RESUMO
Autophagy has been described as a cellular response to stressful stimuli like starvation. One of its primary functions is to recycle amino acids from degraded proteins for cellular survival under nutrient deprived conditions. Autophagy is characterized by double membrane cytosolic vesicles called autophagosomes and prolonged autophagy is known to result in autophagic (Type II) cell death. Beclin-1 is involved in the regulation of autophagy in mammalian cells. This study examined the potential impact of knockdown of beclin-1 in an autophagic response in HT22 neurons challenged with amino acid starvation (AAS). AAS exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in cell bodies as early as 3 h post-AAS in wild type cells. Elevated levels of the autophagosome-targeting LC3-II were also observed following AAS. In addition, neuronal death induced by AAS in HT22-cells led to a moderate activation of caspase-3, a slight upregulation of AIF and did not alter the HtrA2 levels. Autophagy inhibition by a knockdown of beclin-1 significantly reduced AAS-induced LC3-II increase, reduced accumulation of autophagosomes, and potentiated AAS-mediated neuronal death. Collectively, this study shows that the both apoptotic and autophagic machineries are inducible in cultured hippocampal HT22 neurons subjected to AAS. Our data further show that attenuation of autophagy by a knockdown of beclin-1 enhanced neurons susceptibility to proapoptotic signals induced by AAS and underlines that autophagy is per se a protective than a deleterious mechanism.
Assuntos
Aminoácidos/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Autofagia , Hipocampo/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Animais , Fator de Indução de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteína Beclina-1 , Caspase 3/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Hipocampo/patologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/patologia , Interferência de RNA , Serina Endopeptidases/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
The cellular uptake of L-arginine and other cationic amino acids (such as L-lysine and L-ornithine) is mainly mediated by cationic amino acid transporter (CAT) proteins. Despite the important roles of cationic amino acid transporters for normal brain functioning and various brain diseases there is currently only fragmentary knowledge about their cellular and regional distribution patterns in the human brain. We mapped the immunohistochemical localization of human cationic amino acid transporters 1, 2 and 3 (hCAT1, 2, and 3) throughout five adult human brains and found a wide but uneven distribution of these transporters. All three hCAT1s were mainly localized in neurons, but were also found in numerous astrocytes, oligodendrocytes, plexus choroideus epithelial cells, and small blood vessels. The highest density of hCAT expressing neurons was observed in the hypothalamus, in some areas of the cerebral cortex, the thalamic reticular nucleus and the caudate nucleus, whereas weak to moderate expression was detected in the hippocampus, the prefrontal cortex (hCAT1 only), pons, brain stem and cerebellum. In contrast to what has been found in rodent brain, we detected hCAT2 and hCAT3 also in astrocytes. Overall, each hCAT has its characteristic, individual cerebral expression patterns, which, however, overlap with the others.
Assuntos
Encéfalo/metabolismo , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Transportador 2 de Aminoácidos Catiônicos/metabolismo , Adulto , Astrócitos/metabolismo , Transportador 1 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oligodendroglia/metabolismo , Transporte ProteicoRESUMO
OBJECTIVE: Critical size defects (CSDs) of bone are defined as defects that do not heal spontaneously to new bone during the lifetime of an adult individual. In contrast, immature animals are capable to heal defects of identical size. It was our hypothesis that age-related paracrine effects are relevant for this difference in regeneration. METHODS: The pooled supernatant of primary rat calvarial osteoblast-like cell cultures (POBC) derived from prenatal or postnatal donors was concentrated and applied into CSDs of adult recipient organisms (n = 10). In addition, the supernatant of POBC derived from prenatal donors was pooled and purified by reverse-phase chromatography. Each pre-purified fraction was tested in a proliferation indicating bioassay. Peptide fractions containing proliferative activities were re-chromatographed and re-tested in a bioassay. Finally, a proliferative activity was purified, identified by sequence analysis and applied into CSDs of adult recipients. RESULTS: The application of POBC derived from prenatal donors resulted in osseous regeneration of a CSD in adult recipients, while the supernatant of postnatal donors had much smaller effects. The morphologic features resembled the spontaneous osseous healing of calvarial defects of the same size in immature organisms. The polypeptide "tissue inhibitor of metalloproteinases type II"(TIMP-2) was isolated from the supernatant of cultures of POBC derived from prenatal donors by measuring the induction of their proliferation. Additionally, the application of human TIMP-2 injected into calvarial CSDs of adult organisms resulted in osseous healing. CONCLUSION: We conclude that one component responsible for the healing effect of CSDs of POBC supernatants derived from prenatal donors is TIMP-2.
Assuntos
Osso e Ossos/metabolismo , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Envelhecimento , Sequência de Aminoácidos , Animais , Proliferação de Células , Células Cultivadas , Cromatografia/métodos , Modelos Biológicos , Dados de Sequência Molecular , Osteoblastos/metabolismo , Peptídeos/química , Ratos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A set of adenosine 3':5'-monophosphate (cAMP) analogues that combine exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP were tested for their effect on the growth of HL-60 human promyelocytic leukemia cells and LS-174T human colon carcinoma cells. Both diasteromeres of the phosphorothioate derivatives were growth inhibitory, exhibiting a concentration inhibiting 50% of cell proliferation of 3-100 microM. Among the analogues tested, Rp-8-Cl-cAMPS and Sp-8-Br-cAMPS were the two most potent. Rp-8-Cl-cAMPS was 5- to 10-fold less potent than 8-Cl-cAMP while Sp-8-Br-cAMPS was approximately 6-fold more potent than 8-Br-cAMP. The growth inhibition was not due to a block in a specific phase of the cell cycle or due to cytotoxicity. Rp-8-Cl-cAMPS enhanced its growth-inhibitory effect when added together with 8-Cl-cAMP and increased differentiation in combination with N6-benzyl-cAMP. The binding kinetics data showed that these Sp and Rp modifications brought about a greater decrease in affinity for Site B than for Site A of RI (the regulatory subunit of type I cAMP-dependent protein kinase) and a substantial decrease of affinity for Site A of RII (the regulatory subunit of type II protein kinase) but only a small decrease in affinity for Site B of RII, indicating the importance of the Site B binding of RII in the growth-inhibitory effect. These results show that the phosphorothioate analogues of cAMP are useful tools to investigate the mechanism of action of cAMP in growth control and differentiation and may have practical implication in the suppression of malignancy.
Assuntos
AMP Cíclico/análogos & derivados , Receptores de AMP Cíclico/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Isomerismo , Leucemia Promielocítica Aguda/imunologia , Leucemia Promielocítica Aguda/patologia , Proteínas Quinases/biossíntese , Células Tumorais CultivadasRESUMO
Cyclic AMP (cAMP) and cyclic GMP (cGMP) are key second messengers involved in a multitude of cellular events. From the wealth of synthetic analogs of cAMP and cGMP, only a few have been explored with regard to their therapeutic potential. Some of the first-generation cyclic nucleotide analogs were promising enough to be tested as drugs, for instance N(6),O(2)'-dibutyryl-cAMP and 8-chloro-cAMP (currently in clinical Phase II trials as an anticancer agent). Moreover, 8-bromo and dibutyryl analogs of cAMP and cGMP have become standard tools for investigations of biochemical and physiological signal transduction pathways. The discovery of the Rp-diastereomers of adenosine 3',5'-cyclic monophosphorothioate and guanosine 3',5'-cyclic monophosphorothioate as competitive inhibitors of cAMP- and cGMP-dependent protein kinases, as well as subsequent development of related analogs, has proven very useful for studying the molecular basis of signal transduction. These analogs exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity. Furthermore, better understanding of signaling pathways and ligand/protein interactions has led to new therapeutic strategies. For instance, Rp-8-bromo-adenosine 3',5'-cyclic monophosphorothioate is employed against diseases of the immune system. This review will focus mainly on recent developments in cyclic nucleotide-related biochemical and pharmacological research, but also highlights some historical findings in the field.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Receptores de AMP Cíclico/fisiologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Asma/tratamento farmacológico , Ensaios Clínicos como Assunto , Proteínas Quinases Dependentes de AMP Cíclico/farmacocinética , Proteínas Quinases Dependentes de AMP Cíclico/uso terapêutico , GMP Cíclico/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/farmacocinética , Proteínas Quinases Dependentes de GMP Cíclico/uso terapêutico , Humanos , Hipertensão/tratamento farmacológico , Neoplasias/tratamento farmacológico , Transdução de SinaisRESUMO
BACKGROUND: Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS: RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS: GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION: GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.
Assuntos
Guanilato Ciclase/fisiologia , Túbulos Renais Coletores/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Animais , Fator Natriurético Atrial/farmacologia , Membrana Celular/enzimologia , Diuréticos/farmacologia , Feminino , Expressão Gênica , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Túbulos Renais Coletores/enzimologia , Masculino , Fragmentos de Peptídeos/farmacologia , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Membrane permeable derivatives of cAMP are widely used to investigate the role of cAMP in the regulation of cell growth and differentiation. To further investigate the molecular mechanisms, underlying the effects of cAMP analogues on growth control and differentiation, the concentration-dependent action of four structurally related cAMP analogues with substitutions at the N6-position in the adenine moiety, namely N6-benzyl-cAMP (Bn-cAMP), N6-benzoyl-cAMP (Bz-cAMP), N6-butyryl-cAMP (Bt-cAMP) and N6, O2'-cAMP (Bt2-cAMP), on C6 rat glioma cell proliferation was determined. The four analogues tested showed different specificities, and the order of growth inhibitory potency was: Bn-cAMP >> Bt-cAMP = Bt2-cAMP >> Bz-cAMP. Thus, although both derivatives have been described to equally bind and activate cAMP-dependent protein kinase (cAK) isozymes, Bn-cAMP most effectively inhibited C6 glioma cell proliferation with an IC50 of 25 microM, while Bz-cAMP was almost ineffective in C6 cells (IC50 >> 1000 microM). In vivo and in vitro studies using HPLC analysis, revealed that Bn-cAMP was subject to enzymatic degradation and that the metabolite Bn-adenosine (Bn-Ado) exerted growth inhibitory effects at a concentration even below 10 microM. Additionally, C6 glioma cells morphologically differentiated in the presence of Bn-cAMP (100 microM) and of Bn-Ado (10 microM), by extending long cellular processes. The growth inhibitory activity of Bn-Ado was not influenced, when dipyridamole, an inhibitor of adenosine uptake, was added to the incubation medium, indicating that adenosine action was mediated through a receptor-mediated mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
AMP Cíclico/análogos & derivados , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Linhagem Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , DNA/análise , Glioma/metabolismo , Diester Fosfórico Hidrolases , Proteínas/análise , Ratos , Células Tumorais Cultivadas/metabolismoRESUMO
Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogs and ligands that elevate cellular cAMP levels. A marker of this differentiation process is the activation of the decidual PRL (dPRL) promoter. In a primary ES cell culture system we show that relaxin not only acutely but permanently elevates cellular cAMP levels and leads to induction of PRL secretion after 6 days Northern and Western blot analyses revealed that all regulatory subunit isoforms (RI alpha, RI beta, RII alpha, and RII beta) and catalytic subunits C alpha and C beta of protein kinase A (PKA) are expressed in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization but in decidualized ES cells, exposed to relaxin for more than 6 days a significant reduction of RI alpha protein level occurs, whereas levels of all other forms remain unchanged. Reduction of R subunits might result in a net increase in free C subunit activity. This alteration is not due to a change in the mitotic state of the cells, as proliferating cell nuclear antigen is evenly expressed in undifferentiated and differentiated ES cell cultures. In transient transfections of undifferentiated ES cells, the dPRL promoter is activated by 8-bromo-cAMP and the C subunit (C beta) of PKA. This induction as well as the differentiation-dependent activity of the dPRL promoter in transfected decidualized cells are effectively abolished by the coexpression of protein kinase inhibitor. We demonstrate that 332 bp of the dPRL promoter are sufficient to mediate full inducibility by cAMP. Activation of the dPRL promoter by cAMP in ES cells occurs in two steps: an initial weak induction within 12 h and a subsequent, much more pronounced induction after 12 h. The secondary induction is not seen with a control construct driven by a consensus cAMP response element (CRE) linked to a minimal promoter and is absent from a uterine cell line that does not express the endogenous dPRL gene. The early response of the dPRL promoter depends upon a noncanonical CRE at position -12, as mutation of this sequence leads to abolition of the early, but not the delayed, induction. The major activation depends upon a different region within 332 bp of the dPRL promoter; is probably indirect, as it follows different kinetics compared to a classical CRE-mediated response; and is specific to ES cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Decídua/metabolismo , Endométrio/fisiologia , Genes , Prolactina/genética , Transcrição Gênica , Diferenciação Celular , Células Cultivadas , AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endométrio/citologia , Ativação Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Prolactina/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Relaxina/farmacologia , Células Estromais/metabolismo , Células Estromais/fisiologiaRESUMO
Human mammary carcinoma cells (MCF-7) were arrested in late G1-phase after treatment with agents (forskolin, interleukin-1 beta 3-isobutyl-1-methylxanthine) that increased the endogenous concentrations of cAMP. The effect of elevated cAMP was mimicked by microinjected catalytic (C alpha) cAMP-dependent protein kinase (cAK) subunit and reversed by the injection of a dominant negative cAK regulatory mutant (RID199). Further evidence that activation of cAK induced growth arrest was provided by the use of pairs of stable cAMP analogs known to synergistically activate isolated cAK isozymes. Furthermore, the effect of cAMP was not potentiated by serine/threonine phosphatase inhibitors that profoundly restricted MCF-7 growth. Some 8-substituted cAMP analogs, e.g. 8-Cl-cAMP and 8-NH2-cAMP, induced cell death rather than reversible inhibition of growth. Their effect was not synergized with complementary cAMP analogs. Furthermore, their potency was decreased rather than increased in the presence of an inhibitor of degradation (3-isobutyl-1-methylxanthine). Finally, their effect could be mimicked by degradation products unable to activate cAK. We concluded that 8-Cl-cAMP (and 8-NH2-cAMP) induced irreversible growth arrest by a mechanism not involving cAK, whereas activation of cAK resulted in a transient and fully reversible inhibition of cell proliferation.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Neoplasias da Mama/tratamento farmacológico , AMP Cíclico/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA de Neoplasias/biossíntese , Feminino , Fase G1/efeitos dos fármacos , Humanos , Conformação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fase S/efeitos dos fármacos , Timidina/metabolismo , Células Tumorais CultivadasRESUMO
Superoxide anion (O2-.) production from human neutrophils stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 microM) was inhibited by preparations of the inhibitor of cAMP-dependent protein kinase, Rp-cyclic adenosine 3',5'-phosphorothioate (Rp-cAMPS, 100 microM). This effect of Rp-cAMPS was reversed by xanthine amine congener (0.1 microM), an adenosine receptor antagonist, and by low concentrations of adenosine desaminase (0.02 mg/ml). HPLC analysis shows that these preparations of Rp-cAMPS contained concentrations of adenosine which could produce significant inhibition of fMLP-induced O2-. production. These results suggest that Rp-cAMPS should be used with caution in cells or tissues containing adenosine receptors, and that preparations of Rp-cAMPS should be treated with adenosine desaminase before use to avoid activation of adenosine receptors.
Assuntos
Adenosina/análise , AMP Cíclico/análogos & derivados , Tionucleotídeos/análise , Adenosina Desaminase/farmacologia , Cromatografia Líquida de Alta Pressão , AMP Cíclico/análise , AMP Cíclico/farmacologia , Feminino , Humanos , Masculino , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Inibidores de Proteínas Quinases , Superóxidos/sangue , Tionucleotídeos/farmacologiaRESUMO
The mammalian period (Per) genes are components of the circadian clock and appear to be regulated via an autoregulatory feedback loop. Here we show that the human PER1 (hPER1) gene is synergistically activated by protein kinases A and C (PKA, PKC) and cAMP responsive element binding protein. Activators and inhibitors of PKA as well as PKC modulate endogenous hPER1 expression and hPER1 promoter-driven reporter gene activity in a dose-dependent manner. Our results suggest that the hPER1 promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters. This regulation of hPER1 appears to be significant for rapid adaptation to changing environmental conditions.
Assuntos
AMP Cíclico/análogos & derivados , Diclororribofuranosilbenzimidazol/análogos & derivados , Regulação da Expressão Gênica/genética , Genes Reguladores/genética , Proteínas Nucleares/genética , Transdução de Sinais/genética , Proteínas de Ciclo Celular , Linhagem Celular , Ritmo Circadiano/genética , Colforsina/farmacologia , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Diclororribofuranosilbenzimidazol/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reguladores/efeitos dos fármacos , Genes Reporter , Humanos , Proteínas Circadianas Period , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C/farmacologia , RNA Mensageiro/metabolismo , Tionucleotídeos/farmacologia , TransfecçãoRESUMO
Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording dose-response curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to beta-adrenergic stimuli inducing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin. Melatonin concentrations were determined by ELISA. cPKA, phosphorylated CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dose-response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose-response curves from immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quantification and localization of biomolecules, and may even be more sensitive than immunoblotting.
Assuntos
Imuno-Histoquímica/métodos , Glândula Pineal/metabolismo , Proteínas Repressoras , Transdução de Sinais , Animais , Células Cultivadas , AMP Cíclico/antagonistas & inibidores , Modulador de Elemento de Resposta do AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Processamento de Imagem Assistida por Computador , Immunoblotting , Masculino , Melatonina/biossíntese , Fosforilação , Ratos , Ratos Wistar , Reprodutibilidade dos TestesRESUMO
The presence of soluble guanylate cyclase in the pineal and its regulation by adrenergic pathways has been well documented. Recent evidence points to adrenergically stimulated nitric oxide generation as a mechanism for coupling this pathway. To what extent nitric oxide (NO) signalling can influence adrenergically stimulated melatonin synthesis has not been investigated. Cyclic guanosine 3',5'-monophospate (cGMP) signal transduction in the bovine pineal has also received little attention. We describe in the present report: 1) a dose-dependent elevation of cGMP in response to the nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1), 2) a dose-dependent inhibition of melatonin synthesis by SNP and SIN-1, but not by 8-Br-cGMP in both bovine and rat pineal cell cultures, which is not due to cytotoxicity as judged by two different approaches, and 3) immunohistochemical evidence for the presence of nitric oxide synthase (NOS) (EC 1.14.23.-) in the intact bovine pineal gland and in cultured bovine pinealocytes. These data support the view that NOS is a component of the cGMP-generating system in mammalian pinealocytes. Although NO-donor molecules are also potent activators of cGMP accumulation, they may have other important actions in the pineal, namely the inhibition of adrenergic-stimulated melatonin synthesis. As SNP and SIN-1 exerted this inhibitory effect on cells regardless of whether they were stimulated by isoproterenol, forskolin or 8-Br-cAMP it would appear that NO-donors can act 'downstream' from the receptor/adenylate cyclase level.
Assuntos
GMP Cíclico/fisiologia , Melatonina/biossíntese , Molsidomina/análogos & derivados , Óxido Nítrico/metabolismo , Nitroprussiato/farmacologia , Glândula Pineal/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , GMP Cíclico/análogos & derivados , Isoproterenol/farmacologia , Molsidomina/farmacologia , Glândula Pineal/citologia , Ratos , Estimulação QuímicaRESUMO
Here we describe a novel function for members of the well-characterized human natriuretic peptide family. Human "brain-type natriuretic peptide" (hBNP-32) as well as other members of this peptide class are antimicrobially active against Gram-positive and Gram-negative bacteria and yeast in a dose-dependent manner. This activity of natriuretic peptides is comparable to that of known antimicrobial peptides such as casocidins or magainins.
Assuntos
Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Peptídeo Natriurético Encefálico/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Animais , Antibacterianos , Fator Natriurético Atrial/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/farmacologiaRESUMO
The rodent pineal organ transduces a photoneural input into a hormonal output. This photoneuroendocrine transduction leads to highly elevated levels of the hormone melatonin at night-time which serves as a message for darkness. The melatonin rhythm depends on transcriptional, translational and posttranslational regulation of the arylalkylamine-N-acetyltransferase, the key enzyme of melatonin biosynthesis. These regulatory mechanisms are fundamentally linked to two second messenger systems, namely the cAMP- and the Ca(2+)-signal transduction pathways. Our data gained by molecular biology, immunohistochemistry and single-cell imaging demonstrate a time- and substance-specific activation of these signaling pathways and provide a framework for the understanding of the complex signal transduction cascades in the rodent pineal gland which in concert not only regulate the basic profile but also fine-tune the circadian rhythm in melatonin synthesis.
Assuntos
Membrana Celular/fisiologia , Núcleo Celular/fisiologia , Melatonina/biossíntese , Glândula Pineal/fisiologia , Proteínas Repressoras , Transdução de Sinais , Animais , Arilamina N-Acetiltransferase/genética , Cálcio/fisiologia , AMP Cíclico/fisiologia , Modulador de Elemento de Resposta do AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/genética , Sistemas do Segundo Mensageiro , Transcrição GênicaRESUMO
We investigated whether vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), which stimulate melatonin biosynthesis in the mammalian pineal organ, cause phosphorylation of the cyclic AMP response element binding protein (CREB) in rat pinealocytes. Dispersed cells were treated with varying concentrations of VIP and PACAP for 10 to up to 240 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (pCREB). The experiments showed a dose- and time-dependent induction of pCREB immunoreactivity in the nuclei of subpopulations of pinealocytes identified by the S-antigen immunoreaction. Stimulation with VIP elicited pCREB immunoreaction in approximately 50-65% of the S-antigen immunoreactive pinealocytes. The number of PACAP-responsive pinealocytes was often smaller and more variable. Maximal responses to both neuropeptides were seen after 30 min. pCREB immunoreaction gradually declined within 2 h and could not be induced again by an additional stimulation. In contrast, norepinephrine (NE) elicited pCREB immunoreaction in more than 95% of the pinealocytes, and this response lasted as long as 300 min. Treatment of pinealocytes with forskolin or KCl induced pCREB immunoreaction in the vast majority of pinealocytes, showing that in principle elevation of the intracellular concentrations of both cAMP and calcium can cause the response. Immunoblotting analyses confirmed that the immunoreaction elicited by VIP, PACAP and NE is largely due to phosphorylation of a 42-kDa protein corresponding to CREB, but reflects to a minor extent also phosphorylation of two smaller proteins presumably related to ATF-1. Immunocytochemical and immunochemical investigations with an antibody against total CREB showed that stimulation with VIP, PACAP and NE did not affect the level of CREB. All findings indicate that the stimulatory effects of VIP and PACAP on rat pinealocytes involve phosphorylation of transcription factors of the CREB family as holds also true for NE. However, VIP and PACAP affected only subpopulations of pinealocytes and the reponses lasted for a shorter period of time than those to NE. This conforms to previous results showing that both neuropeptides are also less effective than NE in stimulating the melatonin biosynthesis in the rat pineal organ.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Glândula Pineal/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Células Cultivadas , Immunoblotting , Técnicas Imunoenzimáticas , Masculino , Norepinefrina/farmacologia , Fosforilação , Glândula Pineal/citologia , Glândula Pineal/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Ratos WistarRESUMO
Signal transduction processes regulating melatonin production in the light-sensitive trout pineal organ were investigated by immunocytochemical and immunochemical demonstration of phosphorylated cyclic AMP-responsive element-binding protein (pCREB) and measurements of cyclic AMP, melatonin, and calcium levels. Melatonin levels were tightly controlled by light and darkness. Elevation of cyclic AMP levels by 8-bromo-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine increased the levels of pCREB and melatonin in light- or dark-adapted pineal organs in vitro. Without pharmacological treatment, the levels of pCREB and cyclic AMP remained constant for several hours before and after light onset. Inhibition of cyclic AMP-dependent proteasomal proteolysis by lactacystin, MG 132, and calpain inhibitor I did not prevent the rapid, light-induced suppression of melatonin biosynthesis. However, changes in the intracellular calcium concentration by drugs affecting voltage-gated calcium channels of the L type and intracellular calcium oscillations (cobalt chloride, nifedipine, Bay K 8644) had dramatic effects on the rapid, light-dependent changes in melatonin levels. These effects were not accompanied by changes in cyclic AMP levels. Thus, the rapid, light-dependent changes in melatonin levels in the trout pineal organ are regulated apparently by a novel calcium signaling pathway and do not involve changes in cyclic AMP levels, cyclic AMP-dependent proteasomal proteolysis, or phosphorylation of cyclic AMP-responsive element-binding protein.
Assuntos
Sinalização do Cálcio/fisiologia , Melatonina/genética , Oncorhynchus mykiss/fisiologia , Glândula Pineal/fisiologia , Transdução de Sinais/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Arilamina N-Acetiltransferase/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Cisteína Endopeptidases/metabolismo , Feminino , Expressão Gênica/fisiologia , Imuno-Histoquímica , Masculino , Complexos Multienzimáticos/metabolismo , Técnicas de Cultura de Órgãos , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Estimulação Luminosa , Glândula Pineal/química , Glândula Pineal/citologia , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
We have characterized a new polyclonal antibody against heavy chain (H) of neurofilament which can be used to demonstrate neurofilament H in normal brain tissue and oligodendroglioma cells immunocytochemically and immunochemically. Using this antibody we found neurofilament H-immunoreactive tumor cells in 13 oligodendrogliomas (6 WHO grade II, 7 WHO grade III) out of 84 oligodendrogliomas investigated (59 WHO grade II and 25 WHO grade III). Double immunolabeling and confocal laser scanning microscopy showed colocalization of neurofilament H and glial fibrillary acidic protein in certain oligodendroglioma cells. Colocalization of neurofilament and synaptophysin was observed only rarely. The results support the notion that oligodendrogliomas consists of a heterogeneous cell population displaying various stages of differentiation and dedifferentiation. The occurrence of neurofilament H-immunoreactive tumor cells in oligodendrogliomas is not related to the survival of the patients.
Assuntos
Neoplasias Encefálicas/metabolismo , Proteínas de Neurofilamentos/metabolismo , Oligodendroglioma/metabolismo , Adolescente , Animais , Neoplasias Encefálicas/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Oligodendroglioma/patologia , Ratos , Ratos Wistar , Análise de SobrevidaRESUMO
The subcommissural organ (SCO) is an ependymal brain gland that synthesizes and secretes glycoproteins. Very little is known about the signal transduction cascades operating in this organ and their impact on gene expression. An important transcription factor that regulates gene expression in glial cells and neurons is the cyclic-AMP-responsive element binding protein (CREB), which is activated by phosphorylation of the serine residue 133. Here, we analyzed the presence of CREB in bovine SCO cells and its phosphorylation by drugs that activate cyclic-AMP-dependent or calcium-dependent signal transduction pathways. We also investigated the effects of three natural signaling molecules, serotonin (5HT), substance P (SP) and ATP, on CREB phosphorylation and on the second messengers cyclic AMP and calcium. Investigations were performed with cell and explant cultures by using immunocytochemistry, immunoblot, enzyme-linked immunosorbent assay, and the Fura-2 technique. A strong immunosignal for total (phosphorylated and unphosphorylated) CREB was found in virtually all SCO cells. Total CREB levels did not change upon stimulation. Phosphorylated (p)CREB levels were low in unstimulated cells and significantly elevated by drugs that increase the levels of cyclic AMP or free calcium ions. pCREB was also induced by SP and ATP; both substances increased the intracellular calcium concentration but did not affect the formation of intracellular cyclic AMP. 5HT did not influence the phosphorylation of CREB, the intracellular calcium concentration, or the formation of cyclic AMP. Our data identify CREB as an SCO transcription factor that can be activated by the second messengers cAMP and calcium. SP and ATP stimulate the phosphorylation of CREB apparently via a calcium-dependent mechanism and are thus involved in the control of gene expression in the bovine SCO.