RESUMO
Astrocytes take up glucose via the 45â¯kDa isoform of the Glucose Transporter 1 (GLUT-1), and in this work we have investigated whether histamine regulates GLUT-1 expression in rat cerebro-cortical astrocytes in primary culture. Cultured astrocytes expressed histamine H1 and H3 receptors (H1Rs and H3Rs) as evaluated by radioligand binding. Receptor functionality was confirmed by the increase in the intracellular concentration of Ca2+ (H1R) and the inhibition of forskolin-induced cAMP accumulation (H3R). Quantitative RT-PCR showed that histamine and selective H1R and H3R agonists (1â¯h incubation) significantly increased GLUT-1 mRNA to 153⯱â¯7, 163⯱â¯2 and 168⯱â¯13% of control values, respectively. In immunoblot assays, incubation (3â¯h) with histamine or H1R and H3R agonists increased GLUT-1 protein levels to 224⯱â¯12, 305⯱â¯11 and 193⯱â¯13% of control values, respectively, an action confirmed by inmunocytochemistry. The effects of H1R and H3R agonists were blocked by the selective antagonists mepyramine (H1R) and clobenpropit (H3R). The pharmacological inhibition of protein kinase C (PKC) prevented the increase in GLUT-1 protein induced by either H1R or H3R activation. Furthermore, histamine increased ERK-1/2 phosphorylation, and the effect of H1R and H3R activation on GLUT-1 protein levels was reduced or prevented, respectively, by MEK-1/2 inhibition. These results indicate that by activating H1Rs and H3Rs histamine regulates the expression of GLUT-1 by astrocytes. The effect appears to involve the phospholipase C (PLC) â diacylglycerol (DAG)/Ca2+â PKC and PLC â DAG/Ca2+ â PKC â MAPK pathways.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Transportador de Glucose Tipo 1/biossíntese , Agonistas dos Receptores Histamínicos/farmacologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , AMP Cíclico/metabolismo , Histamina/metabolismo , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Receptores Histamínicos H1/efeitos dos fármacos , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H3/efeitos dos fármacos , Receptores Histamínicos H3/metabolismoRESUMO
We have investigated the effect of the local activation of histamine H3 receptors (H3Rs) in the rat prefrontal cortex (PFCx) on the impairment of pre-pulse inhibition (PPI) of the startle response induced by the systemic administration of MK-801, antagonist at glutamate N-Methyl-d-Aspartate (NMDA) receptors, and the possible functional interaction between H3Rs and MK-801 on PFCx dopaminergic transmission. Infusion of the H3R agonist RAMH (19.8â¯ng/1⯵l) into the PFCx reduced or prevented the inhibition by MK-801 (0.15â¯mg/kg, ip) of PPI evoked by different auditory stimulus intensities (5, 10 and 15â¯dB), and the RAMH effect was blocked by the H3R antagonist/inverse agonist ciproxifan (30.6â¯ng/1⯵l). MK-801 inhibited [3H]-dopamine uptake (-45.4⯱â¯2.1%) and release (-32.8⯱â¯2.6%) in PFCx synaptosomes or slices, respectively, and molecular modeling indicated that MK-801 binds to and blocks the rat and human dopamine transporters. However, H3R activation had no effect on the inhibitory action of MK-801 on dopamine uptake and release. In PFCx slices, MK-801 and the activation of H3Rs or dopamine D1 receptors (D1Rs) stimulated ERK-1/2 and Akt phosphorylation. The co-activation of D1Rs and H3Rs prevented ERK-1/2 and Akt phosphorylation, and H3R activation or D1R blockade prevented the effect of MK-801. In ex vivo experiments, the intracortical infusion of the D1R agonist SKF-81297 (37â¯ng/1⯵l) or the H3R agonist RAMH increased Akt phosphorylation, prevented by D1R/H3R co-activation. These results indicate that MK-801 enhances dopaminergic transmission in the PFCx, and that H3R activation counteracts the post-synaptic actions of dopamine.
Assuntos
Maleato de Dizocilpina/farmacologia , Inibição Pré-Pulso/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Histamínicos H3/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Reflexo de Sobressalto/efeitos dos fármacos , Animais , Benzazepinas/administração & dosagem , Benzazepinas/farmacologia , Maleato de Dizocilpina/administração & dosagem , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Agonistas dos Receptores Histamínicos/administração & dosagem , Agonistas dos Receptores Histamínicos/farmacologia , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Masculino , Microinjeções , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Trítio/metabolismoRESUMO
We previously reported that the activation of histamine H3 receptors (H3Rs) selectively counteracts the facilitatory action of adenosine A2A receptors (A2ARs) on GABA release from rat globus pallidus (GP) isolated nerve terminals (synaptosomes). In this work, we examined the mechanisms likely to underlie this functional interaction. Three possibilities were explored: (a) changes in receptor affinity for agonists induced by physical A2AR/H3R interaction, (b) opposite actions of A2ARs and H3Rs on depolarization-induced Ca2+ entry, and (c) an A2AR/H3R interaction at the level of adenosine 3',5'-cyclic monophosphate (cAMP) formation. In GP synaptosomal membranes, H3R activation with immepip reduced A2AR affinity for the agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680) (Ki control 4.53 nM; + immepip 9.32 nM), whereas A2AR activation increased H3R affinity for immepip (Ki control 0.63 nM; + CGS-21680 0.26 nM). Neither A2AR activation nor H3R stimulation modified calcium entry through voltage-gated calcium channels in GP synaptosomes, as evaluated by microfluorometry. A2AR-mediated facilitation of depolarization-evoked [2,3-3H]-γ-aminobutyric acid ([3H]-GABA) release from GP synaptosomes (130.4 ± 3.6% of control values) was prevented by the PKA inhibitor H-89 and mimicked by the adenylyl cyclase activator forskolin or by 8-Bromo-cAMP, a membrane permeant cAMP analogue (169.5 ± 17.3 and 149.5 ± 14.5% of controls). H3R activation failed to reduce the facilitation of [3H]-GABA release induced by 8-Bromo-cAMP. In GP slices, A2AR activation stimulated cAMP accumulation (290% of basal) and this effect was reduced (- 75%) by H3R activation. These results indicate that in striato-pallidal nerve terminals, A2ARs and H3Rs interact at the level of cAMP formation to modulate PKA activity and thus GABA release.
Assuntos
Globo Pálido/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores Histamínicos H3/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Potenciais Evocados/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 µM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.