RESUMO
Nitric oxide generation is related to the activity of certain proteins located at synaptic sites. Previous findings show that NOS activity, nNOS protein expression, respiratory parameters and mitochondrial complex activities are altered in rat cerebral cortex by administration of levocabastine, an antagonist of histamine H1 and neurotensin NTS2 receptors. ATP provision by mitochondria may play an important role in the functional interaction between synaptic proteins NMDA receptor and PSD-95 with NO synthesis. In this context, our purpose was to evaluate the effect of levocabastine administration on protein expression of PSD-95, GluN2B and iNOS, as well as on mitochondrial ATP production. Male Wistar rats received a single (i.p.) dose of levocabastine (50 µg/kg) or saline solution (controls) and were decapitated 18 h later. Mitochondrial and synaptosomal membrane fractions were isolated from cerebral cortex by differential and sucrose gradient centrifugation. Expression of synaptic proteins was evaluated by Western blot assays in synaptosomal membrane fractions. Oxygen consumption, mitochondrial membrane potential and ATP production rate were determined in fresh crude mitochondrial fractions. After levocabastine treatment, protein expression of PSD-95, GluN2B and ß-actin decreased 97, 45 and 55%, respectively, whereas that of iNOS enhanced 3.5-fold versus controls. In crude mitochondrial fractions levocabastine administration reduced roughly 15% respiratory control rate as assayed with malate-glutamate or succinate as substrates, decreased mitochondrial membrane potential (21%), and ATP production rates (57%). Results suggested that levocabastine administration induces alterations in synaptic proteins of the protein complex PSD-95/NMDA receptor/nNOS and in neuron cytoskeleton. Mitochondrial bioenergetics impairment may play a role in the functional link between synaptic proteins and NO synthesis.
Assuntos
Proteína 4 Homóloga a Disks-Large/metabolismo , Antagonistas dos Receptores Histamínicos H1/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinaptossomos/efeitos dos fármacosRESUMO
Neurotensin is known to inhibit neuronal Na+ , K+ -ATPase, an effect that is rescued by nitric oxide (NO) synthase inhibition. However, whether the neurotensinergic and the nitrergic systems are independent pathways, or are mechanistically linked, remains unknown. Here, we addressed this issue and found that the administration of low affinity neurotensin receptor (NTS2) antagonist, levocabastine (50 µg/kg, i.p.) inhibited NO synthase (NOS) activity by 74 and 42% after 18 h in synaptosomal and mitochondrial fractions isolated from the Wistar rat cerebral cortex, respectively; these effects disappeared 36 h after levocabastine treatment. Intriguingly, whereas neuronal NOS protein abundance decreased (by 56%) in synaptosomes membranes, it was enhanced (by 86%) in mitochondria 18 h after levocabastine administration. Levocabastine enhanced the respiratory rate of synaptosomes in the presence of oligomycin, but it failed to alter the spare respiratory capacity; furthermore, the mitochondrial respiratory chain (MRC) complexes I-IV activities were severely diminished by levocabastine administration. The inhibition of NOS and MRC complexes activities were also observed after incubation of synaptosomes and mitochondria with levocabastine (1 µM) in vitro. These data indicate that the NTS2 antagonist levocabastine regulates NOS expression and activity at the synapse, suggesting an interrelationship between the neurotensinergic and the nitrergic systems. However, the bioenergetics effects of NTS2 activity inhibition are likely to be independent from the regulation of NO synthesis.
Assuntos
Encéfalo/efeitos dos fármacos , Antagonistas não Sedativos dos Receptores H1 da Histamina/farmacologia , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/biossíntese , Piperidinas/farmacologia , Animais , Encéfalo/metabolismo , Masculino , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Receptores de Neurotensina/antagonistas & inibidoresRESUMO
Neurotensin behaves as a neuromodulator or as a neurotransmitter interacting with NTS1 and NTS2 receptors. Neurotensin in vitro inhibits synaptosomal membrane Na(+), K(+)-ATPase activity. This effect is prevented by administration of SR 48692 (antagonist for NTS1 receptor). The administration of levocabastine (antagonist for NTS2 receptor) does not prevent Na(+), K(+)-ATPase inhibition by neurotensin when the enzyme is assayed with ATP as substrate. Herein levocabastine effect on Na(+), K(+)-ATPase K(+) site was explored. For this purpose, levocabastine was administered to rats and K(+)-p-nitrophenylphosphatase (K(+)-p-NPPase) activity in synaptosomal membranes and [(3)H]-ouabain binding to cerebral cortex membranes were assayed in the absence (basal) and in the presence of neurotensin. Male Wistar rats were administered with levocabastine (50 µg/kg, i.p., 30 min) or the vehicle (saline solution). Synaptosomal membranes were obtained from cerebral cortex by differential and gradient centrifugation. The activity of K(+)-p-NPPase was determined in media laking or containing ATP plus NaCl. In such phosphorylating condition enzyme behaviour resembles that observed when ATP hydrolyses is recorded. In the absence of ATP plus NaCl, K(+)-p-NPPase activity was similar for levocabastine or vehicle injected (roughly 11 µmole hydrolyzed substrate per mg protein per hour). Such value remained unaltered by the presence of 3.5 × 10(-6) M neurotensin. In the phosphorylating medium, neurotensin decreased (32 %) the enzyme activity in membranes obtained from rats injected with the vehicle but failed to alter those obtained from rats injected with levocabastine. Levocabastine administration enhanced (50 %) basal [(3)H]-ouabain binding to cerebral cortex membranes but failed to modify neurotensin inhibitory effect on this ligand binding. It is concluded that NTS2 receptor blockade modifies the properties of neuronal Na(+), K(+)-ATPase and that neurotensin effect on Na(+), K(+)-ATPase involves NTS1 receptor and -at least partially- NTS2 receptor.