RESUMO
BACKGROUND AND PURPOSE: Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease. EXPERIMENTAL APPROACH: Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg(-1) propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1ß, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro. RESULTS: Propranolol at 0.1 and 5 mg·kg(-1) reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg(-1) reduced IL-1ß, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9. CONCLUSIONS AND IMPLICATIONS: Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Propranolol/administração & dosagem , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/genética , Perda do Osso Alveolar/prevenção & controle , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Catepsina K/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno Tipo I/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Gengiva/metabolismo , Hemodinâmica/efeitos dos fármacos , Inflamação/prevenção & controle , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Fatores de Transcrição NFATC/antagonistas & inibidores , Osteoclastos/patologia , Peptídeos/metabolismo , Ligante RANK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fosfatase Ácida Resistente a Tartarato , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The etiological agent of Chagas disease, Trypanosoma cruzi, is consisted of two phylogenetic lineages. Using live epimastigotes, in this study we have characterized ecto-phosphatase activities of two strains of T. cruzi, one (Y strain) is a member of group T. cruzi I and the other (Colombiana) is a member of group T. cruzi II. About one-third of the total ecto-phosphatase activity from the Y strain was Mg(2+)-dependent, but no such activity was observed with Colombiana. The level of Mg(2+)-independent activity was dramatically different in the two strains, with Colombiana showing more than 15-fold higher activity. Experiments using classical inhibitors of acid phosphatases, as well as inhibitors of phosphotyrosine phosphatase, showed a decrease in these phosphatase activities, with different patterns of inhibition. The Mg(2+)-independent activities of the Colombiana and Y strains decreased inversely with pH, varying from 6.5 to 8.0. On the other hand, the Mg(2+)-dependent activity of the Y strain increased concomitantly with the increase in pH in the same range.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Trypanosoma cruzi/classificação , Trypanosoma cruzi/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/metabolismo , Animais , Cátions Bivalentes/farmacologia , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Especificidade da Espécie , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The FMVI strain of Trichomonas vaginalis was freshly isolated from an asymptomatic patient, and its morphological properties and virulence in vitro compared with the well-established JT strain. The morphological variability of the parasites was assessed by differential interference microscopy and both scanning and transmission electron microscopy. The FMV1 strain presented nearly 20% amoeboid cells whereas the JT strain presented high percentages of ellipsoid but no amoeboid cells. The FMV1 morphotype population was unaltered after at least 1 year of subculturing. Electron microscopy revealed that this strain produced numerous pseudopod structures which mediated intimate contact and interdigitation among trophozoites. Dead FMV1 parasites were often phagocytosed by conspecific cells. We also compared the cytolytic capacity of these two populations against epithelial MDCK cells and its contact dependence. The FMV1 strain rapidly adhered to plastic or glass surfaces and to MDCK monolayers. This strain destroyed about 93% of the epithelial cells in 90 min whereas the cytolytic activity of the JT parasites was very much lower (about 41%). Parasite supernatants displayed no cytolytic activity, indicating contact-mediated lysis. The protozoan virulence in vitro did not correlate well with the clinical observations. The implications of these results are discussed.
Assuntos
Células Epiteliais/patologia , Células Epiteliais/parasitologia , Trichomonas vaginalis/citologia , Trichomonas vaginalis/patogenicidade , Animais , Adesão Celular , Morte Celular , Linhagem Celular , Cães , Feminino , Humanos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Microscopia de Interferência , Pessoa de Meia-Idade , Fagocitose , Pseudópodes/ultraestrutura , Trichomonas vaginalis/crescimento & desenvolvimento , Trichomonas vaginalis/ultraestrutura , VirulênciaRESUMO
In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.
Assuntos
Apirase/isolamento & purificação , Apirase/metabolismo , Trypanosomatina/enzimologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Antígenos CD , Cátions Bivalentes/farmacologia , Ativadores de Enzimas/análise , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Especificidade por Substrato , Suramina/farmacologia , Tripanossomicidas/farmacologiaRESUMO
In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant of living H. m. muscarum.
Assuntos
Fosfatase Ácida/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Moscas Domésticas/parasitologia , Fator de Ativação de Plaquetas/farmacologia , Trypanosomatina/enzimologia , Animais , Meios de Cultura , Trypanosomatina/crescimento & desenvolvimentoRESUMO
The effects of platelet-activating factor (PAF) on the infection of peritoneal mouse macrophages by Leishmania amazonensis were investigated. Prior to the infection, the parasites and/or the macrophages were treated with PAF and/or one of the following modulators: WEB 2086 (PAF antagonist), and the modulators of protein kinase C, phorbol-12-myristate-13-acetate (PMA), and sphingosine. The infection was inhibited when the macrophages or both the parasites and the macrophages were treated with PAF, but stimulated by PAF-treated parasites. WEB 2086 abrogated PAF effects in both systems. The infection was stimulated when the macrophages were treated with sphingosine plus PAF, but inhibited when the macrophages were treated with sphingosine and the parasites with sphingosine plus PAF. The infection was inhibited by sphingosine-treated parasites, either in the presence or in the absence of PAF. Leishmania amazonensis-macrophage infection was inhibited by PMA in all systems tested.
Assuntos
Leishmania/efeitos dos fármacos , Leishmania/patogenicidade , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Fator de Ativação de Plaquetas/farmacologia , Animais , Azepinas/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fator de Ativação de Plaquetas/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Proteína Quinase C/metabolismo , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Triazóis/farmacologiaRESUMO
Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present pro- and opisthomastigotes forms in their life cycles. In this study, we investigated the Ca(2+) transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca(2+), and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca(2+) transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca(2+) release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca(2+) uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca(2+) transport system, attributed to the endoplasmic reticulum, was also detected. Nigericin (1 microM), FCCP (1 microM), or bafilomycin A(1) (5 microM) had no effect on the vanadate-sensitive Ca(2+) transport. These data suggest the absence of a Ca(2+) transport mediated by a Ca(2+)/H(+) antiport. No evidence of a third Ca(2+) compartment with the characteristics of the acidocalcisomes described by A. E. Vercesi et al. (1994, Biochem. J. 304, 227-233) was observed. Thapsigargin and IP(3) were not able to affect the vanadate-sensitive Ca(2+) transport. Ruthenium red was able to inhibit the Ca(2+) uniport of mitochondria, inducing a slow mitochondrial Ca(2+) efflux, compatible with the presence of a Ca(2+)/H(+) antiport. Moreover, this efflux was not stimulated by the addition of NaCl, which suggests the absence of a Ca(2+)/Na(+) antiport in mitochondria.
Assuntos
Cálcio/química , Cálcio/metabolismo , Macrolídeos , Trypanosomatina/química , Difosfato de Adenosina/metabolismo , Animais , Antibacterianos/farmacologia , Antimicina A/farmacologia , Transporte Biológico/efeitos dos fármacos , Calcimicina/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Digitonina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Guanosina Trifosfato/metabolismo , Indicadores e Reagentes/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Membranas Intracelulares/metabolismo , Ionóforos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Oligomicinas/farmacologia , Rutênio Vermelho/farmacologia , Trocador de Sódio e Cálcio/fisiologia , Tapsigargina/farmacologia , Fatores de Tempo , Trypanosomatina/fisiologia , Desacopladores/farmacologia , Vanadatos/farmacologiaRESUMO
The effects of platelet-activating factor (PAF) on the ecto-phosphatase activity of Trypanosoma cruzi were investigated. Living parasites hydrolyzed p-nitrophenyl phosphate (p-NPP) at a rate of 5.71 +/- 0.37 nmol P(i) mg(-1) min(-1). This ecto-phosphatase activity increased to 8.70 +/- 1.12 nmol P(i) mg(-1) min(-1) when the cells were grown in the presence of 10(-9) M PAF. This effect was probably due to stimulation of the release of the ecto-phosphatase and/or the secretion of an intracellular phosphatase to the extracellular medium, as suggested by cytochemical analysis. Modulation of the ecto-phosphatase activity was also observed when PAF was added during the time course of the reaction. WEB 2086, a competitive PAF antagonist, was able to revert PAF effects when both were used at the same concentration. When PAF was added to a membrane enriched fraction preparation of T. cruzi, no alteration on the phosphatase activity was observed. This result suggests an involvement of intracellular signaling, as PAF was only effective on intact cells. Sphingosine and phorbol-12-myristate-13-acetate (PMA) were then used to investigate a possible involvement of protein kinase C (PKC) with PAF-induced phosphatase secretion. Sphingosine by itself stimulated the secretion of a phosphatase but did not significantly interfere with PAF effects on this enzyme. On the other hand, PMA was able to abrogate PAF-induced release of this phosphatase. These data are highly suggestive of a putative involvement of signal transduction mediated by a ligand of mammalian origin (PAF), through PKC and a specific receptor located on the cell surface of the human parasite Trypanosoma cruzi.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Trypanosoma cruzi/efeitos dos fármacos , Animais , Azepinas/farmacologia , Meios de Cultivo Condicionados/química , Fosfoproteínas Fosfatases/análise , Fator de Ativação de Plaquetas/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Triazóis/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
In the present work ecto-phosphatase activity in Herpetomonas muscarum muscarum has been characterized using live parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 4.27 nmol Pi/mg of protein.min. A pH curve was generated, in which these intact flagellates showed the highest phosphatase activity at pH 6.5. Classical inhibitors for acid phosphatase, such as sodium orthovanadate, sodium tartrate, and ammonium molybdate, were used in the experiments and showed different patterns of inhibition. Lithium fluoride, aluminum chloride, and fluoroaluminate complexes were also tested. Although lithium fluoride and fluoroaluminate complexes were capable of inhibiting the phosphatase activity, aluminum chloride stimulated this enzyme. Cytochemical analysis showed the localization of this enzyme on the parasite surface. This ecto-phosphatase activity was also significantly diminished when the parasites were treated with 10(-6) M platelet-activating factor (PAF), a potent phospholipid mediator that promoted cellular differentiation in this parasite.
Assuntos
4-Nitrofenilfosfatase/antagonistas & inibidores , 4-Nitrofenilfosfatase/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Trypanosomatina/enzimologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hidrólise , Cinética , Trypanosomatina/citologia , Trypanosomatina/efeitos dos fármacosRESUMO
The effects of platelet-activating factor (PAF), at doses ranging from 10(-6) M to 10(-10) M, on cell growth and on cell differentiation of Herpetomonas muscarum muscarum were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF did not interfere with the protozoan growth. However, parasites grown in the presence of PAF (10(-6) M) were significantly more differentiated than those grown in the absence of PAF, since the first day of culture. On the first two days of culture, PAF doses ranging from 10(-10) M to 10(-7) M, did not significantly interfere with the differentiation of these parasites, although after the third day of culture, all PAF doses used significantly increased the protozoan differentiation. Specific PAF receptor antagonists totally abrogated (WEB 2086 and WEB 2170) or significantly decreased (BN 52021) PAF effect on cell differentiation. These findings indicate PAF triggers the process of cell differentiation in Herpetomonas muscarum muscarum and suggest these parasites have receptors for PAF.
Assuntos
Fator de Ativação de Plaquetas/farmacologia , Trypanosomatina/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Trypanosomatina/citologiaRESUMO
ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).
Assuntos
Adenosina Trifosfatases/metabolismo , Leishmania tropica/enzimologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , 4-Nitrofenilfosfatase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Leishmania tropica/metabolismo , Magnésio/farmacologia , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Especificidade por SubstratoRESUMO
The effects of platelet-activating factor (PAF), at (10)-6M and (10)-9M, on cell growth and cell differentiation of Trypanosoma cruzi were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF slightly interfered with the protozoan growth. However, parasites growth in the presence of PAF were significantly more differentiated than those grown in the absence of PAF, beginning on the fourth day of culture. A specific PAF receptor antagonist (WEB 2086) totally abrogated PAF effect on cell differentiation. These findings indicate that PAF triggers the process of cell differentiation in T. cruzi and suggest that these parasites have receptors for PAF.
Assuntos
Fator de Ativação de Plaquetas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Azepinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Cinética , Microscopia Eletrônica , Inibidores da Agregação Plaquetária/farmacologia , Triazóis/farmacologia , Trypanosoma cruzi/ultraestruturaRESUMO
The effects of propranolol (10(-3) mM) on the surface anionic groups of Herpetomonas muscarum muscarum were analysed by cell electrophoresis, by ultrastructural cytochemistry and by identification of sialic acids using paper chromatography. Differentiation of H. muscarum muscarum induced by propranolol treatment caused a significant increase in the net negative surface charge. Binding of cationized ferritin (CF) and colloidal iron hydroxide particles was observed at the cell surface of both untreated and propranolol-treated cells. In cells incubated in the presence of the drug the CF particles were distributed in all membrane regions. However, there were small areas where the particles were absent. In H. muscarum muscarum exposed to propranolol the density of residues of sialic acid per cell was higher, and the agglutinating activity with Sendai virus was more intense. However, the pattern of sialic acid, characterized by the presence of N-acetylneuraminic acid derivative, was not modified upon cell interaction with the drug. Treatment of both control and propranolol-treated protozoa with neuraminidase significantly reduced the surface charge. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. muscarum muscarum.