RESUMO
Methods for producing protein patterns with defined spatial arrangement and micro- and nanoscale features are important for studying cellular-level interactions, including basic cell-cell communications, cell signaling, and mechanisms of drug action. Toward this end, a straightforward, versatile procedure for fabricating micropatterns of bioactive nanofilm coatings as multifunctional biological testbeds is demonstrated. The method, based on a combination of photolithography and layer-by-layer self-assembly (LbL), allows for precise construction of nanocomposite films of potentially complex architecture, and patterning of these films on substrates using a modified lift-off (LO) procedure. As a first step in evaluating nanostructures made with this process, "comparison chips," comprising two coexisting regions of square patterns with relevant proteins/polypeptides on a single substrate, were fabricated with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Using neuronal cells as a model biological system, comparison chips were produced with secreted phospholipase A2 (sPLA2), a known membrane-active enzyme for neurons, for direct comparison with gelatin, poly-l-lysine (PLL), or bovine serum albumin (BSA). Fluorescence microscopy, surface profilometry, and atomic force microscopy techniques were used to evaluate the structural properties of the patterns on these chips and show that the patterning technique was successful. Preliminary cell culture studies show that neurons respond and bind specifically to the sPLA2 enzyme embedded in the polyelectrolyte thin films and present as the outermost layer. These findings point to the potential for this method to be applied in developing test substrates for a broad array of studies aimed at identifying important biological structure-function relationships.
Assuntos
Engenharia Biomédica/métodos , Nanotecnologia/métodos , Neurônios/química , Neurônios/citologia , Animais , Células Cultivadas , RatosRESUMO
OBJECTIVES: An epidemiologic study of urinary calculi (N = 1843) was conducted in Western France: distribution according to the main chemical compounds, age and sex. Comparison with the results of a study with national recruitment (N = 10,617) and a study with regional recruitment (N = 1774). METHOD: The study involved 1843 stones characterized beforehand by morphological analysis associated with infra-red spectrophotometry (FTIR). If analysis of the composition of the stones was carried out on the totality of calculi, studies related to age and sex included only 1583 cases. Comparison of percentages was made using chi 2 test. RESULTS: The composition in main compounds of calculi was comparable with the results of other studies; minor significant compounds presented great differences, raising the problem of interpretation of the infra-red spectra of the latter. Hence, our work was directed towards the analysis of the major compounds and we showed, like most authors, that monohydrate calcium oxalate is predominant in male (46%) as well as in females (37%). Calculi average sex-ratio was 2.19 but dehydrated calcium oxalate sex-ratio was 4.42, suggesting that this compound is found mainly in men. Conversely, for the majority of phosphate stones, the sex-ratio was lower or equal to one, indicating that they predominate in women. Infectious calculi (particularly struvite calculi) appeared slightly more frequent in our population than in other studies, whereas the number of uric acid calculi was lower. This, however, remains to be confirmed. CONCLUSION: The population studied was not significantly different from the national population regarding lithiasis, except perhaps for uric acid and struvite calculi, despite specific regional differences in diet and the role of nutritional factors in lithogenesis.
Assuntos
Cálculos Urinários/química , Cálculos Urinários/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Oxalato de Cálcio/análise , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Feminino , França/epidemiologia , Humanos , Lactente , Recém-Nascido , Compostos de Magnésio/análise , Masculino , Pessoa de Meia-Idade , Fosfatos/análise , Fatores Sexuais , Espectrofotometria Infravermelho , Estruvita , Ácido Úrico/análiseRESUMO
By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.
Assuntos
Receptores Nicotínicos/metabolismo , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Acetilcolina/farmacologia , Alanina/química , Animais , Encéfalo/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Citoplasma/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Oócitos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Serina/química , Transfecção , Técnicas do Sistema de Duplo-Híbrido , XenopusRESUMO
The aim of this study was to assess whether the quality of the surgical act could be an important prognostic factor for patients undergoing radical prostatectomy. This study also aims to investigate whether the surgical quality can be assessed by any means. Questionnaires were collected from 23 different institutes including 232 radical prostatectomies (RPr) performed for T1T2 prostate cancer. Blood loss, duration of surgery, margin status, postoperative prostate specific antigen (PSA) and urinary incontinence were analysed and correlated with the yearly number of RPr performed. The mean values obtained for each parameter were very different in the various centres. The outcome in terms of tumour control and incontinence could not be related to a higher or lower number of RPr performed. Quality control of RPr is feasible on the basis of an analysis of a few parameters, such as surgical margins, postoperative PSA and incontinence, that might recognise urologists that perform better or poorer than a proposed average.
Assuntos
Prostatectomia/normas , Neoplasias da Próstata/cirurgia , Controle de Qualidade , Estudos de Viabilidade , Humanos , Masculino , Estadiamento de Neoplasias/métodos , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/radioterapia , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Interleukin-1 beta (IL-1beta) is an inflammatory cytokine whose expression is elevated in brain during seizures, ischemia, and injury. Expression of IL-1beta and its receptor can also be observed in normal brain. Platelet-activating factor (PAF) is also a dual mediator that promotes neuronal plasticity responses as well as inflammation. We have determined the role of PAF in the regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes by IL-1beta in rat primary hippocampal cultures. As assessed by reverse transcriptase/polymerase chain reaction (RT/PCR), recombinant mouse IL-1beta (1 nM) led to an induction of COX-2 mRNA which peaked at 2 hours, declined to baseline levels by 4 hours, began to rise again by 6 hours, and remained elevated at 24 hours post-treatment. iNOS mRNA was also induced, but unlike COX-2, its abundance peaked at 4 hours and decreased by 6 hours to a plateau lasting through 24 hours. Pretreatment with PAF antagonist BN50730 blocked induction of COX-2 mRNA by 2-hour IL-1beta treatment, and 2-hour treatment with the PAF analog mcPAF mimicked the effects of IL-1beta on COX-2 mRNA levels. Following injury, synaptic plasticity changes may be affected by IL-1beta-PAF-COX-2 neuronal signaling.
Assuntos
Hipocampo/enzimologia , Interleucina-1/fisiologia , Isoenzimas/biossíntese , Neurônios/enzimologia , Óxido Nítrico Sintase/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Azepinas/farmacologia , Ciclo-Oxigenase 2 , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II , Fator de Ativação de Plaquetas/análogos & derivados , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Proteínas Recombinantes/farmacologia , Tienopiridinas , Triazóis/farmacologiaRESUMO
Tissue polypeptide antigen (TPA) is a serological tumor marker, measuring cytokeratin 8, 18 and 19, used in the follow-up of non squamous epithelium- and derived neoplasms. It has been demonstrated that TPA is reliable in the monitoring of the efficacy of a curative or palliative treatment of bladder cancer. Recently, a monoclonal antibody-based assay for TPA (TPA-M) has been developed, which seems to be equivalent to the polyclonal-based assay. The aim of the study was to assess the superiority of the monoclonal to the polyclonal test in patients with bladder carcinoma. The value of tissue polypeptide antigen was therefore measured both with TPA and TPA-M IRMA. A correlation coefficient of 0.96 was obtained. Precision testing showed a lower overall variability of TPA-M. Since both tests correlate well and TPA-M testing is more precise, faster and easy to perform, we conclude a superiority of TPA-M and advise the monoclonal test as best suited for clinical use in the follow-up of bladder cancer patients with poorly differentiated superficial, locally advanced or systemic disease after curative or palliative therapy.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células de Transição/diagnóstico , Queratinas/análise , Antígeno Polipeptídico Tecidual/sangue , Neoplasias da Bexiga Urinária/diagnóstico , Anticorpos , Anticorpos Monoclonais , Carcinoma de Células de Transição/sangue , Carcinoma de Células de Transição/terapia , Feminino , Seguimentos , Humanos , Ensaio Imunorradiométrico/métodos , Masculino , Análise de Regressão , Reprodutibilidade dos Testes , Neoplasias da Bexiga Urinária/sangue , Neoplasias da Bexiga Urinária/terapiaRESUMO
Pigment epithelial-derived factor (PEDF) has been shown to be a survival factor for cerebellar granule neurons. Here we investigated the ability of PEDF to enhance the survival of hippocampal neurons in culture, and to protect these neurons against acute glutamate toxicity. Hippocampal neurons prepared from 1- to 3-day postnatal rat brain were cultured for either 7 or 14 days in vitro (DIV). At 14 DIV, neurons were only slightly protected (13% +/- 4%) against 50 microM glutamate toxicity when treated with 1 microg/ml of PEDF for 3 successive days before glutamate exposure as measured by lactate dehydrogenase (LDH) release. In comparison, basic fibroblast growth factor (bFGF) at 10 ng/ml for the same treatment period protected 58% +/- 8% of the neurons against glutamate. Using quantitative image analysis of digitized micrographs, we found that the average size of neurons in young, developing hippocampal cultures (7 DIV), was greatly decreased by treatment with 50 microM glutamate. Treatment for up to 5 successive days with 1 microg/ml of PEDF before glutamate addition dramatically increased the average hippocampal neuron soma size, compared to cells treated with glutamate alone. Thus, PEDF may promote the growth of hippocampal neurons, and, if added to developing hippocampal neurons, can also protect these cells from subsequent injury, such as the excitotoxicity of glutamate.
Assuntos
Proteínas do Olho , Ácido Glutâmico/toxicidade , Hipocampo/citologia , Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Fármacos Neuroprotetores/farmacologia , Proteínas/farmacologia , Serpinas/farmacologia , Animais , Animais Recém-Nascidos , Bovinos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/farmacologia , L-Lactato Desidrogenase , Neurônios/efeitos dos fármacos , Ratos , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
Glutamate receptors modulate multiple signaling pathways, several of which involve mitogen-activated protein (MAP) kinases, with subsequent physiological or pathological consequences. Here we report that stimulation of the N-methyl-D-aspartate (NMDA) receptor, using platelet-activating factor (PAF) as a messenger, activates MAP kinases, including c-Jun NH2-terminal kinase, p38, and extracellular signal-regulated kinase, in primary cultures of hippocampal neurons. Activation of the metabotropic glutamate receptor (mGluR) blocks this NMDA-signaling through PAF and MAP kinases, and the resultant cell death. Recombinant PAF-acetylhydrolase degrades PAF generated by NMDA-receptor activation; the hetrazepine BN50730 (an intracellular PAF receptor antagonist) also inhibits both NMDA-stimulated MAP kinases and neuronal cell death. The finding that the NMDA receptor-PAF-MAP kinase signaling pathway is attenuated by mGluR activation highlights the exquisite interplay between glutamate receptors in the decision making process between neuronal survival and death.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hipocampo/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Azepinas/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Morte Celular , Hipocampo/patologia , Neurônios/metabolismo , Neurônios/patologia , Fosfolipases A/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Proteínas Recombinantes/farmacologia , Sistemas do Segundo Mensageiro , Transdução de Sinais/efeitos dos fármacos , Tienopiridinas , Triazóis/farmacologiaRESUMO
The bioactive lipid platelet-activating factor (PAF) accumulates in brain during injury, seizures and ischemia and may, in addition, be significant in AIDS dementia and in other neurodegenerative diseases. We have used plasma-type recombinant PAF acetylhydrolase (rPAF-AH) to test the hypothesis that PAF accumulation is involved in early events leading to neuronal apoptosis during excitotoxic neuronal injury. Neuronal cultures were labeled with FITC-12-dUTP (TUNEL technique) and propidium iodide, digitized using fluorescence microscopy and a chilled 3CCD color camera, and analyzed with 2D graphics analysis software. N-methyl-D-aspartate (NMDA) (50 microM, 2 hr) induced a 2.5-fold increase in apoptosis of hippocampal neurons compared with controls when analyzed 24 hr after NMDA treatment. Hippocampal neurons receiving rPAF-AH (20 microg/ml) before, during, and after NMDA treatment demonstrated a concentration-dependent neuroprotective effect which resulted in 47% and 30% neuroprotection against 50 and 100 microM NMDA, respectively. The noncompetitive NMDA receptor antagonist MK-801(300 nM) completely inhibited apoptosis caused by NMDA. The neuroprotective effect of rPAF-AH against NMDA-induced apoptosis was confirmed using as additional criteria, histone release, electron microscopy, and DNA laddering. Neuroprotection elicited by rPAF-AH demonstrates that PAF is an injury mediator in NMDA-induced neuronal apoptosis and that the recombinant protein is potentially useful as a therapeutic approach.
Assuntos
Apoptose/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/fisiologia , N-Metilaspartato/farmacologia , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacologia , Fosfolipases A/farmacologia , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Maleato de Dizocilpina/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Concentração Osmolar , Ratos , Proteínas RecombinantesRESUMO
Excitatory amino acids transduce physiological and pathological signals to neurons. Similarly, the neuroactive lipid platelet-activating factor (PAF) has been implicated in modulating long-term potentiation and neuronal survival. Excitatory amino acids and PAF have been shown to increase mitogen-activated protein (MAP) kinases in different cell types. Here, we have investigated the similarities and differences between PAF and kainate in activating MAP kinases in primary hippocampal neurons in vitro. Extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 kinases were activated by kainate or PAF in hippocampal neurons. This activation was blocked by the receptor antagonists CNQX and BN 50730 for kainate and PAF, respectively. The PAF receptor antagonist BN 50730 also blocked kainate activation. CNQX had no effect on PAF activation of the kinases, indicating that PAF is downstream of kainate activation. Coapplication of submaximal concentrations of PAF and kainate resulted in a less than additive activation, suggesting similar routes of activation by the two agonists. Both CNQX and BN 50730 blocked kainate-induced neurotoxicity. These results indicate that PAF and kainate activate similar kinase pathways. Therefore, PAF acts downstream of the kainate subtype of glutamate receptors, and when excessive receptor activation takes place, this bioactive lipid may contribute to neuronal cell death.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Ácido Caínico/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Neurônios/enzimologia , Fator de Ativação de Plaquetas/farmacologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Azepinas/farmacologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , MAP Quinase Quinase 4 , Proteína Quinase 3 Ativada por Mitógeno , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Quinases/metabolismo , Ratos , Tienopiridinas , Triazóis/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
PURPOSE: To investigate the effect of the inflammatory mediator platelet-activating factor (PAF) in the induction of the inducible prostaglandin H synthase-cyclooxygenase-2 (COX-2) gene expression in corneal epithelium. METHODS: Rabbit corneas were incubated in organ culture with or without carbamyl PAF (cPAF, 100 nM). The effects of PAF antagonist BN50730 (10 microM), protein synthesis inhibitor cycloheximide (CHX; 30 micrograms/ml), RNA synthesis inhibitor actinomycin D (50 micrograms/ml), and tumor promoter phorbol ester (TPA); (100 nM) were tested. Total RNA for corneal epithelium was analyzed by Northern blot analysis using mouse COX-2 cDNA fragments labeled with 32P as probes. Western blots were performed using mouse monoclonal antibodies. Primary cultures of rabbit corneal epithelium were loaded with the fluorescent dye fluo-3 AM and changes in intracellular calcium concentration [Ca2+]i were analyzed by laser scanning confocal microscopy. RESULTS: Platelet-activating factor induction of COX-2 expression was detectable by Northern blot analysis at 2 hours, peaked at 4 hours, and remained increased for as long as 8 hours. At 16 hours, there was a marked increase in COX-2 expression. The effect was abolished by the PAF antagonist. TPA also induced COX-2 gene expression. Neither PAF-nor TPA-induced expression was inhibited by CHX. In a Ca(2+)-free medium, there was a 50% inhibition of COX-2 gene induction by PAF. The calcium ionophore A23187 also caused an increase in expression of COX-2 messenger RNA; this did not occur in Ca(2+)-free medium. Confocal microscopy imaging showed that after the addition of PAF, there was a transient increase in [Ca2+]i in corneal epithelial cells that peaked between 30 and 60 seconds. The increase was inhibited in the presence of BN50730 or in a Ca(2+)-free medium. A23187 also caused a transient increase in [Ca2+]i that was not altered in cells previously treated with PAF or BN50730. CONCLUSIONS: PAF may enhance prostaglandin synthesis in the corneal epithelium by increasing COX-2 gene expression. This increase is by means of transcriptional activation of the gene and results in increased COX-2 protein formation. Influx of Ca2+ due to PAF stimulation is required to induce the COX-2 gene. A PAF antagonist abolishes all PAF effects and could be of therapeutic value by modulating ocular inflammation at the level of COX-2 gene expression.
Assuntos
Cálcio/metabolismo , Epitélio Corneano/enzimologia , Expressão Gênica , Isoenzimas/biossíntese , Fator de Ativação de Plaquetas/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Transdução de Sinais/fisiologia , Compostos de Anilina , Animais , Azepinas/farmacologia , Northern Blotting , Western Blotting , Calcimicina/farmacologia , Ciclo-Oxigenase 2 , Indução Enzimática/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Corantes Fluorescentes , Ionóforos/farmacologia , Isoenzimas/genética , Microscopia Confocal , Técnicas de Cultura de Órgãos , Peroxidases/biossíntese , Peroxidases/genética , Fator de Ativação de Plaquetas/antagonistas & inibidores , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/metabolismo , Coelhos , Tienopiridinas , Triazóis/farmacologia , XantenosRESUMO
The effect of neuroprotective sigma ligands possessing a range of relative selectivity for sigma and phencyclidine (PCP) binding sites on N-methyl-D-aspartate (NMDA) and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated calcium flux was studied in 12-15-day-old primary cultures of rat cortical neurons. In approximately 80% of the neurons tested, NMDA (80 microM) caused a sustained increase in intracellular calcium ([Ca2+]i). With the exception of R-(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP) (previously shown not to be neuroprotective) all of the sigma ligands studied significantly altered NMDA-induced calcium dynamics. The primary effect of dextromethorphan, (+)-pentazocine, (+)-cyclazocine, (+)-SKF10047, carbetapentane, 1,3-di(2-tolyl) guanidine (DTG), and haloperidol was to shift the NMDA response from a sustained, to either a biphasic or a transient, calcium event. In contrast to NMDA, the primary response observed in 62% of the neurons treated with trans-ACPD (100 microM) was a transient elevation in [Ca2+]i. Here, however, only the highly selective neuroprotective sigma ligands (i.e., those lacking substantial PCP binding affinity) significantly decreased the number of transient responses elicited by trans-ACPD whereas the PCP-related sigma ligands such as dextromethorphan, (+)-SKF10047 and (+)-cyclazocine were ineffective. Unexpectedly, (+)-3-PPP potentiated trans-ACPD activity. These results demonstrating attenuating effects of sigma ligands on NMDA-stimulated neuronal calcium responses agree with earlier studies using glutamate and KCl and identify a sigma receptor modulation of functional NMDA responsiveness. Furthermore, the ability of sigma ligands to attenuate NMDA-, trans-ACPD- and KCl-evoked neuronal calcium dynamics indicates that the receptor mechanisms mediating sigma neuroprotection comprise complex interactions involving ionotropic, metabotropic, and even voltage-gated calcium signaling processes.
Assuntos
Cálcio/fisiologia , Cicloleucina/análogos & derivados , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Receptores sigma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Cicloleucina/antagonistas & inibidores , Cicloleucina/farmacologia , Ligantes , N-Metilaspartato/antagonistas & inibidores , Ratos/embriologia , Ratos Sprague-DawleyRESUMO
Diabetes produces dramatic changes in retinal microvasculature, triggering endothelial cell proliferation and microaneurysms. Capillaries become weakened, releasing blood into vitreal and retinal spaces. Photoreceptors become occluded and separated from the choriocapillaris, resulting in visual acuity decline, detachment and cell death. Several models have been developed that have proved useful for the study of this disease, resulting in a better understanding of the processes involved. Streptozotocin treatment affects the pancreatic beta cells, rapidly reducing them until insulin is no longer synthesized in sufficient amounts. The galactosemic model shifts metabolism away from glucose, increasing aldose reductase and retinal polyol metabolism. Finally, two weeks of cycled oxygen from high to low tension every 24 hours, followed by return to room air, triggers microangiogenesis in developing retinas. Use of these models, separately or in combination, as well as electroretinographic analysis, has begun to reveal the events taking place as diabetic retinopathy progresses. Endothelial cells become separated from pericytes as basement membranes thicken, and vascular endothelial growth factor increases, triggering their proliferation. Finally, early changes occurring within photoreceptors can now be studied.
Assuntos
Retinopatia Diabética/etiologia , Animais , Antibacterianos , Diabetes Mellitus Experimental , Retinopatia Diabética/induzido quimicamente , Modelos Animais de Doenças , Galactosemias/complicações , Oxigênio/administração & dosagem , Oxigênio/farmacologia , Estreptozocina/farmacologiaRESUMO
Secretory and cytosolic phospholipases A2 (sPLA2 and cPLA2) may contribute to the release of arachidonic acid and other bioactive lipids, which are modulators of synaptic function. In primary cortical neuron cultures, neurotoxic cell death and [3H]arachidonate metabolism was studied after adding glutamate and sPLA2 from bee venom. sPLA2, at concentrations eliciting low neurotoxicity (=100 ng/ml), induced a decrease of [3H]arachidonate-phospholipids and preferential reesterification of the fatty acid into triacylglycerols. Free [3H]arachidonic acid accumulated at higher enzyme concentrations, below those exerting highest toxicity. Synergy in neurotoxicity and [3H]arachidonate release was observed when low, nontoxic (10 ng/ml, 0.71 nM), or mildly toxic (25 ng/ml, 1. 78 nM) concentrations of sPLA2 were added together with glutamate (80 microM). A similar synergy was observed with the sPLA2 OS2, from Taipan snake venom. The NMDA receptor antagonist MK-801 blocked glutamate effects and partially inhibited sPLA2 OS2 but not sPLA2 from bee venom-induced arachidonic acid release. Thus, the synergy with glutamate and very low concentrations of exogenously added sPLA2 suggests a potential role for this enzyme in the modulation of glutamatergic synaptic function and of excitotoxicity.
Assuntos
Ácido Araquidônico/metabolismo , Morte Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Glutamatos/toxicidade , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Fosfolipases A/toxicidade , Animais , Venenos de Abelha/toxicidade , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Sinergismo Farmacológico , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosfolipídeos/metabolismo , RatosRESUMO
Brief 200-Hz stimulation of the corticomedial amygdala (CMA) increases the aggressiveness of male Syrian golden hamsters for about 30 min; the effect peaks 10-15 min after stimulation. This effect is sensitive to stimulation amplitude and frequency. Stimulation at the parameters that reduce attack latency increases flank marking but does not affect copulation latency or general activity. Immunocytochemical analysis suggests that stimulation effects may be coupled to c-fos expression and that unilateral stimulation has bilateral effects. CMA stimulation effects appear to mimic part of the time course of behaviorally induced attack priming. The temporal persistence of aggression may result from long-term potentiation-like changes within CMA-related neural circuitry.
Assuntos
Agressão/fisiologia , Tonsila do Cerebelo/fisiologia , Córtex Cerebral/fisiologia , Comportamento Agonístico/fisiologia , Animais , Nível de Alerta/fisiologia , Mapeamento Encefálico , Cricetinae , Estimulação Elétrica , Masculino , Mesocricetus , Vias Neurais/fisiologia , Tempo de Reação/fisiologia , Comportamento Sexual Animal/fisiologiaRESUMO
Glutamate (Glu)-stimulated phosphatidylinositol (PI) metabolism in primary neuronal cultures was found to be modulated by acute and chronic treatment with two estrogenic compounds. 17 beta-Estradiol 3-benzoate (0.1 and 1 microM), when applied with Glu, significantly reduced Glu (40 microM)-stimulated PI metabolism by 20-36%, an effect not seen with 17 alpha-estradiol. The weak estrogen phenol red (20 microM), had no effect when added immediately before Glu stimulations. Two-week pretreatment with 17 beta-estradiol 3-benzoate (1 microM) resulted in a significant decrease in Glu-stimulated PI metabolism (10-100 microM). Chronic treatment with 20 microM phenol red, at a concentration commonly found in culture medium, resulted in parallel but not statistically significant effects to those observed with chronic estradiol treatment. Estrogenic compounds may modulate the excitatory responses of neurons by both genomic and non-genomic means.
Assuntos
Estradiol/análogos & derivados , Estradiol/farmacologia , Ácido Glutâmico/farmacologia , Neurônios/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Animais , Células Cultivadas , Estudos de Avaliação como Assunto , Neurônios/metabolismo , Fenolsulfonaftaleína , Ratos , Estimulação Química , Fatores de TempoRESUMO
Since unique calcium dynamics have been reported for toxic (40-80 M) and non-toxic (5-10 microM) concentrations of glutamate, we evaluated the effect of neuroprotective sigma ligands on glutamate and potassium chloride (KCl)-stimulated changes in [Ca2+]i using 12-15 day old primary rat neuronal cortical cultures. In approximately 80% of the neurons tested, 80 microM glutamate caused a sustained calcium flux previously shown to be associated with neurotoxicity. The majority of sigma ligands that were evaluated altered glutamate-induced calcium flux. For example, the primary effect of maximally neuroprotective concentrations of the sigma ligands dextromethorphan, (+)-pentazocine, (+)-cyclazocine, (+)-SKF 10047, carbetapentane and haloperidol was a shift from a sustained, to either a biphasic or a monophasic transient calcium response indicative of neuroprotection. (+)-3-PPP, previously shown not to be neuroprotective in this model system, failed to alter glutamate-induced calcium flux. In contrast to glutamate, KCl (50 mM) produced changes in [Ca2+]i which were not neurotoxic to the neurons as measured by LDH release. The primary response observed in 59% of the neurons treated with 50 mM KCl alone was an initial spike in [Ca2+]i which abruptly declined then plateaued above basal levels throughout the 12 min of analysis (modified sustained response). The highly selective sigma ligands produced a shift from the modified sustained response to a monophasic transient calcium response. Again, (+)-3-PPP had no effect on KCl-induced calcium dynamics. Of the PCP-related sigma ligands only (+)-SKF-10047 consistently attenuated the KCl-induced calcium flux. Collectively, these results indicate that modulation of [Ca2+]i through receptor and voltage-gated calcium channels contributes significantly to sigma mediated neuroprotection.
Assuntos
Cálcio/fisiologia , Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores sigma/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Ácido Glutâmico/farmacologia , Ligantes , Cloreto de Potássio/farmacologia , Ratos , Receptores sigma/efeitos dos fármacosRESUMO
Consistent with the neuroprotective effects of the non-opioid antitussive dextromethorphan (DM) described in several models of CNS injury, micromolar concentrations of three novel analogs of DM markedly attenuated the injury produced by glutamate in cultured rat cortical neurons. Furthermore, the neuroprotective actions of the DM analogs correlated with their effects to block glutamate-induced excitotoxic calcium signals and were unrelated to metabolism to the phencyclidine (PCP)-like drug dextrorphan (DX). These observations establish a new class of compounds related to DM which, by virtue of their efficacy to protect neurons against a severe glutamate insult, may possess therapeutic potential as treatment modalities for a number of neurodegenerative diseases.
Assuntos
Cálcio/fisiologia , Dextrometorfano/análogos & derivados , Dextrometorfano/farmacologia , Ácido Glutâmico/toxicidade , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , RatosRESUMO
The present study evaluated the neurotoxic potential of phospholipase A2 (PLA2) in in vitro (primary neuronal cultures) and in vivo (EEG and behavior) rat models of CNS excitability. In vitro, PLA2 (0.0038-5.8 nM) or melittin (a potent activator of endogenous PLA2; 100-5000 nM), were highly neurotoxic, causing approximately 500 units/ml LDH release. The neurotoxic EC50s for PLA2 and melittin were 1.8 (1.4-2.3) and 848 (501-1280) nM, respectively. Neurotoxic concentrations of PLA2 stimulated neuronal release of [3H]AA. Preliminary in vitro experiments evaluating changes in neuronal calcium flux indicated that PLA2 caused transient, and melittin sustained, increases in [Ca2+]i. In vivo, PLA2 (0.5-5 micrograms i.c.v.) or melittin (2.5-20 micrograms i.c.v.) produced nonconvulsive EEG seizures, which generalized to status epilepticus. While the onset of seizure development was markedly delayed for PLA2 (1.5-4.5 h), the seizure inducing effects of melittin were evident within 3.5 +/- 0.2 min and more severe. Both PLA2 and melittin were lethal, exhibiting LD50s of 0.62 micrograms and 8.4 micrograms, respectively. Pretreatment with (+)-MK801 (5 micrograms, i.c.v.) significantly attenuated melittin, but not PLA2, in vivo neurotoxicity. PLA2 induced neuropathology in surviving rats revealed extensive cortical and subcortical injury to forebrain neurons and fibre pathways. Collectively, these results demonstrate the potent neurotoxic potential of PLA2, the delayed clinical nature of its in vivo neurotoxicity and the applicability of these model systems to future studies on mechanisms of PLA2 neurotoxicity and the development of potential PLA2 antagonists.
Assuntos
Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Fosfolipases A/toxicidade , Animais , Cálcio/metabolismo , Células Cultivadas/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiologia , Maleato de Dizocilpina/farmacologia , Eletroencefalografia , Eletrofisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Injeções Intraventriculares , Meliteno/farmacologia , Fosfolipases A2 , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
The role of the putative sigma receptor in mediating neuroprotection against glutamate-induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the selective sigma 1 ligand (+)-3-PPP, all of the sigma ligands tested were neuroprotective, preventing glutamate-induced morphological changes and increases in LDH release. Their rank order of neuroprotective potency (and EC50 values) was as follows: (+)-SKF 10,047 (0.81 microM) > (+)- cyclazocine (2.3 microM) > dextromethorphan (3.1 microM) = haloperidol (3.7 microM) > (+)-pentazocine (8.5 microM) > DTG (42.7 microM) = carbetapentane (46.3 microM). When corrected for relative sigma versus PCP binding affinity, it appears that a positive correlation exists between neuroprotective potency and sigma 1 site affinity. However, there does not appear to be a significant correlation between neuroprotective potency and the sigma 2 site. Critically, none of the sigma ligands were neurotoxic when tested alone at concentrations at least 5-30 times their respective neuroprotective EC50 values. Results from preliminary experiments with the selective sigma 1 ligand (+)-pentazocine indicated that sigma-mediated neuroprotection may involve the buffering of glutamate-induced calcium flux. Collectively, the results of these in vitro experiments demonstrate that sigma ligands are neuroprotective and therefore deserve further exploration as potential therapeutic agents in in vivo models of CNS injury and neurodegenerative disorders.