RESUMO
The spontaneously hypertensive rat (SHR) is an excellent animal model that mimics the behavioral and neurochemical phenotype of attention-deficit/hyperactivity disorder (ADHD). Here, we characterized the striatal GABA transport of SHR and investigated whether caffeine, a non-selective antagonist of adenosine receptors, could influence GABAergic circuitry. For this purpose, ex vivo striatal slices of SHR and Wistar (control strain) on the 35th postnatal day were dissected and incubated with [3H]-GABA to quantify the basal levels of uptake and release. SHR exhibited a reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system. GAT-1 appears to be the primary transporter for [3H]-GABA uptake in SHR striatum, as GAT-1 selective blocker, NO-711, completely abolished it. We also verified that acute exposure of striatal slices to caffeine improved [3H]-GABA uptake and release in SHR, whereas Wistar rats were not affected. GABA-uptake increase and cAMP accumulation promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA). As expected, the pharmacological blockade of cAMP-PKA signaling by H-89 also prevented caffeine-mediated [3H]-GABA uptake increment. Interestingly, a single caffeine exposure did not affect GAT-1 or A1R protein density in SHR, which was not different from Wistar protein levels, suggesting that the GAT-1-dependent transport in SHR has a defective functional activity rather than lower protein expression. The current data support that caffeine regulates GAT-1 function and improves striatal GABA transport via A1R-cAMP-PKA signaling, specifically in SHR. These results reinforce that caffeine may have therapeutic use in disorders where the GABA transport system is impaired.
Assuntos
Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Western Blotting , Corpo Estriado/metabolismo , Feminino , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos WistarRESUMO
Cocaine (COC) is a psychostimulant that acts by increasing catecholaminergic neurotransmission mainly due to its effects on the dopamine transporter (DAT). However, other neurotransmitter systems may also be regulated by COC, including the GABAergic system. Since the effect of COC in modulating gamma-aminobutyric acid (GABA) reuptake is not defined, we investigated the molecular mechanisms related to the increase in GABA uptake induced by acute COC exposure and its effects on locomotor activity in adolescent mice. Behavioral experiments showed that COC increased locomotor activity and decreased immobilization time in mice. A single COC exposure reduced both GABA uptake and GAT-1 protein levels. On the other hand, cyclic adenosine monophosphate (cAMP) levels increased after a COC challenge. The major changes induced by acute COC on behavioral and neurochemical assays were avoided by previous treatment with the selective D1 receptor antagonist SCH-23390 (0.5 mg/kg). Our findings suggest that GABA uptake naturally decreases during mice development from preadolescence until adulthood and that dopamine (DA) D1-like receptors are key players in the regulation of GABA uptake levels following a single COC exposure in adolescent mice.
Assuntos
Cocaína/farmacologia , Dopamina/metabolismo , Lobo Frontal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/efeitos dos fármacos , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/administração & dosagem , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Lobo Frontal/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta-adrenergic agonist, increased [3H]-GABA uptake in mice FC, while alpha1-adrenergic agonist phenylephrine had no effect. As GAT-1 is expressed and fully functional at the FC, addition of NO-711, a GAT-1 inhibitor, dose dependently blocked [3H]-GABA uptake. The increase of [3H]-GABA uptake induced by ISO was also blocked by NO-711. [3H]-GABA release induced by 80â¯mM KCl was reduced by NO-711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212-2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT-1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212-2.
Assuntos
Benzoxazinas/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Lobo Frontal/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de GABA/efeitos dos fármacos , Morfolinas/farmacologia , Naftalenos/farmacologia , Animais , Endocanabinoides/metabolismo , Lobo Frontal/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Camundongos , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/efeitos dos fármacosRESUMO
Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [(3)H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10min of cocaine administration and lasted for up to 30min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC.
Assuntos
Ácido Aspártico/metabolismo , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptores de Dopamina D1/metabolismo , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
l-Glutamate and l-aspartate are the main excitatory amino acids (EAAs) in the Central Nervous System (CNS) and their uptake regulation is critical for the maintenance of the excitatory balance. Excitatory amino acid transporters (EAATs) are widely distributed among central neurons and glial cells. GLAST and GLT1 are expressed in glial cells, whereas excitatory amino acid transporter 3/excitatory amino acid carrier 1 (EAAT3/EAAC1) is neuronal. Different signaling pathways regulate glutamate uptake by modifying the activity and expression of EAATs. In the present work we show that immature postnatal day 3 (PN3) rat retinas challenged by l-glutamate release [3H]-d-Aspartate linked to the reverse transport, with participation of NMDA, but not of non-NMDA receptors. The amount of [3H]-d-Aspartate released by l-glutamate is reduced during retinal development. Moreover, immature retinae at PN3 and PN7, but not PN14, exposed to a single dose of 200 or 500µM caffeine or the selective A2A receptor (A2AR) antagonist 100nM ZM241385 decreased [3H]-d-Aspartate uptake. Caffeine also selectively increased total expression of EAAT3 at PN7 and its expression in membrane fractions. However, both EAAT1 and EAAT2 were reduced after caffeine treatment in P2 fraction. Addition of 100nM DPCPX, an A1 receptor (A1R) antagonist, had no effect on the [3H]-d-Aspartate uptake. [3H]-d-Aspartate release was dependent on both extracellular sodium and Dl-TBOA, but not calcium, implying a transporter-mediated mechanism. Our results suggest that in the developing rat retina caffeine modulates [3H]-d-Aspartate uptake by blocking adenosine A2AR.
Assuntos
Ácido Aspártico/metabolismo , Cafeína/farmacologia , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Ácido Glutâmico/metabolismo , Retina/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Retina/metabolismo , Sódio/metabolismoRESUMO
Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® (Methylphenidate Hydrochloride; 2.5 mg/kg orally), (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [3H]-Dopamine uptake and cAMP accumulation in the prefrontal cortex (PFC) and are not responsive to dopaminergic stimuli in when compared to Wistar rats. After a 24-hour withdrawal of MPD, SHR did not present any alterations in [3H]-Dopamine Uptake, [3H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [3H]-Dopamine uptake, of the quantity of [3H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [3H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [3H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [3H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.