RESUMO
Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1(-/-) or B6.RAG1(-/-)C3(-/-) (H-2(b)) mice received B10.BR (H-2(k)) heart allografts and repeated doses of IgG2a, IgG1 or F(ab')(2) fragments of IgG2a DSA (anti-H-2(k)). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti-NK1.1 reduced significantly DSA-induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ-chain knock out) also showed decreased severity of DSA-induced CAV. Direct NK reactivity to the graft was not necessary. F(ab')(2) DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivo by increased expression of phospho-extracellular signal-regulated kinase (pERK), a response not elicited by F(ab')(2) DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d-negative chronic antibody-mediated rejection in humans.
Assuntos
Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Imunidade Celular , Isoanticorpos/imunologia , Células Matadoras Naturais/imunologia , Animais , Doença Crônica , Modelos Animais de Doenças , Citometria de Fluxo , Rejeição de Enxerto/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
Complement fixation, as evidenced by C4d in the microvasculature, is a widely accepted criterion of antibody-mediated rejection. Complement fixation has been shown to be essential in acute antibody-mediated rejection, but its role in chronic rejection has not been addressed. Previous studies showed that passive transfer of complement fixing monoclonal IgG2a anti-H-2Kk into B6.RAG1-/- KO recipients of B10.BR hearts led to progressive chronic transplant arteriopathy (CTA) over 14-28 days, accompanied by C4d deposition. The present studies were designed to test whether complement was required for these lesions. We report that a noncomplement fixing donor-specific alloantibody (DSA, monoclonal IgG1 anti-H-2Kk) injected into B6.RAG1-/- KO recipients of B10.BR hearts also promotes CTA, without C4d deposition. Furthermore, a passive transfer of DSA (monoclonal IgG2a anti-H-2Kk) initiated endarteritis followed by CTA in B6.RAG1-/- mice genetically deficient in the third component of complement (RAG1-/-C3-/-). These studies indicate that antibody to class I MHC antigens can trigger chronic arterial lesions in vivo without complement participation, in contrast to acute antibody-mediated rejection. This pathway may be relevant to C4d-negative chronic rejection sometimes observed in patients with DSA, and argues that lack of C4d deposition does not exclude antibody-mediated chronic rejection.
Assuntos
Anticorpos/química , Artérias/patologia , Complemento C4b/genética , Proteínas do Sistema Complemento/metabolismo , Endarterite/imunologia , Transplante de Coração/métodos , Fragmentos de Peptídeos/genética , Animais , Anticorpos Monoclonais/metabolismo , Progressão da Doença , Coração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de TempoRESUMO
Monoclonal antibody against the CD45RB protein induces stable transplantation tolerance to multiple types of allograft. We have previously established that this tolerance protocol relies on the regulatory function of B lymphocytes for its effect. B lymphocytes have also been reported to participate in immune regulation in several other settings. In most of these systems, the regulatory function of B lymphocytes depends on the production of IL-10. Therefore, we investigated the role of IL-10 in the anti-CD45RB model of B-cell-mediated transplantation tolerance. Surprisingly, using antibody-mediated neutralization of IL-10, IL-10-deficient recipients and adoptive transfer of IL-10-deficient B lymphocytes, we found that IL-10 actually counter-regulates tolerance induced by anti-CD45RB. Furthermore, neutralization of IL-10 reduced the development of chronic allograft vasculopathy compared to anti-CD45RB alone and reduced the production of graft reactive alloantibodies. These data suggest that the participation of regulatory B lymphocytes in transplantation tolerance may be distinct from how they operate in other systems. Identifying the specific B lymphocytes that mediate transplantation tolerance and defining their mechanism of action may yield new insights into the complex cellular network through which antigen-specific tolerance is established and maintained.
Assuntos
Imunidade Adaptativa , Linfócitos B/imunologia , Interleucina-10/fisiologia , Imunologia de Transplantes , Animais , Sobrevivência de Enxerto , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Testes de NeutralizaçãoRESUMO
Viral infections including those due to cytomegalovirus have been associated with accelerated cardiac allograft vasculopathy (CAV) in clinical trials and some animal models. Evidence demonstrating a direct causal relationship between such infections and de novo formation of coronary vascular lesions is lacking. Heterotopic murine cardiac transplants were performed in a parental to F1 combination in animals lacking both T- and B-lymphocytes (RAG(-/-)). Coronary vasculopathy developed almost exclusively in the presence of recipient infection with lymphocytic choriomeningitis virus but not in uninfected controls. This process was also dependent upon the presence of natural killer (NK) cells as depletion of NK cells abrogated the process. These data show that a viral infection in its native host, and not previously implicated in the production of CAV, can contribute to the development of advanced coronary vascular lesions in cardiac allotransplants in mice. These data also suggest that virus-induced CAV can develop via an NK-cell-dependent pathway in the absence of T- and B-lymphocytes.
Assuntos
Infecções por Arenaviridae/imunologia , Doença das Coronárias/virologia , Transplante de Coração , Hospedeiro Imunocomprometido , Células Matadoras Naturais/virologia , Vírus da Coriomeningite Linfocítica , Animais , Infecções por Arenaviridae/complicações , Linfócitos B/imunologia , Doença das Coronárias/imunologia , Sobrevivência de Enxerto/imunologia , Proteínas de Homeodomínio/genética , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Linfócitos T/imunologia , Imunologia de Transplantes , Transplante HomólogoRESUMO
Several components of the chloroplast protein import machinery have been identified mostly in the C3 dicot plant pea. We describe here two homologous cDNA sequences of Toc34, a regulatory component of the import machinery, from maize, a C4 monocot plant. The deduced amino acid sequences of two maize Toc34 proteins, named ZmToc34-1 and ZmToc34-2, show 95% identity to each other and about 60% to those of previously identified Toc34 proteins. The GTP-binding motif and the presumed carboxyl terminal membrane anchor are also conserved in the ZmToc34 proteins. The maize Toc34, like that of pea, is tightly associated with the outer envelope membrane of chloroplast and exposed to the cytosol.
Assuntos
Cloroplastos/metabolismo , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Zea mays/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Higher plant chloroplasts contain a 21-kDa protein, chaperonin 21 (Cpn21), that is a functional homolog of the chaperonin 10 (Cpn10). The chloroplast Cpn21 polypeptide consists of two Cpn10-like domains fused together in tandem. We describe here the cDNA sequence of the Cpn21 (AtCpn21) precursor protein from Arabidopsis thaliana. The deduced amino acid sequence of the AtCpn21 precursor protein, 253 amino acids long, shows 61% identity with the spinach Cpn21 protein. The AtCpn21 precursor protein contains the typical chloroplast transit peptide of 51 amino acids at its aminoterminus and the two Cpn10-like domains which exhibits 46% sequence identity to each other. The predicted mature-sized polypeptide of AtCpn21 was expressed in Escherichia coli as a soluble 21-kDa protein. Gel-filtration and chemical cross-linking analyses showed that the recombinant mature AtCpn21 protein forms a stable homo-oligomer composed of three or four polypeptides.
Assuntos
Arabidopsis/genética , Chaperoninas/genética , DNA Complementar/química , Precursores de Proteínas/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis , Sequência de Bases , Chaperoninas/biossíntese , Chaperoninas/química , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Chaperoninas do Grupo I , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
We isolated an Arabidopsis thaliana cDNA whose translated product shows sequence similarity to the FtsY, a bacterial homologue of SRP receptor protein. The Arabidopsis FtsY homologue contains a typical chloroplast transit peptide. The in vitro-synthesized 37 kDa FtsY homologue was imported into chloroplasts, and the processed 32 kDa polypeptide bound peripherally on the outer surface of thylakoids. Antibodies raised against the FtsY homologue also reacted with a thylakoid-bound 32 kDa protein. The antibodies inhibited the cpSRP-dependent insertion of the light-harvesting chlorophyll alb-binding protein into thylakoid membranes suggesting that the chloroplast FtsY homologue is involved in the cpSRP-dependent protein targeting to the thylakoid membranes.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cloroplastos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Bacillus subtilis/genética , Transporte Biológico Ativo , Clonagem Molecular , DNA Complementar/genética , DNA de Plantas/genética , Escherichia coli/genética , Dados de Sequência Molecular , Pisum sativum/genética , Pisum sativum/metabolismo , Homologia de Sequência de Aminoácidos , Partícula de Reconhecimento de Sinal/genéticaRESUMO
Previously, we cloned rat MRP3 as a candidate for an inducible transporter for the biliary excretion of organic anions [Hirohashi et al. (1998) Mol. Pharmacol. 53, 1068-10751. In the present study, we cloned human MRP3 (1527 amino acids) from Caco-2 cells. Human MRP3 is predominantly expressed in liver, small intestine and colon; hepatic expression of MRP3 was observed in humans but not in normal rats. In HepG2 cells, the expression of MRP3 was induced by phenobarbital. These results suggest that MRP3 may act as an inducible transporter in the biliary and intestinal excretion of organic anions.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Northern Blotting , Células CACO-2 , Carcinoma Hepatocelular , Colo/química , Expressão Gênica/efeitos dos fármacos , Humanos , Intestino Delgado/química , Fígado/química , Neoplasias Hepáticas , Dados de Sequência Molecular , Fenobarbital/farmacologia , Células Tumorais CultivadasRESUMO
The aim of this nationwide study was to determine the recent incidence and prevalence of inflammatory bowel disease, i.e., Crohn's disease (CD) and ulcerative colitis (UC), in Japan. We mailed out a preliminary examination sheet with diagnostic criteria, asking about the presence of patients with inflammatory bowel disease, to all hospitals in Japan that have more than 200 beds for general use. The rate of reply was 60.93%. A total of 4243 patients with CD were reported. The incidence per 100 000 population per annum was 0.51 (0.71 in males, 0.32 in females). The prevalence per 100 000 population per annum was 5.85 (7.94 in males, 3.83 in females). Peak age at onset was 20-24 years in males and 15-19 years in females. A total of 12559 cases of UC were reported. The incidence per 100 000 population per annum was 1.95 (2.23 in males, 1.68 in females). The prevalence per 100 000 population per annum was 18.12 (18.70 in males, 18.17 in females). Peak age at onset was 20-24 years in males and 25-29 years in females.
Assuntos
Colite Ulcerativa/epidemiologia , Doença de Crohn/epidemiologia , Adolescente , Adulto , Idade de Início , Feminino , Inquéritos Epidemiológicos , Humanos , Incidência , Japão/epidemiologia , Masculino , PrevalênciaRESUMO
To study the effect of laser irradiation on normal lumbar discs, a 2100 nm Holmium (HO)-YAG laser irradiation was applied to the 83 lumbar discs of 23 adult rabbits. The extent of disc vaporization, the temperature changes in the surrounding tissues, and changes in the radiograph and MRI findings were assessed after laser irradiation. When laser irradiation was delivered to the discs, the disc weight decreased linearly with the increase in total laser energy, indicating steady vaporization of disc material. The temperature was highest at the site of the guide needle. Laser irradiation was delivered at 0.5 J/pulse or 1.4 J/pulse x 5 pulses/sec to the intervertebral discs, and radiographs and T2-weighted MRI of the irradiated discs were investigated at 1, 4, and at 12 weeks after irradiation. At 1 week after irradiation at 0.5 and 1.4 J/pulse, the radiographs showed a decrease in the disc height. At 12 weeks after irradiation at 0.5 J/pulse, the disc height had restored to normal, while the decrease was persistent after irradiation at 1.4 J/pulse. At 1 week after irradiation, MRI showed a decrease in the signal intensity of discs treated at 0.5 J/pulse, but the decrease was recovered at 12 weeks. After irradiation at 1.4 J/pulse, the decrease in signal intensity was also recovered by 12 weeks, but the recovery was less than the recovery after treatment at 0.5 J/pulse. Laser irradiation is applicable for the treatment of intervertebral discs, but it is necessary to select the optimal operating conditions. It may also be necessary to change the power of irradiation according to the pathological condition of the disc being treated.
Assuntos
Disco Intervertebral/efeitos da radiação , Terapia a Laser , Vértebras Lombares/efeitos da radiação , Animais , Deslocamento do Disco Intervertebral/radioterapia , Imageamento por Ressonância Magnética , Coelhos , TemperaturaRESUMO
It has been postulated that the process of iron transfer to the human fetus begins with the binding of maternal transferrin-iron complexes to placental transferrin receptors. This presentation deals with the dynamics of transferrin receptors on the cell membrane of trophoblastic cells including normal placenta cells, molar trophoblasts and choriocarcinoma cell lines (GCH-1, GCH-2, ENAMI). Results were as follows. Receptors for transferrin were observed on all the trophoblastic cell membranes tested. The transferrin receptors increased with the gestational weeks. Transferrin receptors on molar trophoblasts were approximately one-half to twice as numerous as those on normal trophoblasts. The transferrin receptors on choriocarcinoma cell lines were five to ten times as numerous as those on normal trophoblast. Thymidine uptake and native hCG secretion in choriocarcinoma cell lines increased with transferrin-iron addition to these mediums (but no increase in the beta-hCG secretion in the medium was observed). On the other hand, the transferrin receptors on these membranes decreased.
Assuntos
Placenta/análise , Receptores de Superfície Celular/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Coriocarcinoma/análise , Gonadotropina Coriônica/análise , Vilosidades Coriônicas/análise , Feminino , Humanos , Placenta/citologia , Gravidez , Receptores de Superfície Celular/fisiologia , Receptores da Transferrina , Trofoblastos/análise , Neoplasias Uterinas/análiseRESUMO
Preprotein translocation across the outer and inner envelope membranes of chloroplasts is an energy-dependent process requiring ATP hydrolysis. Several precursor proteins analyzed so far have been found to be imported into isolated chloroplasts equally well in the dark in the presence of ATP as in the light where ATP is supplied by photophosphorylation in the chloroplasts themselves. We demonstrate here that precursors of two maize (Zea mays L. cv Golden Cross Bantam) ferredoxin isoproteins, pFdI and pFdIII, show distinct characteristics of import into maize chloroplasts. pFdI, a photosynthetic ferredoxin precursor, was efficiently imported into the stroma of isolated maize chloroplasts both in the light and in the dark. In contrast pFdIII, a non-photosynthetic ferredoxin precursor, was mostly mis-sorted to the intermembrane space of chloroplastic envelopes as an unprocessed precursor form in the light but was efficiently imported into the stroma and processed to its mature form in the dark. The mis-sorted pFdIII, which accumulated in the intermembrane space in the light, could not undergo subsequent import into the stroma in the dark, even in the presence of ATP. However, when the mis-sorted pFdIII was recovered and used for a separate import reaction, pFdIII was capable of import into the chloroplasts in the dark. pFNRII, a ferredoxin-NADP+ reductase isoprotein precursor, showed import characteristics similar to those of pFdIII. Moreover, pFdIII exhibited similar import characteristics with chloroplasts isolated from wheat (Pennisetum americanum) and pea (Pisum sativum cv Alaska). These findings suggest that the translocation of precursor proteins across the envelope membranes of chloroplasts may involve substrate-dependent light-regulated mechanisms.
Assuntos
Cloroplastos/metabolismo , Ferredoxinas/metabolismo , Membranas Intracelulares/metabolismo , Precursores de Proteínas/metabolismo , Triticum/metabolismo , Zea mays/metabolismo , Trifosfato de Adenosina/metabolismo , Escuridão , Ferredoxinas/genética , Luz , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Transcrição GênicaRESUMO
Higher plant chloroplasts contain two chaperonin 60 family proteins, Cpn60alpha and Cpn60beta, which are known to have divergent amino acid sequences. Although Cpn60alpha and Cpn60beta are present in roughly equal amounts and copurify in their native states, a heterogeneous ensemble of the chaperonin oligomer has not yet been demonstrated. We separately purified Cpn60alpha and Cpn60beta proteins from spinach leaves as the monomeric form. Either antibody raised against one chaperonin 60 protein could coimmunoprecipitate the other chaperonin 60 protein in their oligomeric state but not in its monomeric state, suggesting that the chloroplast Cpn60alpha and Cpn60beta polypeptides actually reside in the same chaperonin oligomer in the stroma. Moreover, the chaperonin oligomers migrated as at least two distinct bands on the native gel electrophoresis, each of which contained both chaperonin 60 proteins. These results suggest that chloroplast chaperonin oligomers might be composed of at least two distinct sets of two chaperonin proteins.
Assuntos
Chaperonina 60/química , Chaperoninas/química , Cloroplastos/química , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peptídeos/química , Proteínas de Plantas/química , Testes de Precipitina , Conformação Proteica , Isoformas de Proteínas/química , Spinacia oleraceaRESUMO
We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Resistência a Múltiplos Medicamentos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Animais , Células Cultivadas , Glucuronatos/metabolismo , Glutationa/metabolismo , Células HeLa , Humanos , Cinética , Células LLC-PK1 , Ratos , SuínosRESUMO
An enzyme immunoassay (EIA) for hCG-beta CTP was newly developed. Anti hCG-beta CTP antibody was prepared from antiserum to the synthetic peptide corresponding to the carboxyl terminal region by immunization of rabbits with hCG-beta subunit. Specimens to be tested were reacted with the antibody labelled with peroxidase and determined with a spectrophotometer after the addition of o-phenylenediamine. It was found that the sensitivity of the assay for hCG in sera was 0.2miu/ml and that values for the assay were significantly correlated with those for the RIA assay for hCG-beta (r = 0.916, Y = 1.27X + 1.8). Cross reactivity with LH, FSH, hCG-beta or hCG-alpha was observed to be 0.18%, 0.05%, 10.4% or less than 0.011% respectively. The assay has been applied in the management of hydatidiform mole, invasive mole and choriocarcinoma, indicating its usefulness because of the increase in sensitivity roughly 25 times or 10 times as great as hemagglutination or hCG-beta RIA assay.
Assuntos
Gonadotropina Coriônica/sangue , Técnicas Imunoenzimáticas , Fragmentos de Peptídeos/sangue , Neoplasias Trofoblásticas/diagnóstico , Neoplasias Uterinas/diagnóstico , Adulto , Gonadotropina Coriônica Humana Subunidade beta , Citidina Trifosfato/sangue , Feminino , Seguimentos , Humanos , Masculino , GravidezRESUMO
2' ,7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxymethyl ester (BCECF-AM), a fluorescence reagent for the measurement of intracellular pH with a molecular weight of 809 Da, was used to test the hypothesis that the blood-brain barrier (BBB) does not restrict the influx of substrate with a molecular weight greater than 600 Da. Using cultured bovine brain capillary endothelial cells (BCEC), the influx rate of BCECF-AM was found to be 151 +/- 2 microl/min/mg protein and was extrapolated to give 446 +/- 7 microl/min/g brain as a BBB permeability surface area product (PS). No significant saturation was observed for the initial in vitro uptake of BCECF-AM into BCEC at concentrations 0.1, 1.0 and 5.0 microM. The apparent activation energy of the initial uptake of BCECF-AM was found to be 5.09 kcal/mol. These results suggest that BCECF-AM is transported into the BBB by passive diffusion. The in vivo BBB PS value was also found to be 295 +/- 48 microl/min/g brain and 132 +/- 24 microl/min/g brain by the in situ brain perfusion and the carotid artery injection methods, respectively. No significant efflux of BCECF-AM from the brain was observed over a 120 sec washout period, suggesting that BCECF-AM is immediately hydrolyzed to BCECF, a hydrophilic analogue, in the brain after crossing the BBB. The octanol/water partition coefficient of BCECF-AM was found to be 5.66 +/- 0.27. The BBB PS value of BCECF-AM was predicted to be 105 microl/min/g brain, based on the relationship between the BBB PS value and the value of partition coefficient divided by the square root of the molecular weight. These results demonstrate that BCECF-AM transport across the BBB is not restricted despite its large molecular size.
Assuntos
Barreira Hematoencefálica , Endotélio Vascular/metabolismo , Fluoresceínas/farmacocinética , 1-Butanol , 3-O-Metilglucose/metabolismo , Animais , Transporte Biológico , Butanóis/farmacocinética , Calorimetria , Capilares , Artérias Carótidas , Bovinos , Células Cultivadas , Circulação Cerebrovascular , Difusão , Fluoresceínas/administração & dosagem , Injeções Intra-Arteriais , Masculino , Manitol/farmacocinética , Ratos , Ratos WistarRESUMO
We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Trifosfato de Adenosina/metabolismo , Ácidos e Sais Biliares/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Animais , Transporte Biológico , Membrana Celular/metabolismo , Resistência a Múltiplos Medicamentos , Humanos , Cinética , Células LLC-PK1 , Osmose , Ratos , Suínos , TrítioRESUMO
The prostacyclin I1 (PGI1) analogue, SM-10906 and its methyl ester, SM-10902, have been compared with the PGI2 analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. SM-10906 displaced [3H]iloprost binding to the membrane fraction, the IC50 value being 100 nM, but showed very low affinity for the prostaglandin E (PGE) receptor. SM-10906 dose-dependently stimulated GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 35 nM. Furthermore, SM-10906 prevented a thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 300 nM. These IC50 and EC50 values are much lower than those of SM-10902. These results demonstrate that SM-10906, a stable PGI1 derivative, is an agonist for the [3H]iloprost-binding (PGI2) receptor, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells. On the other hand, a methyl ester derivative of PGI1, SM-10902, was inactive in the binding assay, but it seems to be a partial agonist for adenylate cyclase activity [corrected].
Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/biossíntese , Epoprostenol/análogos & derivados , Mastócitos/efeitos dos fármacos , Receptores de Prostaglandina/metabolismo , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Epoprostenol/metabolismo , Epoprostenol/farmacologia , Iloprosta/metabolismo , Iloprosta/farmacologia , Mastócitos/metabolismo , Sarcoma de Mastócitos , Camundongos , Prostaglandinas E/metabolismo , Receptores de Epoprostenol , Receptores de Prostaglandina E/metabolismo , Trombina/farmacologia , Células Tumorais CultivadasRESUMO
The pharmacological characteristics of the 3-oxamethano-prostaglandin I1 compound (+)-methyl [2-[(2R,3aS,4R,5R,6aS)-octahydro-5-hydroxy-4- [(E)-(3S,5S)-3-hydroxy-5-methyl-1-nonenyl]-2-pentalenyl]etho xy] acetate (SM-10902, CAS 139403-31-9), a novel stable analogue of prostacyclin and its free acid, SM-10906, were studied. SM-10902 was rapidly deesterified to its free acid in rabbit and human serum. SM-10902 and SM-10906 exhibited antiplatelet potency against ADP-induced aggregation in rabbit and human platelets. In the presence of diisopropyl fluorophosphate, an esterase inhibitor, the antiplatelet activity of SM-10902 was markedly reduced, to much less than that of SM-10906. SM-10906 inhibited platelet aggregation induced by various inducers in several species and enhanced the cyclic AMP (cAMP) level in human platelets. These activities were nearly equal to those of prostaglandin (PG) E1 and less than those of PGI2. SM-10906 relaxed isolated rabbit mesenteric and bovine coronary arteries, and elevated the cAMP level in bovine coronary arteries. SM-10906 given intravenously exhibited a sustained reduction in blood pressure based on vasodilation in ganglion-blocked, angiotensin II-supported rats. SM-10902 applied to the guinea-pig auricles increased the skin temperature, but SM-10906 and PGI2 showed no such effect. In conclusion, SM-10902, which is considered to be a prodrug of SM-10906, was suggested to exert its anti-platelet and vasodilator activities through the increase of cAMP. Since SM-10902 penetrates well into the skin, it may be useful as an external preparation to improve peripheral circulatory insufficiency.
Assuntos
Epoprostenol/análogos & derivados , Epoprostenol/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Vasodilatadores/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Bovinos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , AMP Cíclico/metabolismo , Cães , Epoprostenol/sangue , Esterases/antagonistas & inibidores , Cobaias , Humanos , Técnicas In Vitro , Masculino , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/sangue , Coelhos , Ratos , Ratos Wistar , Temperatura Cutânea/efeitos dos fármacos , Vasodilatadores/sangueRESUMO
In order to elucidate the mechanism of action of DL-melinamide [DL-MA, N-(alpha-methylbenzyl)linoleamide], an inhibitor of cholesterol absorption, the effect of DL-MA on esterification of cholesterol in the mucosa of rabbit small intestine was studied. DL-MA inhibited acyl CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) activity in the mucosal microsomes, with 50% inhibition occurring at approximately 0.5 microM. On the other hand, DL-MA had no effect on the cholesterol esterase (EC 3.1.1.13) activity in the mucosal cytosol. Kinetic studies indicate that DL-MA is an uncompetitive inhibitor of ACAT. D-MA, one of the two optical isomers of DL-MA, was found to be a more effective inhibitor of ACAT than L-MA, another isomer. This finding indicates that the inhibition of cholesterol absorption by DL-MA depends on the inhibition of ACAT by this compound, in view of the fact that D-MA is a more effective inhibitor of cholesterol absorption than L-MA.