Assuntos
Coristoma , Neoplasias da Coroide , Neovascularização de Coroide , Osteoma , Neoplasias da Coroide/complicações , Neoplasias da Coroide/diagnóstico , Neovascularização de Coroide/diagnóstico , Angiofluoresceinografia , Humanos , Imagem Multimodal , Osteoma/complicações , Osteoma/diagnóstico , Tomografia de Coerência ÓpticaRESUMO
INTRODUCTION: The purpose of this study was to identify anatomic-functional correlations in patients with Birdshot chorioretinopathy in order to better understand the mechanisms of visual loss. MATERIALS AND METHODS: We conducted a single-center observational prospective study at the Nantes University Medical Center including all patients followed for Birdshot chorioretinopathy between January 2019 and July 2019. The parameters studied were visual acuity, microperimetry, standard automated perimetry, SD-OCT, EDI-OCT, OCT-A, RNFL-OCT, wide-field fundus photographs, and fluorescein and ICG angiography. RESULTS: Forty-four eyes were studied. A significant correlation was found between visual acuity and retinal thickness (P=0.002, r=0.44), but not choroidal thickness (P=0.16). A significant correlation was also observed between retinal sensitivity and total macular thickness (P<0.001, r=0.68) as well as inner retinal thickness (P<0.001, r=0.65), while the correlation was weaker with choroidal thickness (P=0.03, r=0.32). There was a weak correlation between retinal sensitivity and superficial capillary density (P=0.02, r=0.42) as well as deep capillary density (P=0.02, r=0.41). The peripheral hypo-autofluorescent zones correspond to the visual field defects, but these defects are larger than the hypo-autofluorescent zones. Patients with paracentral or peripheral field defects had a history of vasculitis more often than hypo-ICG spots. DISCUSSION: We found significant correlations between functional parameters such as visual acuity and retinal sensitivity and retinal anatomic parameters, particularly the inner retina, while there was no correlation shown with choroidal anatomic parameters. CONCLUSION: The main results of our study suggest a retinal rather than choroidal origin of the degradation of visual function in birdshot chorioretinopathy.
Assuntos
Corioide , Tomografia de Coerência Óptica , Coriorretinopatia de Birdshot , Corioide/diagnóstico por imagem , Angiofluoresceinografia , Humanos , Estudos ProspectivosRESUMO
Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fenômenos Fisiológicos Virais , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Genes Reporter , Substâncias de Crescimento/farmacologia , Humanos , Fator Regulador 3 de Interferon , Células Jurkat , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Mutagênese , NF-kappa B/metabolismo , Estresse Oxidativo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases , Estrutura Terciária de Proteína , Respirovirus/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
Angiotensin II (Ang II), the primary effector of the renin-angiotensin system, is a multifunctional hormone that plays an important role in vascular function. In addition to its classical vasoconstrictor action, more recent studies demonstrated that Ang II stimulates the growth of a number of cell types, including vascular smooth muscle cells (SMC) (reviewed in [1-3]). In vivo studies have shown that chronic infusion of Ang II leads to the development of vascular hypertrophy in rats, whereas administration of angiotensin-converting enzyme (ACE) inhibitors or Ang II receptor antagonists prevents or regresses vascular hypertrophy in models of genetic and experimental hypertension [4]. Consistent with in vivo data, several laboratories have shown that Ang II stimulates protein synthesis and induces cellular hypertrophy, but not cell proliferation, in cultured aortic SMC [5-9]. Ang II also induces directed migration (chemotaxis) of vascular SMC [10, 11], although its effect is less prominent than that of platelet-derived growth factor (PDGF). The cellular mechanisms underlying these diverse actions of Ang II are not clearly understood but are likely to involve the activation of distinct signaling pathways.
Assuntos
AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Músculo Liso Vascular/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Angiotensina/fisiologia , Transdução de Sinais/fisiologia , Angiotensina II/fisiologia , Animais , Humanos , Músculo Liso Vascular/citologia , Ratos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de AngiotensinaRESUMO
Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed interferon regulatory factor 3 (IRF-3) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-3 dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-3 transcriptionally up regulates alpha/beta interferon genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF-3(5D)] and dominant negative (IRF-3 DeltaN) forms of IRF-3 that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-3, we observed that IRF-3 is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-3 is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-3(5D) alone is sufficient to induce apoptosis in human embryonic kidney 293 and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-3 expression augments paramyxovirus-induced apoptosis, while expression of IRF-3 DeltaN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and 3 in IRF-3-induced apoptosis. These results suggest that IRF-3, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection.
Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Respirovirus/fisiologia , Fatores de Transcrição/metabolismo , Caspases/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Fator Regulador 3 de Interferon , Interferons/biossíntese , Células Jurkat , Respirovirus/patogenicidade , Fatores de Transcrição/genética , TransgenesRESUMO
Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G(1) cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G(1) phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27(Kip1) expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27(Kip1) protein. The time course of p27(Kip1) decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27(Kip1) synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-beta1 by aortic SMC. These results identify p27(Kip1) as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.
Assuntos
Angiotensina II/farmacologia , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Mitógenos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Supressoras de Tumor , Animais , Aorta/citologia , Becaplermina , Células Cultivadas , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Proteínas Associadas aos Microtúbulos/biossíntese , Músculo Liso Vascular/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro , Ratos , Fase S , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
Interferons are a large family of multifunctional secreted proteins involved in antiviral defense, cell growth regulation and immune activation. Viral infection induces transcription of multiple IFN genes, a response that is in part mediated by the interferon regulatory factors (IRFs). The initially characterized members IRF-1 and IRF-2 are now part of a growing family of transcriptional regulators that has expanded to nine members. The functions of the IRFs have also expanded to include distinct roles in biological processes such as pathogen response, cytokine signaling, cell growth regulation and hematopoietic development. The aim of this review is to provide an update on the novel discoveries in the area of IRF transcription factors and the important roles of the new generation of IRFs--particularly IRF-3, IRF-4 and IRF-7.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Interferons/genética , Interferons/metabolismo , Fosfoproteínas/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Divisão Celular/fisiologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Sistema Imunitário/metabolismo , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Fator Regulador 3 de Interferon , Fator Regulador 7 de Interferon , Fatores Reguladores de Interferon , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Leucemia de Células T/metabolismo , Dados de Sequência Molecular , Fosfoproteínas/química , Proteínas Repressoras/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
We monitored cardiac angiotensin II concentration and AT1-receptor density after long-term blockade of the renin-angiotensin system in inbred control hamsters treated with placebo or losartan (100 mg/kg/day) and cardiomyopathic hamsters treated with placebo, low-(30 mg/kg/day), or high-dose (100 mg/kg/day) losartan or quinapril (100 mg/kg/day). All treatments were started at age 50 days. Angiotensin II-receptor density and affinity were measured by radioligand-binding assays, and ventricular angiotensin II concentration was determined by radioimmunoassay. After 125 and 275 days of treatment, both doses of losartan significantly reduced AT1-receptor density, whereas quinapril had no effect. The administration of both drugs resulted in significant reductions in ventricular angiotensin II concentration. The prolonged administration of losartan was associated with an increase in cardiac hypertrophy, suggesting that angiotensin II signaling is not directly involved or at least does not play a major role in the remodeling process observed in cardiomyopathic hamsters.
Assuntos
Angiotensina II/metabolismo , Cardiomegalia/metabolismo , Receptores de Angiotensina/biossíntese , Sistema Renina-Angiotensina/fisiologia , Antagonistas de Receptores de Angiotensina , Animais , Ligação Competitiva , Cricetinae , Regulação para Baixo , Ventrículos do Coração/metabolismo , Losartan/farmacologia , Masculino , Mesocricetus , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/metabolismoRESUMO
In the present study, we have examined the effect of increased cyclic AMP (cAMP) levels on the stimulatory action of angiotensin II (Ang II) on protein synthesis. Treatment with cAMP-elevating agents potently inhibited Ang II-induced protein synthesis in rat aortic smooth muscle cells and in rat fibroblasts expressing the human AT1 receptor. The inhibition was dose-dependent and was observed at all concentrations of the peptide. To explore the mechanism of cAMP action, we have analyzed the effects of forskolin and 3-isobutyl-1-methylxanthine on various receptor-mediated responses. Elevation of cAMP did not alter the binding properties of the AT1 receptor and did not interfere with the activation of phospholipase C or the induction of early growth response genes by Ang II. Likewise, Ang II-dependent activation of the mitogen-activated protein kinases ERK1/ERK2 and p70 S6 kinase was unaffected by cAMP. In contrast, we found that increased concentration of cAMP strongly inhibited the stimulatory effect of Ang II on protein tyrosine phosphorylation. Specifically, cAMP abolished Ang II-induced tyrosine phosphorylation of the focal adhesion-associated protein paxillin and of the tyrosine kinase Tyk2. These results identify a novel mechanism by which the cAMP signaling system may exert growth-inhibitory effects in specific cell types.
Assuntos
Angiotensina II/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Músculo Liso Vascular/metabolismo , Fosfotirosina , Receptores de Angiotensina/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Aorta , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Fibroblastos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoproterenol/farmacologia , Cinética , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Músculo Liso Vascular/efeitos dos fármacos , Fosforilação , Ratos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a dual-specificity protein phosphatase encoded by an immediate-early gene responsive to growth factors and stress. The MKP-1 protein selectively inactivates MAP kinases in vitro by dephosphorylation of the regulatory Thr and Tyr residues. Little is known on the mechanisms that regulate MKP-1 gene expression. Here, we demonstrate that Ca2+ is both necessary and sufficient for the induction of MKP-1 gene expression. Treatment of Rat1 fibroblasts with the Ca2+ chelating agent BAPTA completely suppressed serum-induced MKP-1 expression in a dose- and time-dependent manner. The inhibitory effect of BAPTA was observed at the level of the protein and the mRNA. Importantly, Ca2+ chelation blocked the induction of MKP-1 expression in response to all stimuli tested and in different cell types. Increasing the intracellular concentration of Ca2+ with the ionophore A23187 was sufficient to induce MKP-1 mRNA and protein expression in rat fibroblasts. We also provide evidence that activation of MAP kinases is not an absolute requirement for induction of the MKP-1 gene. Exposure of rat fibroblasts to A23187 induced MKP-1 expression without activating the JNK and p38 MAP kinase pathways. Also, inhibition of the ERK pathway with the selective MEK inhibitor PD98059 did not interfere with serum-stimulated MKP-1 mRNA expression. These results will help define the regulatory mechanisms that govern MKP-1 gene transcription in target cells.
Assuntos
Cálcio/fisiologia , Proteínas de Ciclo Celular , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno , Fosfoproteínas Fosfatases , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Calcimicina/farmacologia , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Fosfatase 1 de Especificidade Dupla , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Flavonoides/farmacologia , Ionóforos/farmacologia , MAP Quinase Quinase 4 , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso/citologia , Músculo Liso/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Proteínas Quinases/metabolismo , Proteína Fosfatase 1 , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
A common response of cells to mitogenic and hypertrophic factors is the activation of high rates of protein synthesis. To investigate the molecular basis of this action, we have used the recently developed MAP kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD 98059 to examine the involvement of the ERK pathway in the regulation of global protein synthesis by growth factors in rat aortic smooth muscle cells (SMC). Incubation with PD 98059 blocked angiotensin II (AII)-dependent phosphorylation and enzymatic activity of both MEK1 and MEK2 isoforms, leading to inhibition of the phosphorylation and activation of p44(mapk) and p42(mapk). The compound was found to selectively inhibit activation of the ERK pathway by AII, but not the stimulation of p70 S6 kinase, phospholipase C, or tyrosine phosphorylation. Most importantly, treatment of aortic SMC with PD 98059 potently inhibited AII-stimulated protein synthesis with a half-maximal inhibitory concentration of 4.3 microM. The effect of PD 98059 was not restricted to AII, since the compound also blocked to various extent the induction of protein synthesis by growth factors acting through tyrosine kinase receptors, G protein-coupled receptors, or protein kinase C. These results provide strong evidence that activation of ERK isoforms is an obligatory step for growth factor-induced protein synthesis in aortic SMC.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Substâncias de Crescimento/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Músculo Liso Vascular/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Sequência de Aminoácidos , Angiotensina II/antagonistas & inibidores , Angiotensina II/farmacologia , Animais , Aorta , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/farmacologia , Insulina/farmacologia , Cinética , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Fosforilação , Polienos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Ratos , Sirolimo , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Trombina/farmacologiaRESUMO
Desensitization of rat pancreatic acinar cells with 0.1 mM carbamoylcholine (Cch) or 0.5 nM caerulein (CAE), a cholecystokinin (CCK) agonist, altered the subsequent secretory responses to these two agonists. Changes in receptor affinities, shifts in receptor populations, receptor internalization and phosphorylation are the major modifications affecting the muscarinic and CCK receptors in response to desensitization. In this study, post-receptor alterations were examined in order to explain the altered enzyme secretion. Cch or CAE desensitization resulted in decreased Ca2+ release in response to CAE and Cch respectively. Under desensitizing conditions, the biochemical and pharmacological properties of the InsP3 receptor were not affected. Control and desensitized acini had similar Bmax. and KD values. The Ca(2+)-channel property of the InsP3 receptor was not affected, either, since Ca2+ release in response to increasing concentrations of InsP3 remained comparable in both groups of saponin-permeabilized acini. Finally, the quantities of InsP3 formed in response to Cch and CAE, measured by InsP3 radioreceptor assay, were significantly decreased in the Cch- and CAE-desensitized groups, and these decreases were not due to increased InsP3 turnover. These new data indicate that desensitization of acinar cells with Cch and CAE causes post-receptor modifications resulting in decreased InsP3 formation and decreased intracellular Ca2+ mobilization. It is suggested that the attenuated Ca2+ response is related to a decreased formation of InsP3 from PtdInsP2 hydrolysis and that phospholipase C could be the immediate target of this regulation.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Carbacol/farmacologia , Ceruletídeo/farmacologia , Inositol 1,4,5-Trifosfato/biossíntese , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Amilases/metabolismo , Animais , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/metabolismo , Masculino , Pâncreas/citologia , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
Inositol 1,4,5-trisphosphate (InsP3) is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. The properties of InsP3 receptor sites in the rat pancreas were evaluated by binding studies with InsP3 labeled with 3H. Specific binding was very sensitive to pH variations between 7 and 9. Kinetic studies showed that specific binding of InsP3 at 0 degrees C was half-maximal in about 10 min and reached a plateau within 60 min with a K+1 = 3.37 x 10(9) mol-1min-1. Binding was reversible as addition of 10(-6) M unlabeled InsP3 was followed by dissociation of the bound ligand with a K-1 = 0.016 min-1. Scatchard analysis of the binding data was consistent with a single set of high-affinity sites with KD of 9.9 +/- 2.47 nM and a maximal binding capacity of 210 +/- 55 fmol/mg of protein (n = 7). The specificity of [3H]InsP3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1,3,4,5-tetrakisphosphate, phytic acid, fructose 1,6-bisphosphate, and heparin. To assess the functional relevance of the InsP3 binding sites, the Ca(2+)-releasing activity of InsP3 was measured in permeabilized acinar cell preparations. In the presence of oligomycin (10 micrograms/ml), Ca2+ movements were monitored with the fluorescent indicator fura-2 (free acid). Under these conditions, 4 mM ATP caused rapid uptake of Ca2+ by the vesicular component of the acinar cells. Addition of InsP3 (0.1-15 microM) caused a dose-dependent release of Ca2+ with half-maximal effect at 3.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Canais de Cálcio/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Pâncreas/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Heparina/farmacologia , Concentração de Íons de Hidrogênio , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Pâncreas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular stores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosal was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 microM, from bovine adrenal cortex microsomes. Thimerosal-induced Ca2+ release was not prevented by heparin (250 micrograms/ml), ruling out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 microM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the apparent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, in a dose-dependent manner, [3H]InsP3 binding to adrenal cortex microsomes (EC50 = 43.3 +/- 7.6 microM). A similar effect was also observed on [3H]InsP3 binding to solubilized receptors, suggesting a direct modification of the receptor protein by thimerosal. The effects of thimerosal on Ca2+ release and [3H]InsP3 binding were abolished in the presence of the reducing agent dithiothreitol (1 mM), suggesting a modification by thimerosal of specific thiol groups on these microsomal proteins. Scatchard analysis revealed that thimerosal (100 microM) increased InsP3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 association rate constant. The concomitant increases of binding affinity and Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.