RESUMO
In the present study, the expression of the activity of adenosine-3',5'-monophosphate-degrading phosphodiesterases (cAMP-PDEs) was analyzed in rat neocortex homogenates. Following separation by anion-exchange chromatography, the isozymes were characterized by their sensitivity to modulators and by their kinetic properties. We identified the activity of five distinct cAMP-PDE isozymes: two calcium/calmodulin-dependent forms (PDE 1), one PDE 2 isozyme stimulated by guanosine-3',5'-monophosphate (cGMP), one cGMP-inhibited form (PDE 3) and a cAMP-specific, rolipram-sensitive form (PDE 4). Our study provides, for the first time, evidence for the existence of PDE 3 enzyme activity in rat neocortex and predicts the expression of at least two isoforms (splice variants) of PDE 1A in this brain area. The existence of different cAMP-degrading phosphodiesterases modulated by different intracellular second messengers (calcium and cGMP) suggests that the activity of neocortical neurons and glia cells is regulated, inter alia, by a 'crosstalk' between calcium-, cGMP- and cAMP-dependent second messenger pathways.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/isolamento & purificação , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/isolamento & purificação , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Neocórtex/enzimologia , Diester Fosfórico Hidrolases , Fatores Etários , Animais , Resinas de Troca Aniônica , Cálcio/fisiologia , Calmodulina/fisiologia , Cromatografia por Troca Iônica/métodos , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Wistar , RolipramRESUMO
In bovine chromaffin cells forskolin, phorbol ester, or high potassium levels induce a rapid increase of c-fos, c-jun, and junB mRNA levels, which precede an induction of proenkephalin gene expression. Preincubation of the cells with cycloheximide inhibited induction of proenkephalin mRNA levels by each of these agents, indicating that newly synthesized transcription factors are involved. Transient transfection of reporter genes showed that the ENKCRE-2 element of the proenkephalin promoter was sufficient for basal and second messenger-induced expression. Gel mobility shift assays revealed that stimulation increased the binding of nuclear proteins to ENKCRE-2 and AP-1 oligonucleotides but not to CRE oligonucleotides. Western analysis showed that the induction of AP-1 binding activity was associated with Fos protein synthesis. Moreover, cotransfection of c-fos, but not of c-jun or CREB, expression plasmids transactivated the expression of the PENKCAT reporter genes. These results suggest that Fos and/or other components of AP-1 transcription factors, rather than CREB or other preexisting proteins, play a specific role in the induction of the proenkephalin gene in bovine chromaffin cells.
Assuntos
Sistema Cromafim/citologia , Encefalinas/genética , Precursores de Proteínas/genética , Fator de Transcrição AP-1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Bovinos , Células Cultivadas/enzimologia , Células Cultivadas/fisiologia , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , 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/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Genes Precoces/genética , Genes Reporter/fisiologia , Cinética , Dados de Sequência Molecular , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Cloreto de Potássio/farmacologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , Sistemas do Segundo Mensageiro/genética , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/metabolismoRESUMO
The induction of the proenkephalin gene by nicotine has been characterized in bovine adrenal medullary chromaffin cells. Nicotine (10 microM) caused an approximately fourfold increase in the proenkephalin mRNA levels within 24 h. The half-life of the proenkephalin mRNA in nicotine-stimulated cells was similar to that in control cells (about 13 h), indicating that nicotine does not affect mRNA stability but acts at the levels of proenkephalin gene transcription. This was also supported by experiments showing that the expression of a proenkephalin chloramphenicol acetyl transferase reporter gene (PENKCAT-153/+50) containing 153 nucleotides of upstream promoter sequences is increased (about twofold) by nicotine after transient transfection in the chromaffin cells. In addition, nicotine induced a marked elevation of the immediate early gene mRNAs c-fos, c-jun, and jun-B. Maximally increased levels for c-fos mRNA (about 100-fold) were obtained after 20 min. c-jun and jun-B were increased three- to fivefold 60 min after nicotine addition. The expression of PENKCAT-153/+53 and of a proenkephalin gene reporter plasmid which contains a dimer of the enkephalin cAMP responsive element 2 (ENKCRE-2) in front of a minimal promoter was increased by cotransfection of a c-fos expression plasmid, indicating that nicotine may induce the proenkephalin gene in chromaffin cells via c-Fos which binds to the ENKCRE-2 element.
Assuntos
Medula Suprarrenal/efeitos dos fármacos , Encefalinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Genes jun , Nicotina/farmacologia , Precursores de Proteínas/genética , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Animais , Sequência de Bases , Bovinos , Células Cultivadas , Dados de Sequência Molecular , Fator de Transcrição AP-1/fisiologia , Transcrição GênicaRESUMO
A retrospective study of 165 Jehovah's Witnesses and 164 Jehovah's Witnesses and 164 control patients compared the morbidity and mortality associated with major obstetric and gynecologic surgery in the 2 groups. There were no deaths and few complications in either group. There were few differences in preoperative and postoperative hemoglobin values. Medicolegal implications of performing major surgery without blood transfusions are discussed. The study adds evidence that major operative procedures can be carried out on Jehovah's Witness patients without blood transfusions or blood products.
