RESUMO
Dysregulation of Ca(2+) signaling following oxidative stress is an important pathophysiological mechanism of many chronic neurodegenerative disorders, including Alzheimer's disease, age-related macular degeneration, glaucomatous and diabetic retinopathies. However, the underlying mechanisms of disturbed intracellular Ca(2+) signaling remain largely unknown. We here describe a novel mechanism for increased intracellular Ca(2+) release following oxidative stress in a neuronal cell line. Using an experimental approach that included quantitative polymerase chain reaction, quantitative immunoblotting, microfluorimetry and the optical imaging of intracellular Ca(2+) release, we show that sub-lethal tert-butyl hydroperoxide-mediated oxidative stress result in a selective up-regulation of type-2 inositol-1,4,5,-trisphophate receptors. This oxidative stress mediated change was detected both at the transcriptional and translational level and functionally resulted in increased Ca(2+) release into the nucleoplasm from the membranes of the nuclear envelope at a given receptor-specific stimulus. Our data describe a novel source of Ca(2+) dysregulation induced by oxidative stress with potential relevance for differential subcellular Ca(2+) signaling specifically within the nucleus and the development of novel neuroprotective strategies in neurodegenerative disorders.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Degeneração Neural/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Regulação para Cima/fisiologia , Animais , Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Receptores de Inositol 1,4,5-Trifosfato/biossíntese , Receptores de Inositol 1,4,5-Trifosfato/genética , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/fisiologia , Camundongos , Modelos Neurológicos , Degeneração Neural/etiologia , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , terc-Butil Hidroperóxido/toxicidadeRESUMO
Formation of new capillaries, a critical component of tissue growth and repair, is a recognized process in the development, formation, and remodeling of bone. Vascular endothelial growth factor (VEGF), a potent angiogenic factor with specific mitogenic actions on endothelial cells, is produced in a regulated manner by many cell types, including osteoblasts. The aim of the present investigation was to test the hypothesis that insulin-like growth factor I (IGF-I), a known osteogenic factor, modulates VEGF expression in osteoblasts. In human SaOS-2 osteoblast-like cells, 10 nM IGF-I increased the abundance of VEGF messenger RNA (mRNA) by 4-fold above the control value at 2h, and the elevated levels of mRNA returned to near basal by 8 h. IGF-I stimulated VEGF mRNA levels at IGF-I concentrations as low as 1-2 nM. The stability of VEGF mRNA was not increased after IGF-I treatment, and actinomycin D abrogated the enhanced expression of VEGF mRNA by IGF-I, indicating that the action of IGF-I was probably mediated by a transcriptional mechanism. The induction of VEGF mRNA by IGF-I in SaOS-2 cells was associated with an increase in immunoreactive VEGF protein, as detected by immunoblot analysis. IGF-I also increased the expression of VEGF mRNA in primary murine osteoblasts, which confirmed that the actions of IGF-I were not unique to SaOS-2 cells. We conclude that IGF-I enhances osteoblast synthesis of VEGF, which may then act locally on endothelium to stimulate angiogenesis, an essential component of bone growth and remodeling.
Assuntos
Fatores de Crescimento Endotelial/genética , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Linfocinas/genética , Osteoblastos/metabolismo , Animais , Northern Blotting , Linhagem Celular , Dactinomicina/farmacologia , Estabilidade de Medicamentos , Humanos , Camundongos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Uterine leiomyomas or fibroids are common among women of reproductive age, but their biology is poorly understood. The PTH-related protein (PTHrP) has been identified in a number of sites throughout the reproductive tract. We, therefore, examined whether fibroids express PTHrP mRNA and compared their level of expression with that in normal myometrium. Total RNA prepared from fibroid tissue and corresponding normal myometrium from seven patients was examined by RNase protection analysis. In all cases, fibroid and myometrial tissue expressed PTHrP, and in six of seven cases, PTHrP expression was higher in fibroids than in normal myometrium. Cultured fibroid cells from four patients also expressed higher levels of PTHrP mRNA than corresponding cultured normal myometrial cells. Tissue extracts from eight patients and conditioned medium from cultured cells from nine patients were examined for PTHrP immunoreactivity using a two-site immunoradiometric assay. In tissue extracts and conditioned medium, the mean PTHrP concentration was significantly higher in fibroids than normal myometrium. Immunohistochemical staining of fibroid and myometrial tissue was positive for PTHrP. Finally, PTHrP-(1-34) induced a dose-dependent increase in cAMP in fibroid and myometrial cells in vitro. These findings suggest that PTHrP may have an autocrine/paracrine function in regulating myometrial physiology and may play a role in regulating fibroid growth or differentiation.
Assuntos
Expressão Gênica , Leiomioma/genética , Proteínas/genética , Neoplasias Uterinas/genética , AMP Cíclico/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Leiomioma/metabolismo , Leiomioma/patologia , Miométrio/metabolismo , Miométrio/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/farmacologia , Proteínas/metabolismo , Proteínas/farmacologia , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
Insulin-like growth factor-1 (IGF-I), an endocrine and autocrine/paracrine factor that enhances collagen synthesis and bone matrix formation by osteoblasts, has been implicated in the coupling of bone formation with bone resorption. We have found, using SaOS-2 osteoblastic cells, that IGF-I inhibits PTH-stimulated cAMP production. Pretreatment of SaOS-2 cells with IGF-I for 24 h inhibited cAMP production stimulated by PTH with an IC50 of 1 nM and maximal inhibition to 10-20% of control values at an IGF-I concentration of 10 nM. Pretreatment with IGF-I had no effect on vasoactive intestinal peptide-stimulated cAMP production, but it enhanced cAMP production stimulated by prostaglandin E2 (PGE2) by as much as 5-fold at a concentration of 10 nM and with an EC50 of 1 nM. Pretreatment of SaOS-2 cells with IGF-I did not affect cholera toxin- or forskolin-stimulated cAMP accumulation. Taken together, these findings indicated that IGF-I did not affect Gs alpha, coupling of Gs alpha to adenylate cyclase, or adenylate cyclase itself. Binding experiments using [125I] chicken PTH-related peptide (PTHrP)-(1-36)-[Tyr36]NH2 demonstrated that IGF-I reduced PTH/PTHrP receptor number to 25% of the control value without affecting receptor affinity. IGF-I and the related growth factors, insulin and IGF-II, inhibited PTH-stimulated cAMP production with a rank order of potency of IGF-I > or = IGF-II > insulin, indicating that the actions of IGF-I on SaOS-2 cells were probably mediated by the IGF-I receptor. We conclude that physiologically relevant concentrations of IGF-I specifically inhibited PTH-stimulated and enhanced PGE2-stimulated production of cAMP by an action at the level of PTH and PGE2 receptors and/or coupling of the receptors to Gs alpha.
Assuntos
AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , Dinoprostona/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Osteoblastos/metabolismo , Hormônio Paratireóideo/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Insulina/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
PTH receptors on osteoblasts and calcitonin receptors on osteoclasts are coupled to adenylate cyclase. Despite similar transduction mechanisms, these hormones have opposing physiological actions. We investigated the consequences of persistent protein phosphorylation on bone resorption in neonatal mouse calvariae using okadaic acid (OA) and calyculin-A, two inhibitors of protein phosphatase-1 and -2A. These two inhibitors caused different responses in bone at picomolar and low nanomolar concentrations. OA inhibited, in a dose-dependent manner, bone resorption stimulated by PTH, 1,25-Dihydroxyvitamin D3, phorbol ester, and prostaglandin E2 (PGE2). OA did not inhibit the generation of the second messengers cAMP or PGs and did not have nonspecific toxic effects, as measured by protein and RNA synthesis. Thus, OA appeared to mimic the global inhibitory action of calcitonin on bone resorption. Unlike OA, calyculin-A elicited a biphasic dose response. At concentrations of 3.3 nM and greater, calyculin-A inhibited, in a dose-dependent manner, stimulated bone resorption. However, calyculin-A alone, at 0.625 and 2.5 nM, stimulated bone resorption via a PG-independent pathway. In calvariae, OA and calyculin-A increased phosphorylation of a 58- to 60-kilodalton protein. A protein of similar molecular mass was hyperphosphorylated in OA-treated ROS 17/2.8 osteoblast-like cells. We conclude that in addition to hormonal regulation of protein kinase activity, protein dephosphorylation plays a functionally important role in the modulation of bone resorption.
Assuntos
Reabsorção Óssea , Osso e Ossos/fisiologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Éteres Cíclicos/farmacologia , Oxazóis/farmacologia , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Autorradiografia , Osso e Ossos/efeitos dos fármacos , Calcitriol/farmacologia , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Eletroforese em Gel de Poliacrilamida , Cinética , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos , Ácido Okadáico , Técnicas de Cultura de Órgãos , Fosfatos/metabolismo , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Radioisótopos de Fósforo , Biossíntese de Proteínas , Proteína Fosfatase 1 , RNA/biossíntese , Teriparatida , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: Currently there is no regimen for managing the inappropriate behavior seen in Alzheimer's disease that does not cause significant patient sedation. Preliminary evidence suggests selegiline may be effective in behavioral modification without the adverse effects observed with other regimens. The purpose of this study was to document the efficacy of selegiline in Alzheimer's patients with behavior problems. METHOD: Eight Alzheimer's patients (6 women and 2 men) ranging in age from 50 to 82 years (mean +/- SD = 74.0 +/- 10.5) were enrolled in this single-blind study. Patients received selegiline 10 mg each day for 8 weeks. Prior to drug administration and at the end of Weeks 1, 2, 4, 6, and 8, patients were evaluated for behavior (BEHAVEAD), cognitive function (Mini-Mental State Examination), and caregiver stress (Caregiver Burden Scale). RESULTS: Of eight enrolled patients, five were available for analysis. No statistically significant differences were found between mean baseline and mean 8-week scores for any of the three tests. However, clinical significance was noted by improvement in cognition (orientation and recall), caregiver stress, and behavior. Behavior was noted to improve in the areas of paranoid and delusional ideation, hallucinations, activity disturbances, anxiety, and phobias. CONCLUSION: These data suggest that some Alzheimer's patients with behavior problems may benefit from selegiline therapy.
Assuntos
Doença de Alzheimer/psicologia , Transtornos Mentais/tratamento farmacológico , Selegilina/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enfermagem , Assistência Ambulatorial , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/psicologia , Feminino , Assistência Domiciliar , Humanos , Masculino , Transtornos Mentais/psicologia , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Método Simples-CegoRESUMO
Several cultured human squamous cell carcinoma cell lines (SCC-4, SCC-12B2, SCC-12F2, EC-GI-10, and BEN) and one normal keratinocyte line (Epy-1) were investigated for the production of bone resorption-stimulating activity (BRSA). Conditioned medium (CM) from each of the six cell lines stimulated bone resorption in neonatal mouse calvariae in culture. The BRSA of SCC-12F2 and EC-GI-10 was inhibited completely by antibody to interleukin-1 alpha (IL-1 alpha), whereas the BRSA in CM from the BEN, SCC-4, SCC-12B2, and Epy-1 cell lines was only partially inhibited by anti-IL-1 alpha. Addition of indomethacin to the calvarial cultures also partially inhibited the BRSA from EC-GI-10, SCC-4, SCC-12B2, and Epy-1 cells; the BRSA from BEN and SCC-12F2 cells was inhibited completely by indomethacin. cAMP production by calvariae was determined after a 60-min incubation with CM. CM from EC-GI-10, BEN, SCC-4, and Epy-1 stimulated cAMP production by bone. Preincubation of CM from BEN, EC-GI-10, SCC-4, and Epy-1 cells with two antisera against PTH-related protein [PTHrP; one specific for two PTHrP-(1-141), the other recognizing both PTHrP-(1-40) and PTHrP-(1-141)] completely inhibited the cAMP-stimulating activity. Using specific enzyme-linked immunosorbent assays for IL-1 alpha and IL-1 beta, IL-1 alpha was measured in CM of the SCC-4, SCC-12B2, SCC-12F2, and Epy-1 cell lines. IL-1 beta was undetectable (less than 0.1 ng/ml) in CM from all cell lines. Our findings indicate that the BRSA secreted by SCC-12F2 cells can be accounted for largely or entirely by IL-1 alpha, while the activity produced by SCC-12B2 includes IL-1 alpha and another unknown factor(s). The BRSA produced by EC-GI-10, BEN, SCC-4, and Epy-1 cells includes both IL-1 alpha and PTHrP. We conclude that IL-1 alpha may be a more prevalent and biologically significant component of the BRSA produced by SCCs than previously recognized.
Assuntos
Reabsorção Óssea , Osso e Ossos/fisiologia , Carcinoma de Células Escamosas/fisiopatologia , Citocinas/biossíntese , Interleucina-1/fisiologia , Animais , Animais Recém-Nascidos , Anticorpos , Osso e Ossos/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/metabolismo , Citocinas/farmacologia , Humanos , Indometacina/farmacologia , Interleucina-1/farmacologia , Queratinócitos/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Hormônio Paratireóideo/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/farmacologia , Proteínas/farmacologia , Células Tumorais Cultivadas/metabolismoRESUMO
Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.
Assuntos
Reparo do DNA , Escherichia coli/enzimologia , Metiltransferases/metabolismo , Salmonella typhimurium/enzimologia , Cisteína/análogos & derivados , Cisteína/análise , Cisteína/metabolismo , Escherichia coli/fisiologia , Guanina/análogos & derivados , Guanina/análise , Cinética , Metanossulfonato de Metila/farmacologia , Metilnitronitrosoguanidina/farmacologia , Metiltransferases/isolamento & purificação , Peso Molecular , O(6)-Metilguanina-DNA MetiltransferaseRESUMO
Human PTH-related protein (hPTHrP) has been characterized as a product of tumor cells with sequence homology to the biologically active amino-terminal portion of human PTH (hPTH). We measured the relative activities of synthetic amino-terminal sequences of hPTH-(1-34) and hPTHrP-(1-34) to stimulate production of cAMP in intact human SaOS-2 osteosarcoma cells. Both peptides enhanced cAMP production at concentrations of 2.5-7.5 X 10(-10) M, had parallel dose-response curves, and were of essentially equal potency. Preincubation of SaOS-2 cells with hPTH-(1-34) or hPTHrP-(1-34) for 1 or 4 h induced homologous desensitization to a second challenge with the same peptide as well as heterologous desensitization to the other PTH peptide, but had little or no effect on the action of vasoactive intestinal peptide; the magnitudes of homologous and heterologous desensitization induced by the same doses of hPTHrP-(1-34) or hPTH-(1-34) were similar. Bone resorption-stimulating activity was measured using 40Ca2+ release from neonatal mouse calvariae in organ culture after 72 h of incubation. hPTHrP-(1-34) gave a dose-response between 0.2 and 5 ng/ml (5 X 10(-11) and 1.2 X 10(-9) M), was about 3 times more potent than Lilly bovine PTH standard (assuming a SA of 3000 U/mg; 100 U/ml), gave the same maximum response as hPTH-(1-34), and was 20-30% as potent as hPTH-(1-34). Neither hPTH-(1-34) nor hPTHrP-(1-34) enhanced prostaglandin production in mouse calvariae, and indomethacin did not inhibit the bone resorption-stimulating activities of either peptide. We conclude that hPTHrP-(1-34) and hPTH-(1-34) have similar high specific biological activities to stimulate production of cAMP in human osteoblast-like cells, but that hPTHrP-(1-34) is modestly less potent than hPTH-(1-34) to stimulate bone resorption in mouse calvariae.
Assuntos
Reabsorção Óssea/efeitos dos fármacos , Osso e Ossos/metabolismo , Proteínas de Neoplasias/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas , Animais , Animais Recém-Nascidos/metabolismo , Osso e Ossos/efeitos dos fármacos , Cálcio/metabolismo , AMP Cíclico/biossíntese , Dinoprostona/biossíntese , Humanos , Camundongos , Técnicas de Cultura de Órgãos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteossarcoma , Teriparatida , Células Tumorais Cultivadas , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
Osteoarticular sporotrichosis was diagnosed in a dog referred for evaluation of hindlimb lameness. There was radiographic evidence of osteopenia of the fourth tarsal and proximal aspects of the metatarsal bones. The diagnosis was based on histologic findings and results of physical examination, radiography, fungal culturing, and serologic tests. The dog was treated successfully with ketoconazole for 3 1/2 months.