RESUMO
OBJECTIVE: To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, on the secretion of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMCs) and on the generation of reactive oxygen species (ROS) by leukocytes. METHODS: PBMCs and leukocytes were obtained from venous blood samples collected from 20 healthy individuals. VEGF secretion was evaluated using a commercial ELISA kit, while ROS production was determined using a luminol-dependent chemiluminescence assay. RESULTS: Rosiglitazone and calphostin C (a protein kinase C inhibitor) inhibited VEGF secretion by PBMCs by 63.7 and 62.3%, respectively. Both agents reduced ROS production in non-stimulated human leukocytes and down-regulated the enhanced generation of ROS in leukocytes that had been stimulated with the PKC activator phorbol 12,13-dibutyrate. CONCLUSION: The results support the involvement of PKC as a direct, and/or NADPH-oxidase as an indirect, target for the action of rosiglitazone on VEGF secretion by PBMCs and ROS production in human leukocytes.
Assuntos
Leucócitos Mononucleares/citologia , PPAR gama/metabolismo , Tiazolidinedionas/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Humanos , Hipoglicemiantes/farmacologia , Leucócitos/citologia , Luminescência , Pessoa de Meia-Idade , Naftalenos/farmacologia , Dibutirato de 12,13-Forbol/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio , RosiglitazonaRESUMO
Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.
Assuntos
Córtex Entorrinal/fisiologia , Odorantes , Bulbo Olfatório/metabolismo , Condutos Olfatórios/metabolismo , Dibutirato de 12,13-Forbol/metabolismo , Proteína Quinase C/metabolismo , Tonsila do Cerebelo/fisiologia , Animais , Autorradiografia , Cálcio/fisiologia , Ativação Enzimática/fisiologia , Hipocampo/fisiologia , Masculino , Ratos , Ratos Long-EvansRESUMO
Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.
Assuntos
Isoenzimas/metabolismo , Melatonina/farmacologia , Proteína Quinase C/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Filamentos Intermediários/enzimologia , Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Camundongos , Microscopia Eletrônica , Dibutirato de 12,13-Forbol/metabolismo , Proteína Quinase C-alfa , Proteína Quinase C-épsilon , Transporte Proteico , Trítio , Células Tumorais CultivadasRESUMO
The beta-adrenoceptor blocker propranolol stimulated testosterone secretion by rat testicular interstitial cells (Leydig cell-enriched preparation) in vitro at concentrations ranging from 10(-5) M to 10(-4) M. Treatment of these cells with H7 (20 microM), an inhibitor of protein kinase C, reduced the stimulatory effect of L-propranolol on testosterone secretion by about 5-fold. At concentrations ranging from 31.25 microM to 1000 microM, L-propranolol reduced [3H]phorbol 12,13-dibutyrate binding (IC50 = 75 microM) to rat testicular interstitial cells. At similar concentrations, L-propranolol displaced the binding of [3H]phorbol 12,13-dibutyrate to the homogenate of these cells by only 5%. These findings suggest that the effect of L-propranolol on [3H]phorbol 12,13-dibutyrate binding could be indirect, possibly by increasing the concentration of a chemical mediator interacting with the regulatory domain of protein kinase C. At even lower concentrations (10(-9) M to 10(-7) M), propranolol added directly to the reaction mixture with protein kinase C partially purified from rat testicular interstitial cells increases the phosphorylation of histone. This phosphorylation was comparable to that obtained with (25 microg/ml) phosphatidylserine. The D- and L-stereoisomers of propranolol were equally active. A complete reversal of this propranolol effect on histone phosphorylation was achieved with (20 microM) H-7. In the absence of Ca2+, propranolol was not able to phosphorylate the histone. Taken together, these results suggest that protein kinase C could be the putative kinase involved in this reaction and that its activation by propranolol may be due to interaction of the drug with the regulatory domain of the enzyme at a site differing from the site of interaction with phorbol 12,13-dibutyrate. The ability of propranolol to activate the putative protein kinase C could be related to its stimulatory effect on testosterone secretion by Leydig cells.
Assuntos
Histonas/metabolismo , Propranolol/farmacologia , Proteína Quinase C/metabolismo , Testículo/metabolismo , Testosterona/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Cromatografia em Agarose , Inibidores Enzimáticos/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Dibutirato de 12,13-Forbol/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Testículo/efeitos dos fármacos , TrítioRESUMO
It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 microM) and protein kinase C inhibitors (100 microM) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins.
Assuntos
Encéfalo/enzimologia , Melatonina/farmacologia , Proteína Quinase C/metabolismo , Animais , Calmodulina/antagonistas & inibidores , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Cinética , Dibutirato de 12,13-Forbol/metabolismo , Ligação Proteica , RatosRESUMO
Several lines of evidence indicate that protein kinase C (PKC) participates in long-term potentiation (LTP) and in certain forms of learning. Here we describe a rapid, specific and time-dependent increase in [3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding to membrane-associated PKC in selected brain regions of rats submitted to an inhibitory avoidance task. A quantitative film autoradiographic method was used to determine the amount and distribution of membrane-bound PKC in rats sacrificed at various time intervals after training. At 0, 30 and 120 min following training there was a prominent increase (up to 200%) in the binding of [3H]PDBu throughout the hippocampus relative to naive, shocked or habituated control groups. No significant changes in [3H]PDBu binding in any brain region were found at 180 min after training. Similar training-specific increments in the binding of [3H]PDBu were observed in the frontal, parietal and entorhinal cerebral cortices, amygdala and cerebellum. The maximal effect was seen at 30 min in the CA2 region of the hippocampus (+200%) and at 30 and 120 min after training in the amygdala (+170%) in comparison to naive control values. No alterations in [3H]PDBu binding were found in the other brain regions studied. The present findings, together with previous data reporting a similar temporal course in the effects of intrahippocampal or intraamygdala infusion of specific PKC inhibitors on memory, suggest that PKC activation plays a role in the acquisition and consolidation of an inhibitory avoidance learning.