RESUMO
NFAT (nuclear factor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-kappaB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaN in vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.
Assuntos
Compostos de Anilina/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Interleucina-2/biossíntese , Proteínas Nucleares , Pirazóis/farmacologia , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Sequência de Aminoácidos , Compostos de Anilina/química , Animais , Sequência de Bases , Células COS , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-2/genética , Células Jurkat , Teste de Cultura Mista de Linfócitos , Dados de Sequência Molecular , Peso Molecular , Fatores de Transcrição NFATC , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Pirazóis/química , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
ABT-770 [(S)-N-[1-[[4'-trifluoromethoxy-[1,1'-biphenyl]-4-yl]oxy]methyl-2-(4,4-dimethyl-2,5-dioxo-1-imidazolidinyl)ethyl]-N-hydroxyformamide], a matrix metalloproteinase inhibitor (MMPI), produced generalized phospholipidosis in rats. Phospholipid accumulation was accompanied by retention of drug-related material and was associated with increased mortality. Generation of a successful drug candidate depended upon understanding the cause of the phospholipidosis and redesigning the chemical structure accordingly. ABT-770 and other MMPIs, plus several metabolites of each, were assayed for their ability to induce phospholipidosis in primary cultured rat and human hepatocytes. Phospholipid accumulation was detected by following the incorporation of a fluorescent phospholipid analogue into intracytoplasmic inclusion bodies characteristic of phospholipid storage disorders. At 24 and 48 hr, none of the parent compounds induced phospholipidosis in vitro in rat or human hepatocytes. Phospholipidosis was associated primarily with an amine metabolite of ABT-770. The amine metabolite of another MMPI, ABT-518 ([S-(R*,R*)]-N-[1-(2,2-dimethyl-1,3-dioxol-4-yl)-2-[[4-[4-(trifluoromethoxy)-phenoxy]phenyl]sulfonyl]ethyl]-N-hydroxyformamide), produced little phospholipidosis in rat and human hepatocytes even at concentrations up to 100 microM. The presence or absence of phospholipidosis in the in vitro assay correlated well with ultrastructural findings and drug accumulation in rat tissues. ABT-770, which produced phospholipidosis associated with its amine metabolite in vitro and in vivo, also generated a higher tissue to plasma distribution of metabolites particularly in tissues where phospholipidosis was observed. ABT-518 and its amine metabolite, however, produced low tissue to plasma ratios and induced little to no phospholipidosis in vitro or in vivo. These results demonstrate that the phospholipidosis observed for ABT-770 could be attributed to a cationic metabolite, and that altering the properties of such a metabolite, by modification of the parent compound, alleviated the disorder.
Assuntos
Compostos de Bifenilo/efeitos adversos , Hepatócitos/efeitos dos fármacos , Ácidos Hidroxâmicos/efeitos adversos , Lipidoses/induzido quimicamente , Inibidores de Metaloproteinases de Matriz , Animais , Compostos de Bifenilo/metabolismo , Formamidas/metabolismo , Formamidas/farmacologia , Inibidores da Protease de HIV/efeitos adversos , Inibidores da Protease de HIV/metabolismo , Hepatócitos/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Endothelins (ETs) are 21 amino acid peptides which bind to ET(A)- and ET(B)-receptors to evoke diverse physiological responses. This report studies the internalization of ET(A)-receptor in Chinese hamster ovary (CHO) cells which were stably transfected with ET(A)-receptor cDNA. ET-1 binding induced ET(A) internalization in a time-dependent manner with 40% of ET(A)-receptors internalized at 37 degrees C after 30 min. To localize internalized ET(A)-receptor, cells were immunostained using a polyclonal antibody against the extracellular loop between IV and V transmembrane segments of the ET(A)-receptor. To examine the fate of internalized ET-1, cells were treated with 10 nM biotinylated ET-1 coupled with Texas Red-labeled streptavidin. In the absence of ET-1, a majority of ET(A) was localized on the surface of cells. After ET-1 treatment for 60 min, internalized ET(A)-receptors were localized in a perinuclear structure. ET-1 remained bound to ET(A)-receptor after internalization for up to 60 min and then dissociated from the receptor. After dissociation, ET-1 possibly became degraded and ET(A) recycled back to the cell surface. Protein kinase inhibitors such as KT5926 and staurosporine partially inhibited ET(A)-receptor internalization. The results of this study may facilitate the understanding of pathways involved in ET-1-induced receptor internalization.
Assuntos
Receptores de Endotelina/metabolismo , Animais , Células CHO , Cricetinae , Endotelina-1/metabolismo , Proteínas Quinases/fisiologia , Receptor de Endotelina A , Receptores de Endotelina/análiseRESUMO
Balloon angioplasty has become an important intervention in clinical cardiology; however, the technique is associated with a high incidence of restenosis, requiring repeated procedures. Endothelin-1 (ET-1), specifically through its action on ET(A) receptors, has been implicated in the cell proliferation and subsequent neointimal formation that leads to restenosis. Therefore we examined a potent antagonist of the ET(A) receptor, A127722.5, in a pig model of balloon angioplasty in iliac and carotid arteries. Ten pigs received A-127722.5 (7.5 mg/kg b.i.d.) orally, starting 3 days before angioplasty and continuing for 4 weeks; 10 additional pigs were treated with the same dosing regimen of the angiotensin-converting enzyme (ACE) inhibitor captopril (3.0 mg/kg b.i.d.), while a third group of 10 animals received placebo. At 2 and 4 weeks after the start of treatment, these doses of the ET(A) receptor antagonist and ACE inhibitor blocked the presser responses induced by big ET-1 and angiotensin I, respectively. In the iliac arteries, neointimal formation, neointimal/medial ratio, and maximal neointimal thickness were all significantly reduced, and the residual lumen area was significantly increased in pigs treated with the ET(A) receptor antagonist compared with placebo and captopril-treated groups. Medial collagen content, collagen deposition, and medial growth also were significantly reduced relative to the placebo group. Beneficial effects also were observed in the carotid arteries, although the results were less striking. Captopril was ineffective in protecting against the effects of balloon angioplasty in both vessels. Our results indicate that an orally active and potent antagonist of the ET(A) receptor inhibits cell proliferation and synthesis of extracellular matrix in pigs and may provide an important therapeutic approach to the prevention of restenosis.
Assuntos
Angioplastia com Balão/efeitos adversos , Antagonistas dos Receptores de Endotelina , Músculo Liso Vascular/efeitos dos fármacos , Pirrolidinas/farmacologia , Angiotensina I/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Atrasentana , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Lesões das Artérias Carótidas , Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Colágeno/biossíntese , Endotelina-1/sangue , Endotelina-1/farmacologia , Hiperplasia , Artéria Ilíaca/efeitos dos fármacos , Artéria Ilíaca/lesões , Artéria Ilíaca/metabolismo , Artéria Ilíaca/patologia , Masculino , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Pirrolidinas/sangue , Receptor de Endotelina A , Suínos , Porco Miniatura , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologiaRESUMO
We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by NG-methyl-L-arginine (NMA), and the inhibition was reversed by L-arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2',5'-ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca2+/calmodulin dependent and required NADPH and tetrahydrobiopterin as cofactors. Superoxide dismutase protected and oxyhemoglobin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155-kDa protein band. Immunohistochemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine-beta-hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine-induced stimulation of soluble guanylyl cyclase.
Assuntos
Aminoácido Oxirredutases/metabolismo , Gânglios Simpáticos/enzimologia , Acetilcolina/farmacologia , Animais , Western Blotting , Bovinos , Cromatografia de Afinidade , GMP Cíclico/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Feminino , Gânglios Simpáticos/citologia , Imuno-Histoquímica , Masculino , Neurônios/enzimologia , Óxido Nítrico SintaseRESUMO
Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.
Assuntos
Aminoácido Oxirredutases/metabolismo , Cerebelo/enzimologia , Guanilato Ciclase/metabolismo , Isoenzimas/metabolismo , NADPH Desidrogenase/metabolismo , Neurônios/enzimologia , Transdução de Sinais , Animais , Western Blotting , Bovinos , Cerebelo/anatomia & histologia , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Óxido Nítrico Sintase , Testes de Precipitina , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.