RESUMO
BACKGROUND: Single applications of sustained-release local anaesthetics may provide prolonged pain relief without requiring indwelling catheters, but have not yet been investigated for epidural postoperative pain management. We synthesized injectable sustained-release lidocaine particles (SRLPs) from biodegradable polymers and examined their effect in a rat model of postoperative pain. METHODS: Two types of polylactic acid particles, SRLP-10 and SRLP-25, containing 10% or 25% lidocaine, respectively, were generated and the lidocaine release was evaluated in vitro for 14 days. The SRLPs were then injected epidurally in the male Sprague-Dawley rats immediately before they received a hindpaw incision (the postoperative pain model), and hindpaw hypersensitivity was evaluated with the von Frey test. Motor paralysis and coordination were also assessed using a paralysis score and rota-rod test. Neurotoxicity and inflammation of the spinal cord, cauda equina, and tissue surrounding the injection site were histologically evaluated. RESULTS: In vitro, SRLP-10 and SRLP-25 released lidocaine over 7 and 3 days, respectively. The in vivo injection of SRLP-10 (80 mg) produced anti-hypersensitivity with no evidence of motor paralysis for 7 days after the paw incision, and SRLP-25 (60 mg) inhibited postoperative hypersensitivity for 7 days. Temporary motor paralysis (15 min) was observed after the injection of SRLP-25 (even with 40 mg). Foreign body reactions were observed around the SRLP injection site at 1 and 4 weeks after injection. No histopathological changes were observed at 1 or 4 weeks. CONCLUSIONS: The epidural injection of SRLPs produced prolonged anti-hypersensitivity in a rat model of postoperative pain with no major complications.
Assuntos
Anestésicos Locais/farmacologia , Lidocaína/farmacologia , Dor Pós-Operatória/tratamento farmacológico , Análise de Variância , Animais , Preparações de Ação Retardada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Injeções Epidurais , Masculino , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , TempoRESUMO
BACKGROUND: Recently described neuronal intermediate filament inclusion disease (NIFID) shows considerable clinical heterogeneity. OBJECTIVE: To assess the spectrum of the clinical and neuropathological features in 10 NIFID cases. METHODS: Retrospective chart and comprehensive neuropathological review of these NIFID cases was conducted. RESULTS: The mean age at onset was 40.8 (range 23 to 56) years, mean disease duration was 4.5 (range 2.7 to 13) years, and mean age at death was 45.3 (range 28 to 61) years. The most common presenting symptoms were behavioral and personality changes in 7 of 10 cases and, less often, memory loss, cognitive impairment, language deficits, and motor weakness. Extrapyramidal features were present in 8 of 10 patients. Language impairment, perseveration, executive dysfunction, hyperreflexia, and primitive reflexes were frequent signs, whereas a minority had buccofacial apraxia, supranuclear ophthalmoplegia, upper motor neuron disease (MND), and limb dystonia. Frontotemporal and caudate atrophy were common. Histologic changes were extensive in many cortical areas, deep gray matter, cerebellum, and spinal cord. The hallmark lesions of NIFID were unique neuronal IF inclusions detected most robustly by antibodies to neurofilament triplet proteins and alpha-internexin. CONCLUSION: NIFID is a neuropathologically distinct, clinically heterogeneous variant of frontotemporal dementia (FTD) that may include parkinsonism or MND. Neuronal IF inclusions are the neuropathological signatures of NIFID that distinguish it from all other FTD variants including FTD with MND and FTD tauopathies.
Assuntos
Encéfalo/patologia , Demência/classificação , Demência/patologia , Filamentos Intermediários/patologia , Neurônios/patologia , Adulto , Idade de Início , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Demência/fisiopatologia , Diagnóstico Diferencial , Progressão da Doença , Evolução Fatal , Feminino , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Lobo Frontal/fisiopatologia , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Proteínas de Filamentos Intermediários , Filamentos Intermediários/metabolismo , Masculino , Pessoa de Meia-Idade , Doença dos Neurônios Motores/etiologia , Doença dos Neurônios Motores/patologia , Doença dos Neurônios Motores/fisiopatologia , Neurônios/metabolismo , Transtornos Parkinsonianos/etiologia , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Fenótipo , Estudos Retrospectivos , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are multi-functional peptides derived from the same precursor, proadrenomedullin. We have studied the regulatory mechanism of expression of these peptides during neuronal differentiation of rat pheochromocytoma PC12 cells by nerve growth factor (NGF). The cellular levels of the peptides increased slightly, and then progressively decreased below the control by NGF. Immunoreactive (ir)-AM in the medium was transiently increased by NGF. Cytochemical staining showed that ir-AM and ir-PAMP were abundantly present in cytoplasm in the undifferentiated cells, and were decreased during culture with NGF. There was no preferential localization of ir-AM or ir-PAMP in neurites in comparison with in cytoplasm in the differentiated cells. Northern blot analysis showed that mRNA encoding these peptides, as detected as a band of 1.6 kb, increased more than three-fold at 1 h after the addition of NGF and then progressively decreased to one fifth of the control during 72 h. Degradation rate of the mRNA was slowed by NGF even when mRNA level is decreased after 72 h of NGF treatment. The transcription rate of their gene increased transiently and then decreased by the long-term treatment with NGF. These results demonstrate that expression of AM and PAMP is regulated by NGF along with time-dependent differentiation: AM gene transcription is transiently activated by NGF, whereas it was suppressed during neuronal differentiation of the cells.
Assuntos
Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Peptídeos/metabolismo , Biossíntese de Proteínas , Precursores de Proteínas/biossíntese , Adrenomedulina , Animais , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Neurônios/citologia , Neurônios/metabolismo , Células PC12 , Biossíntese Peptídica/efeitos dos fármacos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/efeitos dos fármacos , Peptídeos/efeitos dos fármacos , Feocromocitoma/metabolismo , Precursores de Proteínas/efeitos dos fármacos , Proteínas/efeitos dos fármacos , RNA Mensageiro/análise , Radioimunoensaio , Ratos , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
A-52-year-old woman was admitted to a hospital because of 2-year history of abnormal behavior and impaired visual acuity. Magnetic resonance imaging delineated a sizable mass at the suprasellar region. The partially removed tumor was arranged in irregular lobules composed of an admixture of clusters of cobblestone-like small cells and process-bearing cells with ovoid nuclei, surrounded by a fine, neuropil-like matrix. The Zellballen structure was inconspicuous, and mitosis was absent. Immunohistochemically, the tumor cells were positive for chromogranin A, synaptophysin, class III beta-tubulin and neurofilament, while negative for glial fibrillary acidic protein, cytokeratin and all 6 pituitary hormones. S100 protein expression was limited to cells adjacent to stroma. The MIB-1 labeling index was 0.5%. Histopathological diagnosis was paraganglioma of abortive architecture. Ultrastructurally, numerous dense-cored vesicles were found within the processes and cytoplasm. Synapse formation was not demonstrated. Interestingly, crystalloids up to 3 microm in size were frequently found. They had hexagonal or quadrilateral architecture without limiting membranes. The interval between periodically arranged fibrils was variable, ranging from approximately 20 - 50 nm. Retrospective examination by light microscopy failed to reveal corresponding structures. Crystalloids are rare manifestation of paragangliomas, yet undescribed in those of intracranial origin. Furthermore, the ultrastructure of the present case differs from those of previous cases.
Assuntos
Biomarcadores Tumorais/análise , Corpos de Inclusão/ultraestrutura , Neoplasias Meníngeas/patologia , Paraganglioma/patologia , Neoplasias Hipofisárias/patologia , Biópsia , Cromogranina A , Cromograninas/análise , Diagnóstico Diferencial , Feminino , Humanos , Imageamento por Ressonância Magnética , Neoplasias Meníngeas/diagnóstico , Microscopia Eletrônica , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/análise , Paraganglioma/diagnóstico , Neoplasias Hipofisárias/diagnóstico , Sela Túrcica/patologia , Sinaptofisina/análise , Tubulina (Proteína)/análiseRESUMO
The patient was a 47-year-old male, who visited Hidaka Hospital with a chief complaint of swelling in the right inguinal region and the scrotum. With a diagnosis of a right spermatic cord tumor, right high orchiectomy was performed. Since an inflammatory type of malignant fibrous histiocytoma (MFH) was diagnosed from histopathological findings, chemotherapy and radiation therapy were performed as postoperative treatment. Malignant fibrous histiocytoma with the primary focus of the spermatic cords is a rare disease. To our knowledge, this is the 20th case of MFH of the spermatic cord in Japan (the 42nd in the world) and it is the second case of inflammatory type of MFH in Japan.
Assuntos
Neoplasias dos Genitais Masculinos/patologia , Histiocitoma Fibroso Benigno/patologia , Cordão Espermático/patologia , Terapia Combinada , Diagnóstico Diferencial , Neoplasias dos Genitais Masculinos/terapia , Histiocitoma Fibroso Benigno/terapia , Humanos , Masculino , Pessoa de Meia-Idade , OrquiectomiaRESUMO
The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM >> amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.
Assuntos
Amiloide/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , AMP Cíclico/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/biossíntese , Oligodendroglia/metabolismo , Peptídeos/fisiologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Medula Suprarrenal/citologia , Adrenomedulina , Amiloide/farmacologia , Animais , Comunicação Autócrina , Neoplasias Encefálicas/patologia , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina , Bovinos , Células Cultivadas , Glioma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Comunicação Parácrina , Fragmentos de Peptídeos/farmacologia , Peptídeos/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/biossíntese , Receptores da Calcitonina/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/efeitos dos fármacos , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas , Receptores de Peptídeos/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
A potential novel variant of meningioma is reported. The tumor was solid, hard, white-colored, well circumscribed in a fibrous capsule and fixed to the dura, showing no invasion into the brain parenchyma. Histopathological study presented a sparsely cellular tumor composed of cells with fine reticular or mesh-like cytoplasm, each containing an oval nucleus. Mitotic figures were rarely seen. Immunohistochemical studies of tumor cells showed positive immunoreactivity for vimentin and epithelial membrane antigen but were negative for GFAP, desmin, neurofilament, keratin, S-100, CD34 and CEA. Bipolar neoplastic cells and long processes were noted on ultrastructural observation; these were attached side by side to each other by desmosomes, resulting in a mesh-like configuration. Perinuclear cytoplasm and processes were rich in intermediate filaments and rough endoplasmic reticulum. These microscopic and ultrastructural features have never before been reported among the variants of meningioma. The name 'meshy meningioma' is proposed for this novel variant.
Assuntos
Aracnoide-Máter/patologia , Encéfalo/patologia , Neoplasias Meníngeas/patologia , Meningioma/patologia , Aracnoide-Máter/metabolismo , Aracnoide-Máter/ultraestrutura , Dura-Máter/patologia , Feminino , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/ultraestrutura , Meningioma/metabolismo , Meningioma/ultraestrutura , Microscopia Eletrônica , Pessoa de Meia-Idade , Mucina-1/metabolismo , Vimentina/metabolismoRESUMO
The middle ear cavity is an air-filled space that must be maintained for effective sound transmission to the inner ear. To examine the mechanisms of water homeostasis in the middle ear, we investigated whether aquaporins (AQPs), a family of water-permeable channels, were expressed in the middle ear. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that mRNAs encoding AQP1, 4 and 5 (but not 2 or 3) subtypes were expressed in rat middle ear epithelium; AQP1, 4 and 5 were detected as 28-, 30- and 30-kDa proteins, respectively. Immunohistochemical analysis showed that AQP1 was localized at capillary endothelial cells and fibroblasts in lamina propria mucosae; AQP4 was present solely at the basolateral membrane of ciliated cells, whereas AQP5 was on the apical surface of ciliated cells as well as of flat and columnar epithelial cells. The characteristic different localizations of AQP1, 4 and 5 subtypes in the middle ear suggest that middle ear water homeostasis requires the coordinated operation of these AQPs.
Assuntos
Aquaporinas/metabolismo , Orelha Média/metabolismo , Proteínas de Membrana , Animais , Aquaporina 1 , Aquaporina 4 , Aquaporina 5 , Epitélio/metabolismo , Immunoblotting , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Patients with neurofibromatosis type 1 (NF1) often have gliomas as a complication, most of which are benign pilocytic astrocytomas which have arisen in optic pathways. In the present case, a 17-year-old girl (at death) with stigmata of NF1, initially had a bulky tumor mass in the left thalamus, developing into the lateral ventricle, at 13 years of age. Partially resected tissue samples showed pleomorphic astrocytoma with abundant xanthoma cells and degenerative structures such as Rosenthal fibers (RF) and eosinophilic granular bodies. Fine eosinophilic granules identical to RF, both immunophenotypically and ultrastructurally, were also seen. The residual tumor was subtotally resected 6 months later, and the tumor histology was essentially similar as before, accompanying the regenerative structures; this was believed to be a good prognostic indicator. However, several anaplastic features such as mitosis, necrosis and vascular proliferation appeared even in areas rich in the regenerative structures. After a 2-year, disease-free interval, multiple tumor relapse occurred in June 1997. Partially resected tumor tissues were composed of monotonous small anaplastic cells with prominent proliferative activity. Surprisingly, the tumor cells had retained eosinophilic granules within the cell bodies. Postoperative chemotherapy with procarbazine, MCNU and vincristine (PCV) suppressed the residual tumor dramatically, but the regrowing tumor finally became uncontrollable, leading to the patient's death. TP53 mutation was not detected, while p27 immunopositivity was constantly high during malignant progression, suggesting acquisition of proliferative activity to overcome p53 and p27 inhibitory functions. A review of previously published reports failed to reveal any cases of this type.
Assuntos
Astrocitoma/patologia , Neoplasias do Ventrículo Cerebral/patologia , Proteínas Musculares , Neurofibromatose 1/patologia , Tálamo/patologia , Adolescente , Antígenos Nucleares , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Astrocitoma/diagnóstico por imagem , Astrocitoma/tratamento farmacológico , Astrocitoma/cirurgia , Biomarcadores Tumorais/análise , Neoplasias do Ventrículo Cerebral/diagnóstico por imagem , Neoplasias do Ventrículo Cerebral/tratamento farmacológico , Neoplasias do Ventrículo Cerebral/cirurgia , Primers do DNA/química , DNA de Neoplasias/análise , Evolução Fatal , Feminino , Humanos , Proteínas dos Microfilamentos/análise , Recidiva Local de Neoplasia , Segunda Neoplasia Primária/patologia , Compostos de Nitrosoureia/uso terapêutico , Proteínas Nucleares/análise , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Procarbazina/uso terapêutico , Radiografia , Tálamo/diagnóstico por imagem , Proteína Supressora de Tumor p53/análise , Vincristina/uso terapêuticoRESUMO
A 25-year-old man was admitted to the Department of Neurology, Gunma University Hospital, in June 1997. He had an intellectual disability and had suffered from repeated infection since childhood. Cerebellar ataxia had developed at 19 years of age and he had been clinically diagnosed with ataxia telangiectasia (AT) comprising cerebellar ataxia and oculocutaneous telangiectasia at 24 years of age. He died from pneumonia and renal failure at 26 years of age. Neuropathological examination revealed Purkinje cell loss and atrophy of the dentate nuclei in the cerebellum, anterior horn-cell atrophy and demyelination of the gracile fasciculi in the spinal cord, and the existence of nucleocytomegalic cells in the anterior pituitary gland. These neuropathological findings correlated with previously reported cases of AT. In addition, spongy degeneration was found, predominantly around the blood vessels in the cerebral cortex. Diffuse spongy degeneration and multiple foci of coagulative necrosis with calcification were noted in the white matter. Abnormal vasculature was noted in both degenerative and necrotic areas in the cerebral cortex and in the white matter. The vessels at the center of the areas of spongy degeneration in the cerebral cortex had irregularly arranged and enlarged smooth-muscle-cell nuclei and a distorted, narrow lumen. The vessels present in the white matter were hyalinized. To our knowledge, these vascular abnormalities in the brain parenchyma have not been reported previously.
Assuntos
Ataxia Telangiectasia/patologia , Circulação Cerebrovascular , Adulto , Ataxia Telangiectasia/diagnóstico , Vasos Sanguíneos/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Evolução Fatal , Humanos , Imageamento por Ressonância Magnética , Masculino , Medula Espinal/patologia , Tomografia Computadorizada por Raios XRESUMO
Treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by 56% in a time (t(1/2) = 15.2 h)- and concentration (EC(50) = 2.9 microM)-dependent manner but did not change the K(d) value. In CsA-treated cells, veratridine-induced (22)Na(+) influx was augmented with no change in the EC(50) of veratridine; also, alpha- and beta-scorpion venom and Ptychodiscus brevis toxin-3 enhanced veratridine-induced (22)Na(+) influx in a more than additive manner, as in nontreated cells. CsA treatment for 1 to 24 h inhibited calcineurin activity, measured by the in vitro assay, with the IC(50) of 0.6 microM but did not alter cellular level of calcineurin. FK506 or rapamycin elevated [(3)H]STX binding by 36 or 25%, whereas GPI-1046, an immunophilin ligand incapable to inhibit calcineurin, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had no increasing effect. The rise of [(3)H]STX binding by CsA was attenuated by the coincident treatment with brefeldin A (BFA), an inhibitor of vesicular exit from the trans-Golgi network. The internalization rate of cell surface Na(+) channels, as determined in the presence of BFA, was decreased in CsA (but not rapamycin)-treated cells (t(1/2) = 20.3 h), compared with nontreated cells (t(1/2) = 13.7 h). CsA treatment, however, did not elevate cellular levels of Na(+) channel alpha-subunit and Na(+) channel alpha- and beta(1)-subunit mRNAs. In CsA-treated cells, veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels and catecholamine secretion were enhanced, whereas high K(+)-induced (45)Ca(+) influx was not. Thus, the inhibition of calcineurin or rapamycin-binding protein causes up-regulation of cell surface functional Na(+) channels via modulating externalization and internalization of Na(+) channels, thus enhancing Ca(2+) channel gating and catecholamine secretion.
Assuntos
Glândulas Suprarrenais/metabolismo , Células Cromafins/efeitos dos fármacos , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Oxocinas , Sirolimo/farmacologia , Canais de Sódio/efeitos dos fármacos , Tacrolimo/farmacologia , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Northern Blotting , Calcineurina/metabolismo , Cálcio/metabolismo , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Immunoblotting , Toxinas Marinhas/farmacologia , Bloqueadores Neuromusculares/farmacologia , Venenos de Escorpião/farmacologia , Sódio/metabolismo , Regulação para Cima/efeitos dos fármacos , Veratridina/farmacologiaRESUMO
An 83-year-old man with pigmented Paget's disease of the breast is reported. He had a blackish swollen right nipple with bloody discharge that lasted for 8 months. Histopathology of the lesion disclosed intraepidermal and dermal atypical cells forming small clusters and underlying intraductal carcinoma. Melanocytes were dispersed in the epidermis. Dermal melanophages were also detected. The atypical cells were positive for carcinoembryonic antigen, human-milk fat globule protein and a recently described new antigen, RCAS-1, but negative for S-100 or HMB-45. We diagnosed this case as Paget's disease of the male breast. Paget's disease of the breast is usually nonpigmented and occurs almost exclusively in women. Pigmented Paget's disease of the male breast is extremely rare, and only a few cases have been reported. Albeit rare, pigmented Paget's disease has to be included in the differential diagnosis of pigmented lesions of the nipple.
Assuntos
Neoplasias da Mama Masculina/patologia , Doença de Paget Mamária/patologia , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama Masculina/diagnóstico , Diagnóstico Diferencial , Humanos , Masculino , Mamilos/patologia , Doença de Paget Mamária/diagnóstico , Pigmentação da PeleRESUMO
Long-term (> or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+]c down-regulates cell surface IR by retarding externalization of IR from the TGN.
Assuntos
Glândulas Suprarrenais/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Células Cromafins/metabolismo , Inibidores Enzimáticos/farmacologia , Receptor de Insulina/metabolismo , Retículo Sarcoplasmático/enzimologia , Tapsigargina/farmacologia , Glândulas Suprarrenais/citologia , Animais , Brefeldina A/farmacologia , Cálcio/metabolismo , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Quelantes/farmacologia , Citoplasma/metabolismo , Regulação para Baixo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Insulina/metabolismo , Concentração Osmolar , Fatores de TempoRESUMO
1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.
Assuntos
Cálcio/metabolismo , Oxocinas , Canais de Sódio/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Brefeldina A/farmacologia , Calcimicina/farmacologia , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Calpaína/antagonistas & inibidores , Carbazóis/farmacologia , Bovinos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Hidroquinonas/farmacologia , Indóis/farmacologia , Ionóforos/farmacologia , Toxinas Marinhas/farmacologia , Subunidades Proteicas , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Saxitoxina/metabolismo , Venenos de Escorpião/farmacologia , Sódio/metabolismo , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , Tapsigargina/farmacologia , Fatores de Tempo , Trítio , Veratridina/farmacologiaRESUMO
We studied whether a novel proadrenomedullin derived peptide was present and what was its physiological function in cultured bovine adrenal chromaffin cells. We found a high level of proadrenomedullin N-terminal 12 peptide (PAMP-12) which consists of a peptide from 9th amino acid to 20th amino acid of proadrenomedullin N-terminal 20 peptide (PAMP-20). PAMP-12 was released from the cells along with catecholamine upon stimulation of nicotinic cholinergic receptors. When PAMP-12 was added in the incubation medium, this peptide inhibited nicotinic receptor-mediated catecholamine release and influx of Na(+) and Ca(2+) into the cells. PAMP-12 did not affect catecholamine release evoked by histamine or by depolarization by high concentration of potassium. PAMP-12 also inhibited synthesis of catecholamines as well as the activation of tyrosine hydroxylase by nicotinic stimulation. Thus, PAMP-12 is an endogenous peptide that regulates release and synthesis of catecholamines by acting on nicotinic cholinergic receptors in an autocrine manner in adrenal chromaffin cells.
Assuntos
Células Cromafins/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos , Proteínas/metabolismo , Receptores Nicotínicos/metabolismo , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Adrenomedulina , Animais , Anticorpos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Carbacol/farmacologia , Radioisótopos de Carbono , Catecolaminas/metabolismo , Bovinos , Agonistas Colinérgicos/farmacologia , Células Cromafins/citologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Proteínas/imunologia , Proteínas/farmacologia , Sódio/metabolismoRESUMO
AMPA type-glutamate receptor channels (AMPARs) assembled without the GluR2 (GluR-B) subunit are characterized by high Ca2+ permeability, and are expressed abundantly in cerebellar Bergmann glial cells. Here we show that the morphology of cultured Bergmann glia-like fusiform cells derived from the rat cerebellum was changed by manipulating expression of Ca2+-permeable AMPARs using adenoviral vector-mediated gene transfer. Converting endogenous Ca2+-permeable AMPARs into Ca2+-impermeable channels by viral-mediated transfer of GluR2 gene induced retraction of glial processes. In contrast, overexpression of Ca2+-permeable AMPARs markedly elongated glial processes. The process extension was blocked by 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX), a specific antagonist of AMPAR. These results indicate that glutamate regulates the morphology of glial processes by activating Ca2+-permeable AMPARs.
Assuntos
Cálcio/metabolismo , Neuroglia/metabolismo , Receptores de AMPA/metabolismo , Adenoviridae/genética , Animais , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas , Cerebelo/citologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Expressão Gênica/fisiologia , Ácido Glutâmico/farmacologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/genéticaRESUMO
We investigated the expression of aquaporin (AQP) subtypes in the rat cerebral microvessels by reverse transcription-polymerase chain reaction, immunoblotting and immunohistochemistry. mRNA for AQP4, but not for AQP1, 2, 3 or 5, was detected in the microvessels. Immunoblot analysis showed that AQP4 protein was detected as a 30 kDa band with higher molecular weight bands. Immunohistochemical staining showed that AQP4 was located on cell surface of the cerebral microvessels. These results suggest that AQP4 in the cerebral microvessels is involved in the regulation of water transport between blood and brain.
Assuntos
Aquaporinas/biossíntese , Aquaporinas/classificação , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Proteínas de Membrana , Microcirculação/metabolismo , Animais , Aquaporina 1 , Aquaporina 2 , Aquaporina 3 , Aquaporina 4 , Aquaporina 5 , Aquaporina 6 , Aquaporinas/genética , Córtex Cerebral/citologia , Imuno-Histoquímica , Masculino , Microcirculação/citologia , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
To characterize transmembrane signaling of adrenomedullin (AM) in the choroid plexus, we studied the effects of AM on cyclic AMP (cAMP) and cyclic GMP (cGMP) levels as well as expression of mRNA for AM receptor in the rat choroid plexus slices. AM or calcitonin gene-related peptide (CGRP) increased cAMP (but not cGMP) level in a concentration-dependent manner, with AM being much more potently than CGRP. AM mRNA as well as calcitonin-receptor-like receptor mRNA and receptor-activity-modifying protein 2 mRNA, were highly expressed in the choroid plexus. Our biochemical and pharmacological studies may raise the possibility that choroid plexus secretes AM into the cerebrospinal fluid, and AM regulates choroid plexus function in an autocrine/paracrine manner via acting on AM-specific receptors.
Assuntos
Plexo Corióideo/metabolismo , Receptores de Peptídeos/metabolismo , Adrenomedulina , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina , Plexo Corióideo/citologia , Plexo Corióideo/efeitos dos fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Nitroprussiato/farmacologia , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Proteínas Modificadoras da Atividade de Receptores , Receptores de Adrenomedulina , Receptores da Calcitonina/biossíntese , Receptores da Calcitonina/genética , Receptores de Peptídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
Oligodendrogliomas are characterized by frequent loss of heterozygosity (LOH) on chromosomes 1p and 19q, but additional genetic alterations are likely to be involved. In this study, we screened 28 oligodendrogliomas (WHO grade II) and 20 anaplastic oligodendrogliomas (WHO grade III) for alterations in the RB1/CDK4/p16INK4a/p15INK4b and TP53/p14ARF/MDM2 pathways. In oligodendrogliomas, hypermethylation of RB1 (1 case) and p14ARF (6 cases) were the only detectable genetic changes (7/28, 25%). In anaplastic oligodendrogliomas, the RB1/CDK4/p16INK4a/p15INK4b signaling pathway regulating the G1 -->3 S transition of the cell cycle was altered in 13/20 (65%) cases, by either RBI alteration, CDK4 amplification, or p16IN4a/p15INK4b homozygous deletion or promoter hypermethylation. Further, 50% (10/20) of anaplastic oligodendrogliomas showed alterations in the TP53 pathway through promoter hypermethylation or homozygous deletion of the p14ARF gene and, less frequently, through TP53 mutation or MDM2 amplification. Of 13 anaplastic astrocytomas with an altered RB1 pathway, 9 (69%) also showed a dysregulated TP53 pathway. Thus, simultaneous disruption of the RB1/CDK4/p16INK4a/p15INK4b and the TP53/p14ARF/MDM2 pathways occurs in 45% (9/20) of anaplastic oligodendrogliomas, suggesting that these phenomena contribute to their malignant phenotype.
Assuntos
Genes p53/fisiologia , Oligodendroglioma/genética , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Deleção de Genes , Inativação Gênica , Homozigoto , Humanos , Oligodendroglioma/metabolismo , Oligodendroglioma/patologia , Proteína do Retinoblastoma/metabolismoRESUMO
Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are peptides having multiple physiological functions and are most abundantly expressed in the adrenal medulla. In addition to PAMP, PAMP12, a 12 amino acid peptide with sequence identity to PAMP between amino acids 9-20, has also been shown to be expressed in the adrenal medulla. AM, PAMP and PAMP12 are released along with catecholamines by regulated exocytosis upon stimulation of adrenal chromaffin cells. PAMP and PAMP12 regulate catecholamine release and synthesis by interfering with nicotinic cholinergic receptors in these chromaffin cells. AM may also cause gradual release of catecholamine from these cells. AM, PAMP and PAMP12 are endogenous peptides that modulate chromaffin cell function via different mechanisms.