RESUMEN
Excitatory and inhibitory ionotropic receptors are regulated by protein kinases and phosphatases, which are localized to specific subcellular locations by one of several anchoring proteins. One of these is the A-kinase anchoring protein (AKAP150), which confers spatial specificity to protein kinase A and protein phosphatase 2B in the rat brain. The distribution of AKAP150 was examined at rat hippocampal CA1 pyramidal cell asymmetric and symmetric post-synaptic densities and with respect to the distribution of markers of excitatory (vesicular glutamate transporter 1, glutamate receptor subunit 1) and inhibitory receptors (vesicular GABA transporter, GABA receptor type A beta2/3 subunits, gephyrin) and the Golgi marker, trans-Golgi network glycoprotein 38. AKAP150 was close to asymmetric synapses, consistent with numerous molecular and biochemical studies suggesting its interaction with components of the excitatory postsynaptic density. In contrast, we did not find AKAP150-immunoreactivity associated with inhibitory synapses in rat CA1 neurons, despite reports demonstrating an in vitro interaction between AKAP150 and GABA receptor type A receptor beta subunits, and the reported co-localization of these proteins in rat hippocampal cultures. There was some overlap between AKAP150 and GABA receptor type A receptor beta2/3-immunoreactivity intracellularly in perinuclear clusters. These findings support previous work indicating the integration of kinase and phosphatase activity at excitatory synapses by AKAP150, but do not support a role for selective targeting of AKAP150 and its accompanying proteins to inhibitory synapses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hipocampo/citología , Células Piramidales/metabolismo , Sinapsis/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteínas de Anclaje a la Quinasa A , Animales , Proteínas Portadoras/metabolismo , Inmunohistoquímica/métodos , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica/métodos , Células Piramidales/citología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de GABA-A/metabolismo , Fracciones Subcelulares/metabolismo , Sinapsis/ultraestructura , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
Benzodiazepines are used to treat the anxiety associated with cocaine withdrawal, as well as cocaine-induced seizures. Since cocaine exposure was shown to affect BZ binding density, abuse liability, subjective hypnotic actions and seizure susceptibility, we assessed whether chronic cocaine alters diazepam's anxiolytic and anticonvulsant actions. Changes in GABA(A) receptor subunit protein expression were also assessed as they may relate to BZ activity at the receptor. Male Sprague-Dawley rats were injected with cocaine-HCl (15 mg/kg, i.p.) or saline once daily for 14 days. One day after the last injection, DZP (1 mg/kg i.p.) significantly increased time spent on and entries into open arms of an elevated plus maze in both saline- and cocaine-treated groups, yet the effect was greater in cocaine-treated rats. Eight days after cessation of treatment DZP did not have a significant anxiolytic effect in either group. To assess the effect of cocaine on DZP's anticonvulsant actions, PTZ was infused at a constant rate via the lateral tail vein and clonus onset was recorded in the presence and absence of DZP (5 mg/kg, i.p). DZP significantly elevated seizure threshold in both groups of rats. Chronic cocaine also had no effect on the beta-CCM seizure threshold. Quantitative immunohistochemistry of GABA(A) receptor subunit protein demonstrated significant regulation of alpha2 (-10%) and beta3 (+9%) subunits in the hippocampal dentate gyrus and CA1 regions, respectively. Small changes in GABAR subunit expression in specific brain areas may relate to DZP's enhanced anxiolytic effectiveness whereas it's anticonvulsant actions likely remain intact following cocaine administration.
Asunto(s)
Ansiolíticos/uso terapéutico , Anticonvulsivantes/uso terapéutico , Ansiedad/tratamiento farmacológico , Cocaína/farmacología , Diazepam/uso terapéutico , Inhibidores de Captación de Dopamina/farmacología , Receptores de GABA-A/efectos de los fármacos , Convulsiones/tratamiento farmacológico , Animales , Convulsivantes , Interacciones Farmacológicas , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Pentilenotetrazol , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Convulsiones/inducido químicamenteRESUMEN
One week oral flurazepam (FZP) administration in rats results in anticonvulsant tolerance in vivo, tolerance measured in vitro in hippocampal CA1 pyramidal cells, and regulation of hippocampal gamma-aminobutyric acid(A)-receptor subunit protein expression. A single injection (4 or 20 mg/kg i.p) of the benzodiazepine antagonist flumazenil (FLM) was given 1 day after FZP treatment, and tolerance and subunit protein expression were evaluated 1 day later. In vivo tolerance was measured by a reduced ability of the alpha(1)-subunit-selective agonist zolpidem to suppress pentylenetetrazole-induced seizures. This tolerance was reversed by 20 but not 4 mg/kg FLM. In in vitro hippocampal slices, there was tolerance to the effect of zolpidem to prolong the decay of pyramidal cell miniature inhibitory postsynaptic currents, which was reversed by FLM (4 mg/kg) pretreatment. A reduction in miniature inhibitory postsynaptic current amplitude ( approximately 50%) was also restored by FLM injection. [(3)H]Zolpidem binding measured 0, 2, and 7 days after FZP treatment was significantly decreased in the hippocampus and cortex at 0 days but not thereafter. Changes in alpha(1)- and beta(3)-subunit protein expression were examined via quantitative immunohistochemical techniques. alpha(1)-Subunit protein levels were down-regulated in the CA1 stratum oriens and beta subunit levels were up-regulated in the stratum oriens and stratum radiatum of the CA3 region. Chronic FZP effects on alpha(1)- and beta(3)-subunit protein levels were also reversed by prior FLM injection. FLM's effect on both functional and structural correlates of benzodiazepine tolerance suggests that each of these measures plays an interdependent role in mediating benzodiazepine tolerance.