RESUMO
Bisphosphonates (BPs), potent inhibitors of bone resorption which inhibit osteoclasts, have also been shown to act on osteocytes and osteoblasts preventing apoptosis via connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. We previously demonstrated the presence of a saturable, specific and high affinity binding site for alendronate (ALN) in osteoblastic cells which express Cx43. However, cells lacking Cx43 also bound BPs. Herein we show that bound [(3)H]-alendronate is displaced by phosphatase substrates. Moreover, similar to Na3VO4, ALN inhibited the activity of transmembrane and cytoplasmic PTPs, pointing out the catalytic domain of phosphatases as a putative BP target. In addition, anti-phospho-tyrosine immunoblot analysis revealed that ALN stimulates tyrosine phosphorylation of several proteins of whole cell lysates, among which the major targets of the BP could be immunochemically identified as Cx43. Additionally, the transmembrane receptor-like PTPs, RPTPµ and RPTPα, as well as the cytoplasmic PTP1B, are highly expressed in ROS 17/2.8 cells. Furthermore, we evidenced that Cx43 interacts with RPTPµ in ROS 17/2.8 and ALN decreases their association. These results support the hypothesis that BPs bind and inhibit PTPs associated to Cx43 or not, which would lead to the activation of signaling pathways in osteoblasts.
Assuntos
Alendronato/farmacologia , Conservadores da Densidade Óssea/farmacologia , Conexina 43/metabolismo , Inibidores Enzimáticos/farmacologia , Osteoblastos/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Animais , Células Cultivadas , Células HeLa , Humanos , Osteoblastos/metabolismo , Fosforilação/efeitos dos fármacos , RatosRESUMO
Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. Previously, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. However, using whole cell binding assays with [(3)H]-alendronate, herein we demonstrated the presence of saturable, specific and high affinity binding sites in the Cx43-expressing ROS 17/2.8 osteoblastic cells, authentic osteoblasts and MLO-Y4 cells expressing Cx43 or not, as well as in HeLa cells lacking Cx43 expression and ROS 17/2.8 cells pretreated with agents that disassemble Cx channels. In addition, both BPs and the PTP inhibitor Na(3)VO(4) increased proliferation of cells expressing Cx43 or not. Furthermore, although BPs are internalized and inhibit intracellular enzymes in osteoclasts, whether the drugs penetrate non-resorptive bone cells is not known. To clarify this, we evaluated the osteoblastic uptake of AF-ALN, a fluorescently labeled analog of alendronate. AF-ALN was rapidly internalized in cells expressing Cx43 or not indicating that this process is not mediated via Cx43 hemichannels. Altogether, these findings suggest that although required for triggering intracellular survival signaling by BPs, Cx43 is dispensable for cellular BP binding, its uptake, as well as the proliferative effects of these agents.
Assuntos
Alendronato/farmacocinética , Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacocinética , Proliferação de Células/efeitos dos fármacos , Conexina 43/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteócitos/metabolismo , Alendronato/farmacologia , Animais , Conservadores da Densidade Óssea/farmacologia , Conexina 43/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Canais Iônicos/metabolismo , Camundongos , Osteócitos/citologia , Vanadatos/farmacologiaRESUMO
INTRODUCTION: There is currently a growing interest for conducting studies into the electrical and neurochemical activity of the pedunculopontine nucleus (PPN) due to the privileged position occupied by this structure in the flow of information to and from the cortex. This nucleus acts as a relay, not only for the motor information that is processed in the basal ganglia but also for information of an emotional type, whose main centre is the nucleus accumbens. It is also strongly linked with the aspects that determine the mechanisms governing addiction to certain drugs. DEVELOPMENT: We conduct a detailed analysis of the main findings from studies of the role played by the PPN in the physiopathology of Parkinsonism, namely the study of metabolic activity, immunohistochemical studies with different tracers, electrophysiological studies that have confirmed the immunohistochemical observations, as well as deep electrical stimulation carried out in non human primates. Furthermore, we also examine the part played by this structure in the processing of emotional information associated with different learning tasks. CONCLUSIONS: Overall, the authors grant the PPN a privileged position in the physiopathology of the axial disorders related to Parkinson s disease; its most important afference, stemming from the subthalamic nucleus, appears to play a key role in the understanding of the part played by the PPN in Parkinsonism.
Assuntos
Emoções , Atividade Motora/fisiologia , Núcleo Tegmental Pedunculopontino/fisiologia , Animais , Humanos , Núcleo Accumbens/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Núcleo Tegmental Pedunculopontino/anatomia & histologiaRESUMO
INTRODUCTION: Microdialysis cerebral technique has been widely employed in order to study the neurotransmitter release. DEVELOPMENT: This technique present numerous advantages such as: allow to work with sample in vivo from animals freely moving, by means of microdialysis can be infused simultaneously different drugs in different points implanted probes in several coordinates; also allow carry out pharmacokinetics studies that show correlation with behavior patter as well as to study metabolic changes, which is not possible when this determination are carry out in tissue, in post mortem stadio. CONCLUSIONS: In the present work is carry out a review about the main results obtaining in the sensitization and abstinence to several drug study and approach to biochemical characteristics to Parkinson s disease (PD) by means of microdialysis technique. In relation to sensibilization studies, the temporal changes in different behavior pattern that modify after amphetamine administration have been studied. Microdialysis study allowed correlationate dopamine concentration with behavior pattern above mentioned. In relation with PD, dopamine concentration after systemic and central (intracerebral) administration of levodopa and another dopaminergic drugs have been studied in different nucleus of basal ganglia as well as its respectively behavior correlates.