RESUMO
Chronic exposure to stress hormones has an impact on brain structures relevant to cognition. Nicotinic acetylcholine receptors (AChRs) are involved in numerous cognitive processes including learning and memory formation. In order to better understand the molecular mechanisms of chronic stress-triggered mental disease, the effect of corticosterone (CORT) on the biology of AChRs was studied in the neuronal cell line CNh. We found that chronic treatment with CORT reduced the expression levels of the α7-type neuronal AChR and, to a lesser extent, of α4-AChR. CORT also delayed the acquisition of the mature cell phenotype in CNh cells. Chronic nicotine treatment affected the differentiation of CNh cells and exerted a synergistic effect with CORT, suggesting that AChR could participate in signaling pathways that control the cell cycle. Overexpression of α7-AChR-GFP abolished the CORT effects on the cell cycle and the specific α7-AChR inhibitor, methyllycaconitine, mimicked the proliferative action exerted by CORT. Whole-cell voltage-clamp recordings showed a significant decrease in nicotine-evoked currents in CORT-treated cells. Taken together, these observations indicate that AChRs, and the α7-AChR in particular, could act as modulators of the differentiation of CNh cells and that CORT could impair the acquisition of a mature phenotype by affecting the function of this AChR subtype.
Assuntos
Córtex Cerebral/fisiologia , Corticosterona/metabolismo , Neurogênese/fisiologia , Neurônios/fisiologia , Receptores Nicotínicos/metabolismo , Aconitina/análogos & derivados , Aconitina/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Córtex Cerebral/efeitos dos fármacos , Camundongos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Receptores Nicotínicos/genéticaRESUMO
Islet transplantation is a potential cure for type 1 diabetes, but clinical results have been disappointing. Currently, islet isolation is by enzymatic digestion of the pancreas which has significant pitfalls: warm ischemia exposure, collagenase-induced damage to the islet mass and viability, poor reproducibility, high cost, a relatively low number of islets obtained per whole pancreas, and selection of islets for collagenase resistance rather than for glucose responsiveness. In the present study we performed a series of experiments in a porcine model to demonstrate the feasibility of a new isolation method based on selective osmotic shock (SOS) using very high glucose solutions, doubling or tripling physiological osmotic strength. The SOS method can be carried out at room temperature or in the cold eliminating warm ischemia time which damages the islets. The SOS method does not depend on the texture of the pancreas so all pancreases can be processed identically and the process can be fully automated. The SOS method isolates all the islets of the pancreas regardless of size and shape allowing a greater number of islets to be harvested. The SOS method avoids exposure to toxins in collagenase solutions, is inexpensive and selects for islets with high concentrations of Glut 2 transporters, representing the best glucose responding islets. The SOS method showed a comparable recovery of islets from young pig pancreas and the islets showed improved viability. We conclude that the selective osmotic shock (SOS) method of separating islets from the pancreatic tissue is superior to the collagenase method.
Assuntos
Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Animais , Morte Celular , Separação Celular/métodos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/cirurgia , Glucose/farmacologia , Transportador de Glucose Tipo 2/análise , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Insulina/uso terapêutico , Secreção de Insulina , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Pressão Osmótica , SuínosRESUMO
The last therapeutic alternative in severe postsurgical hypoparathyroidism is allotransplantation of microencapsulated parathyroid cells. With this technique, it is possible to implant cells or tissue of parathyroid origin to replace them in such patients, without immusupression. We report an allotransplant of parathyroid tissue in a patient with continous endovenous requirement of calcium to survive. The microencapsulation was carried out with a commercial sodium alginate. We implant 23 microspheres in the nondominant forearm and 40 microspheres in the leg in a second attempt. In this article, we show functionality of the graft for at least 20 months without requirement of endovenous calcium. We report this procedure as a therapeutical alternative in severe hypoparathyroidism.
Assuntos
Hipoparatireoidismo/cirurgia , Glândulas Paratireoides/transplante , Adulto , Criopreservação , Composição de Medicamentos/métodos , Feminino , Bócio/cirurgia , Humanos , Hipocalcemia/etiologia , Complicações Pós-Operatórias/cirurgia , Neoplasias da Glândula Tireoide/cirurgia , Tireoidectomia , Transplante HomólogoRESUMO
Monoamine oxidase-A (MAO-A) [amiflamine (AMF) and 4-methylthioamphetamine (MTA)] and MAO-B (L-deprenyl) inhibitors were found to be cytotoxic in a concentration-dependent manner for RCHT cells derived from adult rat hypothalamus. The cytotoxic effects were increased when the inhibitors were co-incubated with dicoumarol and especially with 25 micro M AMF+100 micro M dicoumarol (2.5-fold; P <0.001). The treatment of RCHT cells solely with AMF induced a marked decrease in the expression of DT-diaphorase mRNA.
RESUMO
The endogenous dopamine-derived neurotoxin salsolinol was found to decrease survival in the dopaminergic neuronal cell line RCSN-3, derived from adult rat substantia nigra in a concentration-dependent manner (208 microM salsolinol induced a 50% survival decrease). Incubation of RCSN-3 cells with 100 micro;M dicoumarol and salsolinol significantly decreased cell survival by 2.5-fold (P < 0.001), contrasting with a negligible effect on RCHT cells, which exhibited nearly a 5-fold lower nomifensine-insensitive dopamine uptake. The levels of catalase and glutathione peroxidase mRNA were decreased when RCSN-3 cells were treated with 100 microM salsolinol alone or in the presence of 100 microM dicoumarol. In vitro oxidation of salsolinol to o-quinone catalyzed by lactoperoxidase gave the quinone methide and 1,2-dihydro-1-methyl-6,7-isoquinoline diol as final products of salsolinol oxidation as determined by NMR analysis. Evidence of the formation of salsolinol o-semiquinone radical has been provided by ESR studies during one-electron oxidation of salsolinol catalyzed by lactoperoxidase.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Dopamina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Indolquinonas , Indóis/farmacologia , Isoquinolinas/farmacologia , Neurônios/efeitos dos fármacos , Quinonas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Catalase/genética , Linhagem Celular , Dicumarol/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Glutationa Peroxidase/genética , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Substância Negra/citologia , Superóxido Dismutase/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu-dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu-dopamine complex mediates the uptake of (64)CuSO(4) into the Raúl Caviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m M) (63%, p < 0.001) and nomifensine (2 microM) (77%, p < 0.001). Copper sulfate (1 m M) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyrroline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO(4) and dicoumarol. A decrease in the expression of CuZn-superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN-3 cells were treated with CuSO(4), dopamine, or CuSO(4) and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO(4), dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO(4) increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu-dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminochrome.
Assuntos
Sulfato de Cobre/toxicidade , Dopamina/farmacologia , Indolquinonas , Indóis/metabolismo , Transporte de Íons/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Substância Negra/citologia , Animais , Catalase/biossíntese , Catalase/genética , Linhagem Celular , Sulfato de Cobre/metabolismo , Sulfato de Cobre/farmacologia , Dicumarol/toxicidade , Espectroscopia de Ressonância de Spin Eletrônica , Indução Enzimática/efeitos dos fármacos , Glutationa Peroxidase/biossíntese , Glutationa Peroxidase/genética , Metalotioneína/metabolismo , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/genética , Neurônios/metabolismo , Nomifensina/farmacologia , Oxirredução , Estresse Oxidativo , Doença de Parkinson/metabolismo , RNA Mensageiro/biossíntese , Ratos , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genéticaRESUMO
We established two immortalized cell lines from cerebral cortex of normal (CNh) and trisomy 16 (CTb) mouse fetuses, an animal model of human trisomy 21. Those cells loaded with the fluorescent Ca2+ dyes, Indo-1 and Fluo-3, exhibited increments of intracellular Ca2+ ([Ca2+]i) in response to external glutamate, NMDA, AMPA and kainate. CTb cells exhibited higher basal Ca2+ concentrations and had higher amplitude and slower time-dependent kinetics in the decay than CNh cells, suggesting an impaired Ca2+ buffering capacity in the trisomy 16-derived cell line. Nicotine also induced increments of [Ca2+]i. The CTb cell line could represent a model for studying cellular alterations related to Down syndrome.
Assuntos
Sinalização do Cálcio/fisiologia , Córtex Cerebral/fisiologia , Cromossomos Humanos Par 16 , Trissomia , Animais , Cálcio/metabolismo , Linhagem Celular Transformada , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/embriologia , Feto/citologia , Ácido Glutâmico/farmacologia , Humanos , Membranas Intracelulares/metabolismo , Ácido Caínico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nicotina/farmacologia , Concentração Osmolar , Receptores de Glutamato/metabolismo , Valores de Referência , Trissomia/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.