RESUMO
3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 µM and a pure necrotic response at 60 µM. Low concentrations of 3-BrP (10-20 µM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 µM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Leucemia Mieloide/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases/metabolismo , Piruvatos/farmacologia , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/agonistas , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Glicólise/efeitos dos fármacos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Necrose/induzido quimicamente , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Quinases/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Non-small cell lung cancer (NSCLC) is the most frequent form of lung cancer and represents a set of histological entities that have an ominous long-term prognosis, for example, adenocarcinoma, squamous carcinoma and large cell carcinoma. Both small cell and non-small cell lung cancer are the main causes of oncological death and the oncological diseases with the highest incidence worldwide. With regard to clinical approaches for NSCLC, several advances have been achieved in diagnosis and treatment; the analysis of different molecular markers has led to the development of new targeted therapies that have improved the prognosis for selected patients. Despite this, most patients are diagnosed in an advanced stage, presenting a limited life expectancy with an ominous short-term prognosis. Numerous molecular alterations have been described in recent years, allowing for the development of therapies directed against specific therapeutic targets. The correct identification of the expression of different molecular markers has allowed for the individualization of treatment throughout the disease course, expanding the available therapeutic arsenal. The purpose of this article is to summarize the main characteristics of NSCLC and the advances that have occurred in the use of targeted therapies, thus explaining the limitations that have been observed in the management of this disease.
RESUMO
B-cell chronic lymphocytic leukemia (B-CLL) is an abnormal neoplasic proliferation of B cells, which accumulate mainly in the bone marrow and blood preventing both B cells development in the lymph nodes and the ability to fight against infection. The antitumor agents used in chemotherapy are aimed at inducing malignant cell death, thus limiting the growth and spreading of these cells. However, the lack of specificity for tumor cells exhibited by these agents causes undesirable side effects that have led to the investigation of new therapeutic strategies designed to specifically target malignant cells and thus trigger selective cell destruction. Dequalinium (DQA) is an antitumoral agent that selectively accumulates in the mitochondria and has been shown to display anticancer activity in cells from different malignancies. In the present study, the DQA-induced cytotoxicity in B-CLL cells was analyzed by measuring cell viability and cell death, either by necrosis or apoptosis. Our results support the importance of DQA as a selective and potential antileukemic drug with a higher cytotoxic effect on peripheral blood mononuclear cells from B-CLL patients than in those from healthy donors and encourage the performance of further studies in combination with other agents.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Dequalínio/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Dequalínio/toxicidade , Humanos , Técnicas In Vitro , Leucemia Linfocítica Crônica de Células B/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Necrose/induzido quimicamenteRESUMO
The discovery of novel protein biomarkers in melanoma is crucial. Our introduction of formalin-fixed paraffin-embedded (FFPE) tumor protocol provides new opportunities to understand the progression of melanoma and open the possibility to screen thousands of FFPE samples deposited in tumor biobanks and available at hospital pathology departments. In our retrospective biobank pilot study, 90 FFPE samples from 77 patients were processed. Protein quantitation was performed by high-resolution mass spectrometry and validated by histopathologic analysis. The global protein expression formed six sample clusters. Proteins such as TRAF6 and ARMC10 were upregulated in clusters with enrichment for shorter survival, and proteins such as AIFI1 were upregulated in clusters with enrichment for longer survival. The cohort's heterogeneity was addressed by comparing primary and metastasis samples, as well comparing clinical stages. Within immunotherapy and targeted therapy subgroups, the upregulation of the VEGFA-VEGFR2 pathway, RNA splicing, increased activity of immune cells, extracellular matrix, and metabolic pathways were positively associated with patient outcome. To summarize, we were able to (i) link global protein expression profiles to survival, and they proved to be an independent prognostic indicator, as well as (ii) identify proteins that are potential predictors of a patient's response to immunotherapy and targeted therapy, suggesting new opportunities for precision medicine developments.
RESUMO
Activation of NMDA receptors leads to nitric oxide (NO) synthesis by NO synthase (NOS) from L-arginine. Neuronal NOS colocalizes with somatostatinergic (SRIF) neurons and there is growing evidence of an interaction between NO and the cerebral SRIFergic system in several neurological diseases. Our aim was to study the effect of L-arginine on the regulation of the SRIFergic system in the frontoparietal cortex of male Sprague-Dawley rats. Intraperitoneal administration of L-arginine (150 mg/Kg), twice-daily during eight days, induced a decrease in SRIF receptor density, which was accompanied by a reduction in the capacity of SRIF to stimulate inositol-1,4,5-triphosphate (IP3) accumulation and SRIF-like immunoreactivity (SRIF-LI) levels. To determine if these changes were related to L-arginine-derived NO synthesis, a NOS inhibitor, Nω-nitro-L-arginine methyl ester was coadministered with L-arginine. Its coadministration prevented the reduction in the SRIF receptor density, accumulation of IP3 and SRIF-LI content. These findings indicate that L-arginine induces a deleterious effect on the cortical somatostatinergic system and that the inhibition of NOS could be helpful in some neurological disorders where this neurotransmitter system is affected.
Assuntos
Regulação para Baixo/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Somatostatina/efeitos dos fármacos , Animais , Arginina/farmacologia , Inibidores Enzimáticos/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Ratos Sprague-Dawley , Somatostatina/metabolismoRESUMO
If melatonin or its analogs are to be used therapeutically in humans, their chronic effects on responsiveness of melatonin target cells need to be assessed. We have previously demonstrated that acute melatonin treatment regulates the somatostatinergic system in the rat hippocampus. In the present study, we have investigated the effects of subchronic and chronic daily treatment with melatonin on the somatostatinergic system in the rat hippocampus. Male Wistar rats (200-250 g) were injected with melatonin (25 microg/kg body weight, subcutaneously) daily for 4, 7 or 14 days and sacrificed 24 h after the last injection. Melatonin administration for 4 days induced a decrease in the hippocampal somatostatin (SRIF)-like immunoreactivity content as well as a decrease in the number of SRIF receptors and an increase in their apparent affinity. The decreased number of SRIF receptors in the melatonin (4 days)-treated rats was associated with a decreased capacity of SRIF to inhibit both basal and forskolin-stimulated adenylyl cyclase activity. These melatonin-induced effects reversed to control values after 7 or 14 days of treatment. Hippocampal membranes from control and melatonin-treated rats showed similar Gi and Gs activities. Melatonin treatment altered neither the functional Gi activity nor the Gialpha 1 or Gialpha 2 levels at any of the time periods studied. The present results suggest that chronic exposure to melatonin results in a tolerance of the hippocampus to this hormone.
Assuntos
Antioxidantes/administração & dosagem , Hipocampo/efeitos dos fármacos , Melatonina/administração & dosagem , Somatostatina/análogos & derivados , Somatostatina/metabolismo , Adenilil Ciclases/metabolismo , Análise de Variância , Animais , Antioxidantes/farmacologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Western Blotting/métodos , Relação Dose-Resposta a Droga , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanilil Imidodifosfato/farmacologia , Hipocampo/metabolismo , Imuno-Histoquímica/métodos , Masculino , Melatonina/farmacologia , Radioimunoensaio/métodos , Ratos , Ratos Wistar , Receptores de Somatostatina/efeitos dos fármacos , Receptores de Somatostatina/metabolismo , Somatostatina/farmacocinética , Fatores de TempoRESUMO
Fatty acid synthesis and oxidation are frequently exacerbated in leukemia cells, and may therefore represent a target for therapeutic intervention. In this work we analyzed the apoptotic and chemo-sensitizing action of the fatty acid oxidation inhibitor etomoxir in human acute myeloid leukemia cells. Etomoxir caused negligible lethality at concentrations up to 100 µM, but efficaciously cooperated to cause apoptosis with the anti-leukemic agent arsenic trioxide (ATO, Trisenox), and with lower efficacy with other anti-tumour drugs (etoposide, cisplatin), in HL60 cells. Etomoxir-ATO cooperation was also observed in NB4 human acute promyelocytic cells, but not in normal (non-tumour) mitogen-stimulated human peripheral blood lymphocytes. Biochemical determinations in HL60 cells indicated that etomoxir (25-200 µM) dose-dependently inhibited mitochondrial respiration while slightly stimulating glycolysis, and only caused marginal alterations in total ATP content and adenine nucleotide pool distribution. In addition, etomoxir caused oxidative stress (increase in intracellular reactive oxygen species accumulation, decrease in reduced glutathione content), as well as pro-apoptotic LKB-1/AMPK pathway activation, all of which may in part explain the chemo-sensitizing capacity of the drug. Etomoxir also cooperated with glycolytic inhibitors (2-deoxy-D-glucose, lonidamine) to induce apoptosis in HL60 cells, but not in NB4 cells. The combined etomoxir plus 2-deoxy-D-glucose treatment did not increase oxidative stress, caused moderate decrease in net ATP content, increased the AMP/ATP ratio with concomitant drop in energy charge, and caused defensive Akt and ERK kinase activation. Apoptosis generation by etomoxir plus 2-deoxy-D-glucose was further increased by co-incubation with ATO, which is apparently explained by the capacity of ATO to attenuate Akt and ERK activation. In summary, co-treatment with etomoxir may represent an interesting strategy to increase the apoptotic efficacy of ATO and (with some limitations) 2-deoxy-D-glucose which, although clinically important anti-tumour agents, exhibit low efficacy in monotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Arsenicais/uso terapêutico , Desoxiglucose/uso terapêutico , Compostos de Epóxi/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Óxidos/uso terapêutico , Trióxido de Arsênio , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Humanos , Indazóis/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacosRESUMO
Myelodysplastic syndromes (MDS) are characterized by impaired proliferation and differentiation of hematopoietic stem cells. The participation of toll-like receptor (TLR)-mediated signaling in MDS is well documented. Increased TLR signaling leads to the constitutive activation of NF-κB, which mediates inflammation, cell proliferation and apoptosis. In addition, the TLR pathway induces the expression of miRNAs which participate in the fine-tuning of the inflammatory response. miRNAs also regulate other biological processes, including hematopoiesis. miR-125a and miR-125b are known modulators of hematopoiesis and are abnormally expressed in several hematologic malignancies. However, little is known about their role in MDS. NF-κB-activating ability has been described for both miRNAs. We studied the role of miR-125a/miR-125b in MDS and their relationship with TLR signaling and hematopoietic differentiation. Our results indicate that miR-125a is significantly overexpressed in MDS patients and correlates negatively with patient survival. Expression of miR-99b, which is clustered with miR-125a, is also directly correlated with prognosis of MDS. Both miR-125a and miR-99b activated NF-κB in vitro; however, we observed a negative correlation between miR-99b expression and the levels of TLR2, TLR7 and two downstream genes, suggesting that NF-κB activation by the miRNA cluster occurs in the absence of TLR signaling. We also show that TLR7 is negatively correlated with patient survival in MDS. In addition, our data suggest that miR-125a may act as an NF-κB inhibitor upon TLR stimulation. These results indicate that miR-125a is involved in the fine-tuning of NF-κB activity and that its effects may depend on the status of the TLR pathway. Furthermore, we observed that miR-125a inhibits erythroid differentiation in leukemia and MDS cell lines. Therefore, this miRNA could serve as a prognostic marker and a potential therapeutic target in MDS.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Eritropoese , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/genética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , NF-kappa B/metabolismo , Antígenos CD34/metabolismo , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Citarabina/farmacologia , Ativação Enzimática , Eritropoese/efeitos dos fármacos , Eritropoese/genética , Regulação da Expressão Gênica , Humanos , Células K562 , Modelos Biológicos , Família Multigênica , Síndromes Mielodisplásicas/mortalidade , Fator 88 de Diferenciação Mieloide , Prognóstico , Transdução de Sinais , Receptores Toll-Like/metabolismoRESUMO
This study examined the phytochemical profile and the in vitro anti-proliferative effects of a hot water mycelial extract from the edible mushroom Pleurotus sp. on NB4 human leukemia cells. Flow-cytometry analyses were used to measure cell viability, cell cycle, and apoptosis in cells incubated 24 h with the extract at doses of 100 and 200 µg/mL. Pleurotus sp. extract reduced cell viability, particularly at the concentration of 200 µg/mL to 82% compared to control cells, and induced apoptosis demonstrated by an increase in the number of annexin V-FITC+ cells (25% at 200 µg/mL). The NB4 cells were arrested in the G2/M phase thus supporting a cell-cycle dependent anticancer mechanism. Although carbohydrates (76.8%, w/w) appear to be the most important antitumor compound, secondary metabolites-like phenolics would also contribute to the anti-proliferative activity. The results indicate that Pleurotus sp. mycelia obtained by submerged fermentation may be an interesting renewable resource for developing functional foods and new antitumor therapeutic agents.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Leucemia/fisiopatologia , Micélio/química , Extratos Vegetais/farmacologia , Pleurotus/química , Verduras/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cuba , Inibidores do Crescimento/química , Inibidores do Crescimento/isolamento & purificação , Humanos , Leucemia/tratamento farmacológico , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificaçãoRESUMO
Delocalized lipophilic cations, such as dequalinium (DQA), selectively accumulate in mitochondria and display anticancer activity in cells from different malignancies. Previous studies in K562 human leukemic cells indicate that DQA causes cell damage as a consequence of an early disturbance in the mitochondrial function, inducing oxidative stress. These cells turned out to be resistant to apoptosis and died by necrosis when treated with high DQA concentrations (20 µmol/L) for long time periods (48 h). Resistance of K562 cells to DQA-induced apoptosis could be eliminated by inhibition of the kinase activity of the Bcr-Abl protein with imatinib. In this paper, we have studied the effect of DQA on the Raf/MEK/ERK1/2 and PI3K/Akt signal transduction pathways in K562 cells. Our data suggest a DQA downregulatory activity on both ERK1/2 and PI3K protein kinase activity supporting an interaction between both proteins. Moreover, inhibition of ERK1/2 with U0126 enhanced the ability of DQA to potentiate imatinib-induced apoptosis, suggesting a role of the Raf/MEK/ERK pathway and the Bcr-Abl tyrosine kinase in the K562 cell survival. This study contributes to a better understanding of the action mechanism of DQA on K562 cells and encourages the study of DQA in combination with other agents for improving the efficacy of targeted therapies and overcoming resistance to chemotherapeutic agents.
Assuntos
Apoptose , Dequalínio/toxicidade , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Quinases raf/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Tirosina Quinases/metabolismoRESUMO
While the anti-tumor efficacy of 2-deoxy-D-glucose (2-DG) is normally low in monotherapy, it may represent a valuable radio- and chemo-sensitizing agent. We here demonstrate that 2-10 mM 2-DG cooperates with arsenic trioxide (ATO) and other antitumor drugs to induce apoptosis in human myeloid leukemia cell lines. Using ATO and HL60 as drug and cell models, respectively, we observed that 2-DG/ATO combination activates the mitochondrial apoptotic pathway, as indicated by Bid-, and Bax-regulated cytochrome c and Omi/HtrA2 release, XIAP down-regulation, and caspase-9/-3 pathway activation. 2-DG neither causes oxidative stress nor increases ATO uptake, but causes inner mitochondria membrane permeabilization as well as moderate ATP depletion, which nevertheless do not satisfactorily explain the pro-apoptotic response. Surprisingly 2-DG causes cell line-specific decrease in LKB-1/AMPK phosphorylation/activation, and also causes Akt/mTOR/p70S6K and MEK/ERK activation, which is prevented by co-treatment with ATO. The use of kinase-specific pharmacologic inhibitors and/or siRNAs reveals that apoptosis is facilitated by AMPK inactivation and restrained by Akt and ERK activation, and that Akt and ERK activation mediates AMPK inhibition. Finally, 2-DG stimulates IGF-1R phosphorylation/activation, and co-treatment with IGF-1R inhibitor prevents 2-DG effects on Akt, ERK and AMPK, and facilitates 2-DG-provoked apoptosis. In summary 2-DG elicits IGF-1R-mediated AMPK inactivation and Akt and ERK activation, which facilitates or restrain apoptosis, respectively. 2-DG-provoked AMPK inactivation increases the apoptotic efficacy of ATO, while in turn ATO-provoked Akt and ERK inactivation may increase the efficacy of 2-DG as anti-tumor drug.
Assuntos
Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Desoxiglucose/farmacologia , Leucemia/patologia , Óxidos/farmacologia , Proteínas Quinases/metabolismo , Receptor IGF Tipo 1/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Trifosfato de Adenosina/metabolismo , Trióxido de Arsênio , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Leucemia/enzimologia , Leucemia/metabolismo , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidoresRESUMO
Lonidamine is a safe, clinically useful anti-tumor drug, but its efficacy is generally low when used in monotherapy. We here demonstrate that lonidamine efficaciously cooperates with the anti-leukemic agent arsenic trioxide (ATO, Trisenox) to induce apoptosis in HL-60 and other human leukemia cell lines, with low toxicity in non-tumor peripheral blood lymphocytes. Apoptosis induction by lonidamine/ATO involves mitochondrial dysfunction, as indicated by early mitochondrial permeability transition pore opening and late mitochondrial transmembrane potential dissipation, as well as activation of the intrinsic apoptotic pathway, as indicated by Bcl-X(L) and Mcl-1 down-regulation, Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release to the cytosol, XIAP down-regulation, and caspase-9 and -3 cleavage/activation, with secondary (Bcl-2-inhibitable) activation of the caspase-8/Bid axis. Lonidamine stimulates reactive oxygen species production, and lonidamine/ATO toxicity is attenuated by antioxidants. Lonidamine/ATO stimulates JNK phosphorylation/activation, and apoptosis is attenuated by the JNK inhibitor SP600125. In addition, lonidamine elicits ERK and Akt/mTOR pathway activation, as indicated by increased ERK, Akt, p70S6K and rpS6 phosphorylation, and these effects are reduced by co-treatment with ATO. Importantly, co-treatment with MEK/ERK inhibitor (U0126) and PI3K/Akt (LY294002) or mTOR (rapamycin) inhibitors, instead of ATO, also potentiates lonidamine-provoked apoptosis. These results indicate that: (i) lonidamine efficacy is restrained by drug-provoked activation of MEK/ERK and Akt/mTOR defensive pathways, which therefore represent potential therapeutic targets. (ii) Co-treatment with ATO efficaciously potentiates lonidamine toxicity via defensive pathway inhibition and JNK activation. And (iii) conversely, the pro-oxidant action of lonidamine potentiates the apoptotic efficacy of ATO as an anti-leukemic agent.
Assuntos
Arsenicais/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Indazóis/farmacologia , Óxidos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Leucemia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Estresse Oxidativo/efeitos dos fármacos , Transdução de SinaisRESUMO
alpha(1)-Adrenoceptor stimulation prolongs the duration of the cardiac action potentials and leads to positive inotropic effects by inhibiting the transient outward K(+) current (I(to)). In the present study, we have examined the role of several protein kinases and the G protein involved in I(to) inhibition in response to alpha(1)-adrenoceptor stimulation in isolated adult rat ventricular myocytes. Our findings exclude the classic alpha(1)-adrenergic pathway: activation of the G protein G(alphaq), phospholipase C (PLC), and protein kinase C (PKC), because neither PLC, nor PKC, nor G(alphaq) blockade prevents the alpha(1)-induced I(to) reduction. To the contrary, the alpha(1)-adrenoceptor does not inhibit I(to) in the presence of protein kinase A (PKA), adenylyl cyclase, or G(alphas) inhibitors. In addition, PKA and adenylyl cyclase activation inhibit I(to) to the same extent as phenylephrine. Finally, we have shown a functional coupling between the alpha(1)-adrenoceptor and G(alphas) in a physiological system. Moreover, this coupling seems to be compartmentalized, because the alpha(1)-adrenoceptor increases cAMP levels only in intact cells, but not in isolated membranes, and the effect on I(to) disappears when the cytoskeleton is disrupted. We conclude that alpha(1)-adrenoceptor stimulation reduces the amplitude of the I(to) by activating a G(alphas) protein and the cAMP/PKA signaling cascade, which in turn leads to I(to) channel phosphorylation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Norepinefrina/farmacologia , Técnicas de Patch-Clamp , Fenilefrina/farmacologia , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismoRESUMO
Melatonin and somatostatin are known to exert similar effects on locomotor activity. We have previously demonstrated that acute melatonin treatment regulates somatostatin receptor function in the rat frontoparietal cortex. However, the effects of subchronic and chronic melatonin treatment on the somatostatin receptor-G protein-adenylyl cyclase system in the rat frontoparietal cortex are unknown. Melatonin was administered subcutaneously at a daily dose of 25 microg/kg for 4 days, 1 wk or 2 wk. Twenty-four hours after the last injection, the animals were sacrificed. Melatonin did not alter the somatostatin-like immunoreactivity content in the frontoparietal cortex from control and melatonin-treated rats during any of the previously indicated periods. Four days of melatonin administration induced both an increase in the number of [(125)I]-Tyr11-somatostatin receptors and a decrease in the affinity of somatostatin for its receptors in frontoparietal cortical membranes. The increased number of somatostatin receptors in the melatonin-treated rats was associated with an increased capacity of somatostatin to inhibit basal and forskolin-stimulated adenylyl cyclase activity. Melatonin administration for 4 days induced a higher adenylyl cyclase activity both under basal conditions and after direct stimulation of the enzyme with forskolin. No significant differences were observed in the function of Gi proteins in the 4-day melatonin-treated rats. Western blot analyses showed that the 4-day melatonin treatment reduced Gialpha(2) levels, without altering the amount of Gialpha(1). These melatonin-induced changes reverted to control values after 7 or 14 days of treatment. Altogether, the present findings suggest that subchronic melatonin treatment modulates the somatostatin receptor/effector system in the rat frontoparietal cortex.