to have a great homology to mammalian calmodulin. The role of the plasma membrane of T. cruzi in the regulation of [Ca2+]i has been studied using fura 2-loaded epimastigotes or plasma membrane vesicles prepared from epimastigotes. Plasma membrane vesicles transport Ca2+ in the presence of Mg2+ and have a high affinity, vanadate-sensitive (Ca(2+)-Mg2+)-ATPase with an apparent Km for free Ca2+ of 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Calmodulin-like activity in mycobacteria.

Calmodulin-like activity has been reported for the first time in mycobacterial species, namely Mycobacterium tuberculosis BCG and M. smegmatis ATCC 14468. The activity was mainly located in the soluble fraction of the mycobacterial cells, Radioimmunoassay revealed maximum levels of calmodulin in young growing cells (early logarithmic phase of growth). Calmodulin-dependent phosphodiesterase activation assay revealed low activity (22%) of partially purified calmodulin either due to insufficient amount of calmodulin to activate phosphodiesterase or due to the presence of some factors interfering with the assay. Calmodulin antagonists, viz. trifluoperazine and phenothiazine, significantly inhibited the 32Pi incorporation into mycobacterial phospholipids. Similar inhibition was observed when EGTA (which removes calcium) was added to the medium. Significant inhibition of 32Pi incorporation in the presence of calmodulin antagonists suggested the involvement of calmodulin in mycobacterial phospholipid metabolism.

Effect of anticalmodulin and antitubulin on LDL-receptor synthesis in aortic smooth muscle cells cultured in vitro.

Effect of trifluoperazine and colchicine on LDL-receptor synthesis in smooth muscle cells exposed to hypercholesterolemic medium in vitro have been studied. While trifluoperazine at 25 microM concentration caused stimulation of LDL-receptor synthesis, colchicine acted as a dose-dependent inhibitor of LDL-receptor synthesis. Thus calmodulin down regulates LDL-receptor synthesis independent of microtubular involvement.

Preventive effect of trifluoperazine on atherosclerosis induced by cholesterol & adrenaline in rabbits.

Studies on the preventive role of trifluoperazine on cholesterol and adrenaline-induced experimental atherosclerosis in rabbits, revealed that trifluoperazine completely prevented the development of atherosclerotic lesions in both the aorta and coronary arteries of animals administered atherogenic diet and adrenaline (im) despite the fact that this drug had no significant effect on the elevated serum lipid profile induced by atherogenic diet. These findings confirm earlier observations of the authors that trifluoperazine has an inherent capacity to prevent atherogenesis.

Effect of verapamil and trifluoperazine upon glucose-induced calcium distribution within B cells

Empleando la preparación de páncreas de rata perfundido, se estudió la secreción de insulina y la distribución de calcio en la célula B (técnica del piroantimoniato de potasio) en respuesta a distintas concentraciones de glucosa (3.3 y 16.6 mM) solas o asociadas con verapamil o trifluorperazina (TFP). El número total de precipitados de piroantimoniato de calcio (CPP) de las células B, al igual que el de CPP unidos a cada una de sus organelas, fue mayor en los páncreas perfundidos con glucosa 16.6 mM en cada uno de los tiempos estudiados. Al comienzo del estímulo con glucosa 16.6 mM, los CPP se ubicaron predominantemente en el halo claro de los gránulos de secreción, mientras que más tarde aparecieron ubicados en la membrana plasmática. El verapamil y la TFP disminuyeron significativamente la segunda fase de la secreción de insulina desencadenada por la glucosa 16.6 mM, modificando además el patrón de distribución del calcio en las células B. Los mayores cambios consistieron en una disminución del número total de CPP en todos los tiempos de perfusión estudiados y una alteración de su distribución en las diferentes organelas de la célula B. En esta última, lo más destacado fue la disminución inicial del porcentaje de CPP unido a los gránulos y la disminución de los unidos a la membrana plasmática y de las mitocondrias al final de la perfusión. Estos resultados sugieren que durante el estímulo con glucosa, la disponibilidad de calcio libre intracelular es controlada en forma secuencial...