RESUMEN
A fast excitatory synaptic potential mediated by 5-hydroxytryptamine (5-HT) was recorded in rat lateral amygdala neurons in brain slices. The synaptic potential has brief duration (tens of milliseconds), is mimicked by 5-HT, is potentiated by a 5-HT uptake inhibitor, and is blocked by selective 5-HT3 receptor antagonists. The underlying synaptic current reversed polarity at about 0 mV. This is an example of fast neurotransmission in the mammalian brain mediated by an amine rather than an amino acid. The antiemetic, anxiolytic, and perhaps antipsychotic actions of 5-HT3 antagonists might result from blockade of such synapses.
Asunto(s)
Amígdala del Cerebelo/fisiología , Receptores de Serotonina/fisiología , Serotonina/farmacología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona , Potenciales de Acción/efectos de los fármacos , Animales , Estimulación Eléctrica , Imidazoles/farmacología , Indoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ondansetrón , Quinoxalinas/farmacología , Ratas , Antagonistas de la Serotonina/farmacología , Sinapsis/fisiología , Tropisetrón , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.
Asunto(s)
Calcio/farmacología , ADN/genética , Drosophila melanogaster/genética , Expresión Génica , Canales de Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Clonación Molecular , Conductividad Eléctrica , Datos de Secuencia Molecular , Oocitos/metabolismo , Reacción en Cadena de la Polimerasa , Canales de Potasio/química , Canales de Potasio/fisiología , ARN Mensajero/genética , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Transcripción Genética , Transfección , XenopusRESUMEN
The subthalamic nucleus (STN) plays a pivotal role in normal and abnormal motor function. We used patch pipettes to study effects of 5-HT on synaptic currents evoked in STN neurons by focal electrical stimulation of rat brain slices. 5-HT (10 microM) reduced glutamate-mediated excitatory postsynaptic currents (EPSCs) by 35+/-4%. However, a much higher concentration of 5-HT (100 microM) was required to inhibit GABA-mediated inhibitory postsynaptic currents (IPSCs) to a comparable extent. Concentration-response curves showed that the 5-HT inhibitory concentration 50% (IC50) for inhibition of IPSCs (20.2 microM) was more than fivefold greater than the IC50 for inhibition of EPSCs (3.4 microM). The 5-HT-induced reductions in EPSCs and IPSCs were accompanied by increases in paired-pulse ratios, indicating that 5-HT acts presynaptically to inhibit synaptic transmission. The 5-HT1B receptor antagonist NAS-181 significantly antagonized 5-HT-induced inhibitions of EPSCs and IPSCs. These studies show that 5-HT inhibits synaptic transmission in the STN by activating presynaptic 5-HT1B receptors.
Asunto(s)
Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Serotonina/farmacología , Núcleo Subtalámico/citología , Transmisión Sináptica/efectos de los fármacos , Animales , Benzamidas/farmacología , Benzopiranos/farmacología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Masculino , Morfolinas/farmacología , Oxadiazoles/farmacología , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Sprague-Dawley , Antagonistas de la Serotonina/farmacologíaRESUMEN
Firing patterns of subthalamic nucleus (STN) neurons influence normal and abnormal movements. The STN expresses multiple 5-HT receptor subtypes that may regulate neuronal excitability. We used whole-cell patch-clamp recordings to characterize 5-HT receptor-mediated effects on membrane currents in STN neurons in rat brain slices. In 80 STN neurons under voltage-clamp (-70 mV), 5-HT (30 microM) evoked inward currents in 64%, outward currents in 17%, and biphasic currents in 19%. 5-HT-induced outward current was caused by an increased K(+) conductance (1.4+/-0.2 nS) and was blocked by the 5-HT(1A) antagonist WAY 100135. The 5-HT-evoked inward current, which was blocked by antagonists at 5-HT(2C) and/or 5-HT(4) receptors, had two types of current-voltage (I-V) relations. Currents associated with the type 1 I-V relation showed negative slope conductance at potentials <-110 mV and were occluded by Ba(2+). In contrast, the type 2 I-V relation appeared linear and had positive slope conductance (0.64+/-0.11 nS). Type 2 inward currents were Ba(2+)-insensitive, and the reversal potential of -19 mV suggests a mixed cation conductance. In STN neurons in which 5-HT evoked inward currents, 5-HT potentiated burst firing induced by N-methyl-d-aspartate (NMDA). But in neurons in which 5-HT evoked outward current, 5-HT slowed NMDA-dependent burst firing. We conclude that 5-HT receptor subtypes can differentially regulate firing pattern by modulating multiple conductances in STN neurons.
Asunto(s)
Neuronas/fisiología , Receptores de Serotonina/fisiología , Núcleo Subtalámico/citología , Animales , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Sprague-Dawley , Serotonina/farmacología , Serotoninérgicos/farmacologíaRESUMEN
Whole-cell recordings were made from neurons of the rat locus coeruleus in a tissue slice removed from rat brain. Substance P caused an inward current in cells voltage-clamped at -60 mV. The effect of substance P was concentration-dependent (30 nM-3 microM) and was mimicked by similar concentrations of substance K and neuromedin K. The inward current resulted predominantly from an increase in membrane cation conductance; in potassium-free solutions it reversed polarity at about 12 mV. Substance P also reduced the conductance of an inwardly rectifying potassium current; this action was studied with low external sodium concentration. It is concluded that substance P excites rat locus coeruleus neurons by activating an intracellular transduction pathway leading to both cation conductance increase and potassium conductance decrease.
Asunto(s)
Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Locus Coeruleus/fisiología , Neuronas/fisiología , Canales de Potasio/efectos de los fármacos , Sustancia P/farmacología , Animales , Calcio/farmacología , Cationes , Cloruros/farmacología , Técnicas In Vitro , Canales Iónicos/fisiología , Magnesio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Neuroquinina A/farmacología , Neuroquinina B/farmacología , Neuronas/efectos de los fármacos , Potasio/farmacología , Canales de Potasio/fisiología , Ratas , Tetrodotoxina/farmacologíaRESUMEN
Effects of baclofen on synaptic transmission were studied in rat subthalamic neurons using whole-cell patch clamp recording from brain slices. Focal electrical stimulation of the brain slice evoked GABAergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents. Baclofen reduced the amplitude of evoked inhibitory postsynaptic currents in a concentration-dependent manner with an IC(50) of 0.6+/-0.2 microM. Evoked excitatory postsynaptic currents were also reduced by baclofen concentration-dependently (IC(50) of 1.6+/-0.2 microM), but baclofen was more potent at reducing the GABA(A) receptor inhibitory postsynaptic currents. The GABA(B) receptor antagonist CGP 35348 blocked these inhibitory effects of baclofen on evoked inhibitory and excitatory postsynaptic currents. Baclofen increased the paired-pulse ratios of evoked inhibitory and excitatory postsynaptic currents. Furthermore, baclofen reduced the frequency of spontaneous miniature excitatory postsynaptic currents, but had no effect on their amplitude. These results provide evidence for presence of presynaptic GABA(B) receptors that modulate both GABA and glutamate release from afferent terminals in the subthalamus.
Asunto(s)
Inhibición Neural/fisiología , Terminales Presinápticos/metabolismo , Receptores de GABA-B/fisiología , Núcleo Subtalámico/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Baclofeno/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Agonistas del GABA/farmacología , Ácido Glutámico/fisiología , Técnicas In Vitro , Masculino , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Núcleo Subtalámico/citología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Whole-cell patch clamp recordings were made from the subthalamic nucleus in rat brain slice preparations to examine the effect of adenosine on inhibitory and excitatory synaptic transmission. Adenosine reversibly inhibited both GABA-mediated inhibitory and glutamate-mediated excitatory postsynaptic currents. Adenosine at 100 microM reduced the amplitude of inhibitory and excitatory postsynaptic currents by 42+/-5% and 34+/-6%, respectively. Reductions in the amplitude of both inhibitory and excitatory postsynaptic currents were accompanied by increases in paired-pulse ratios. In addition, adenosine decreased the frequency of spontaneous miniature excitatory postsynaptic currents but had no effect on their amplitude. These results are consistent with a presynaptic site of action. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine completely reversed the adenosine-induced attenuation of inhibitory and excitatory postsynaptic currents, but 8-cyclopentyl-1,3-dipropylxanthine alone had no effect on synaptic currents evoked at 0.1 Hz. However, 8-cyclopentyl-1,3-dipropylxanthine inhibited a time-dependent depression of excitatory postsynaptic currents that was normally observed in response to a 5 Hz train of stimuli, suggesting that endogenous adenosine could be released during higher frequencies of stimulation. These results suggest that adenosine inhibits synaptic release of GABA and glutamate by stimulation of presynaptic A(1) receptors in the subthalamic nucleus.
Asunto(s)
Adenosina/fisiología , Núcleo Subtalámico/fisiología , Transmisión Sináptica , Adenosina/farmacología , Animales , Electrofisiología , Ácido Glutámico/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
In Parkinson's disease, there is evidence of impaired mitochondrial function which reduces the capacity to synthesize ATP in dopamine neurons. This would be expected to reduce the activity of the sodium pump (Na+/K+ ATPase), causing increased intracellular levels of Na+. Patch pipettes were used to introduce Na+ (40 mM in pipette solutions) into dopamine neurons in the rat midbrain slice in order to study the electrophysiological effects of increased intracellular Na+. We found that intracellular Na+ loading evoked 100-300 pA of outward current (at -60 mV) and increased whole-cell conductance; these effects developed gradually during the first 10 min after rupture of the membrane patch. Extracellular Ba2+ reduced most of the outward current evoked by Na+ loading; this Ba(2+)-sensitive current reversed direction at the expected reversal potential for K+ (EK), and was also blocked by extracellular tetraethylammonium (30 mM) and intracellular Cs+ (which replaced K+ in pipette solutions). The sulfonylurea drugs glipizide (IC50 = 4.9 nM), tolbutamide (IC50 = 23 microM) and glibenclamide (1 microM) were as effective as 300 microM Ba2+ in reducing the K+ current evoked by Na+ loading. When recording with "control" pipettes containing 15 mM Na+, diazoxide (300 microM) increased chord conductance and evoked outward current at -60 mV, which also reversed direction near EK. Effects of diazoxide were blocked by glibenclamide (1 microM) or glipizide (300 nM). Diazoxide (300 microM) and baclofen (3 microM), which also evoked K(+)-mediated outward currents recorded with control pipettes, caused little additional increases in outward currents during Na+ loading. Raising ATP concentrations to 10 mM in pipette solutions failed to significantly reduce currents evoked by diazoxide or Na+ loading, suggesting that these currents may not be mediated by ATP-sensitive K+ channels. Finally, Na+ loading using pipettes containing Cs+ in place of K+ evoked a relatively small outward current (50-150 pA at -60 mV), which developed gradually over the first 10 min after rupturing the membrane patch. This current was reduced by dihydro-ouabain (3 microM) and a low extracellular concentration of K+ (0.5 mM instead of 2.5 mM), but was not affected by Ba2+. We conclude that intracellular Na+ loading evokes a current generated by Na+/K+ ATPase in addition to sulfonylurea-sensitive K+ current. This Na(+)-dependent K+ current is unusual in its sensitivity to sulfonylureas, and could protect dopamine neurons against toxic effects of intracellular Na+ accumulation.
Asunto(s)
Dopamina/metabolismo , Mesencéfalo/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Sodio/farmacología , Urea/farmacología , Animales , Bario/farmacología , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Effects of L-arginine in the nervous system are often attributed to nitric oxide. Using whole-cell patch pipettes to record membrane currents in voltage-clamp from dopamine neurons in the rat midbrain slice, the present studies found that L-arginine potentiates GABA-dependent membrane currents via a nitric oxide-independent mechanism. L-Arginine (0.3-10 mM) increased the peak amplitude, half-width duration and time constant of decay of GABA(B) receptor-mediated inhibitory postsynaptic currents in a concentration-dependent manner. In the presence of CGP 35348 (300 microM), a GABA(B) receptor antagonist, L-arginine also prolonged the duration of inhibitory postsynaptic currents mediated by GABA(A) receptors, but their amplitudes were reduced. L-Arginine (10 mM) also evoked 17+/-3 pA of outward current (at -60 mV) which was significantly increased in the presence of exogenous GABA (100 microM). Pressure-ejection of GABA from micropipettes produced outward currents mediated by GABA(B) receptors (recorded in bicuculline) or GABA(A) receptors (recorded in CGP 35348); both types of receptor-mediated currents were increased by L-arginine (10 mM). In contrast, outward currents evoked by baclofen, a GABA(B) receptor agonist, were not potentiated by L-arginine. The GABA transport inhibitors NO 711 (1 microM) and nipecotic acid (1 mM) significantly increased the half-width duration and time-constant of decay of GABA(B)-mediated inhibitory postsynaptic currents, thus mimicking effects of L-arginine. However, nitric oxide donors failed to mimic effects of L-arginine on GABA(B) inhibitory postsynaptic currents, and inhibitors of nitric oxide synthesis failed to selectively block the action of L-arginine. These findings suggest that L-arginine potentiates GABA synaptic transmission by a nitric oxide-independent mechanism. Similarities between effects of L-arginine, NO 711 and nipecotic acid suggest that L-arginine inhibits a GABA transporter.
Asunto(s)
Arginina/farmacología , Dopamina/metabolismo , Neuronas/efectos de los fármacos , Óxido Nítrico/farmacología , Receptores de GABA/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratas , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
1. The alpha 2-adrenoceptors on cell bodies of submucosal neurones, on presynaptic cholinergic nerve terminals innervating submucosal neurones, and on presynaptic sympathetic fibres innervating submucosal arterioles were characterized in functional studies by use of subtype selective ligands. 2. Both membrane hyperpolarization and presynaptic inhibition of nicotinic excitatory synaptic potentials (e.p.s.ps) produced by UK 14304 were similarly antagonized by idazoxan, yohimbine. SKF 104078, WB 4101 and ARC-239. Antagonism was competitive and dissociation equilibrium constants were the same for both effects. 3. Vasoconstriction of submucosal arterioles in response to stimulation of the sympathetic nerves (20 Hz for 2 s) was inhibited by UK 14304 and clonidine: concentrations producing half-maximum responses were 6 nm and 10 nM respectively. Idazoxan, yohimbine, WB 4101 and SKF 104078 antagonized this action, with dissociation constants similar to those for antagonism of the postsynaptic membrane hyperpolarization and presynaptic inhibition of nicotinic e.p.s.ps. 4. Oxymetazoline was a partial agonist when membrane hyperpolarization or presynaptic inhibition of nicotinic e.p.s.ps were measured but a full agonist when presynaptic inhibition of sympathetically-mediated arteriolar vasoconstriction was measured. As an agonist, oxymetazoline produced half maximum responses at 80-120 nM; the dissociation constant for oxymetazoline as an antagonist was 130 nM. 5. Neither prazosin nor chlorpromazine (up to 30 microM) altered any of the three responses to alpha 2-adrenoceptor agonists. 6. It is concluded that alpha 2-adrenoceptors present on submucosal neuronal cell bodies, on presynaptic cholinergic nerve terminals and on presynaptic sympathetic nerve terminals are the alpha 2A subtype. However, functional characterization of this subtype differs from that provided by ligand binding studies.
Asunto(s)
Mucosa Intestinal/metabolismo , Neuronas/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Sinapsis/metabolismo , Acetilcolina/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Animales , Cobayas , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Neurotransmisores/metabolismo , Oximetazolina/farmacología , Receptores Nicotínicos/efectos de los fármacos , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiologíaRESUMEN
We used patch pipettes to record whole-cell currents from single dopamine neurons in slices of rat midbrain. Pharmacological methods were used to isolate EPSCs evoked by focal electrical stimulation. Baclofen was significantly more potent for inhibiting NMDA receptor-mediated EPSCs (IC50=0.24 microM) compared with inhibition of EPSCs mediated by AMPA receptors (IC50=1.72 microM). The increased potency of baclofen for inhibiting the NMDA component persisted in superfusate that contained zero Mg2+ and when postsynaptic K+ conductances were reduced by Cs+ and QX-314. Effects of baclofen on EPSCs were blocked by the GABA(B) receptor antagonist CGP-35348. Adenosine was 20 fold more potent for reducing the NMDA component of transmission (IC50=31 microM) compared with inhibition of AMPA receptor-mediated EPSCs (IC50=654 microM). Effects of adenosine on EPSCs were blocked by the A1 receptor antagonist DPCPX. Both baclofen and adenosine significantly increased the ratio of EPSCs in paired-pulse studies, suggesting presynaptic sites of action. Although adenosine (1 mM) did not reduce currents evoked by exogenous NMDA (10 microM), baclofen (1 microM) reduced NMDA currents by 29%. Neither baclofen nor adenosine altered currents evoked by exogenous AMPA (1 microM). We conclude that adenosine acts at presynaptic A1 receptors to cause a preferential reduction in the NMDA component of synaptic transmission. In contrast, baclofen preferentially reduces NMDA EPSCs by acting at both pre- and postsynaptic GABA(B) receptors. By regulating NMDA receptor function, A1 and GABA(B) receptors may play important roles in regulating the excitability of dopamine neurons.
Asunto(s)
Mesencéfalo/fisiología , Neuronas/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/fisiología , Adenosina/farmacología , Animales , Baclofeno/farmacología , Cesio/farmacología , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Agonistas del GABA/farmacología , Técnicas In Vitro , Lidocaína/análogos & derivados , Lidocaína/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mesencéfalo/efectos de los fármacos , N-Metilaspartato/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-B/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
5-Nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (ACEA-1328) was characterized in vitro for antagonism of excitatory amino acid receptors, and subsequently tested in vivo and compared with MK-801 for phencyclidine (PCP)-like motor stimulation, antinociception, and effects on morphine tolerance in mice. Assayed on rat cerebral cortical glutamate receptors expressed in Xenopus oocytes ACEA-1328 showed potent (Kb approximately 40 nM) antagonism at NMDA receptor/glycine sites and moderate (Kb approximately 3 microM) antagonism at non-NMDA receptors. In both cases inhibition was predominantly competitive. ACEA-1328 was weak, or inactive, at NMDA receptor glutamate recognition sites, metabotropic receptors and opioid binding sites. In the formalin and rotarod tests ACEA-1328 and MK-801 produced both antinociception and disturbances of motor coordination. MK-801 caused a PCP-like motor stimulatory effect, whereas ACEA-1328 was devoid of such an effect. In tolerance studies, ACEA-1328 and MK-801 each blocked morphine tolerance in the formalin test, the effect of ACEA-1328 was dose-dependent. Our data contribute to a growing body of evidence which suggests that activation of NMDA receptors is critical for the development of opioid tolerance, and that antagonism at NMDA receptor/glycine sites may have potential as a means of diminishing tolerance with no PCP-like motor stimulatory side effects.
Asunto(s)
Tolerancia a Medicamentos , Morfina/farmacología , Narcóticos/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Conducta Animal/efectos de los fármacos , Unión Competitiva/fisiología , Química Encefálica , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Formaldehído , Expresión Génica/fisiología , Locomoción/efectos de los fármacos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Nociceptores/efectos de los fármacos , Oocitos/fisiología , Quinoxalinas/farmacología , Ensayo de Unión Radioligante , Ratas , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevisRESUMEN
Whole-cell patch recordings were made from dopamine-containing neurons in the ventral tegmental area (VTA) and substantia nigra zona compacta (SNC). Isoguvacine evoked an outward current (at -60 mV) in a concentration-dependent manner with an EC50 of 62+/-8 microM. The gamma-aminobutyric acid (GABA) uptake inhibitor 1-(2(((diphenylmethylene)imino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711) (3 microM) shifted the isoguvacine concentration-response curve to the left, with a new EC50 of 22+/-4 microM. L-Arginine (3 mM) also shifted the isoguvacine concentration-response curve to the left, with a new EC50 of 29+/-5 microM. L-Arginine (3 mM) increased the currents evoked by GABA (100 microM) and muscimol (1 microM) by 208% and 261%, respectively. The GABA uptake inhibitor 4,5,6,7,-tetrahydroisoxazolo[4,5-c]-pyridin-3-ol hydrobromide (THPO) (300 microM) not only mimicked but also occluded the ability of L-arginine (3 mM) to potentiate currents evoked by isoguvacine. Equimolar replacement of Na+ with choline increased GABA-evoked currents, suggesting that a low Na+ concentration has an inhibitory effect on GABA transport. Low Na+ concentration (25 mM) inhibited isoguvacine currents but still occluded the potentiating effects of L-arginine. We conclude that GABA uptake inhibitors potentiate the actions of the GABA(A) receptor agonists, isoguvacine and muscimol, probably because they are effective substrates for GABA transporters in the ventral midbrain.
Asunto(s)
Agonistas del GABA/farmacología , Agonistas de Receptores de GABA-A , Proteínas de Transporte de Membrana , Mesencéfalo/metabolismo , Inhibidores de la Captación de Neurotransmisores/farmacología , Transportadores de Anión Orgánico , Ácido gamma-Aminobutírico/metabolismo , Animales , Proteínas Portadoras/metabolismo , Sinergismo Farmacológico , Electrofisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Técnicas In Vitro , Ácidos Isonicotínicos/farmacología , Isoxazoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mesencéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ácidos Nipecóticos/farmacología , Oximas/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Sodio/fisiología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/efectos de los fármacos , Núcleo Supraquiasmático/metabolismoRESUMEN
Recent studies indicate that competitive and non-competitive NMDA receptor antagonists can block the development of morphine tolerance. Since glycine is considered to be a co-agonist for activation of NMDA receptors we examined the effect of a novel bioavailable NMDA receptor/glycine site antagonist, 5-nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (ACEA-1328), on the development of morphine tolerance. Administration of ACEA-1328 (20 mg/kg) completely blocked tolerance to morphine-induced antinociception in the tail flick test in CD-1 mice, without affecting the basal nociceptive response or potentiating morphine-induced antinociceptive effects. These data suggest that inhibition of NMDA receptor activity via blockade of the glycine co-agonist site is potentially viable as a therapeutic approach for preventing development of morphine tolerance.
Asunto(s)
Morfina/farmacología , Quinoxalinas/farmacología , Receptores de Glicina/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Analgésicos/farmacología , Animales , Tolerancia a Medicamentos , Cinética , Masculino , Ratones , Oocitos/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Ratas , XenopusRESUMEN
Excitatory amino acid receptor antagonists show potential for the treatment of ischemic stroke and head trauma. In search of novel antagonists, a series of alkyl- and alkoxyl-substituted 1, 4-dihydro-2,3-quinoxalinediones were synthesized and assayed for inhibition of glutamate receptors. We report on the pharmacological characterization of one such compound, 7-chloro-6-methyl-5-nitro-1,4-dihydro-2, 3-quinoxalinedione (ACEA-1416). Electrophysiological assays showed that ACEA-1416 is a potent antagonist of rat brain NMDA receptors expressed in Xenopus oocytes, and NMDA receptors expressed by cultured rat cortical neurons. Antagonism is via competitive inhibition at glycine co-agonist sites (Kb = 7.9 nM in oocytes, Kb = 11 nM in neurons). ACEA-1416 also antagonizes AMPA receptors, though potency is considerably lower (Kb = 3.5 microM in oocytes, Kb = 1.6 microM in neurons). Oocyte assays indicated that ACEA-1416 is weak or inactive as an antagonist at NMDA receptor glutamate binding sites (Kb > 5.9 microM) and metabotropic glutamate receptors (Kb > 57 microM). Many NMDA receptor glycine site antagonists show poor penetration of the blood-brain barrier. Systemic bioavailability of ACEA-1416 was assessed by measuring the ability of the compound to protect against electroshock-induced seizures in mice. Protective effects of ACEA-1416 had rapid onset following i.v. administration. Peak efficacy was at approximately 2 min and the biological half-time of protection was approximately 60 min. The ED50 measured at peak efficacy was approximately 1.5 mg/kg. Our results show that ACEA-1416 is a high potency systemically active NMDA receptor glycine site antagonist and a moderate potency AMPA receptor antagonist. Separate studies indicate that ACEA-1416 is efficacious as a neuroprotectant in a rat model of focal cerebral ischemia. Taken together, our results suggest that ACEA-1416 has potential for clinical development as a neuroprotectant.
Asunto(s)
Anticonvulsivantes/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glicina/metabolismo , Quinoxalinas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
Our lab recently showed that N-methyl-D-aspartate (NMDA) evokes ATP-sensitive K(+) (K-ATP) currents in subthalamic nucleus (STN) neurons in slices of the rat brain. Both K-ATP channels and 5'-adenosine monophosphate-activated protein kinase (AMPK) are considered cellular energy sensors because their activities are influenced by the phosphorylation state of adenosine nucleotides. Moreover, AMPK has been shown to regulate K-ATP function in a variety of tissues including pancreas, cardiac myocytes, and hypothalamus. We used whole-cell patch clamp recordings to study the effect of AMPK activation on K-ATP channel function in STN neurons in slices of the rat brain. We found that bath or intracellular application of the AMPK activators A769662 and PT1 augmented tolbutamide-sensitive K-ATP currents evoked by NMDA receptor stimulation. The effect of AMPK activators was blocked by the AMPK inhibitor dorsomorphin (compound C), and by STO609, an inhibitor of the upstream AMPK activator CaMKKß. AMPK augmentation of NMDA-induced K-ATP current was also blocked by intracellular BAPTA and by inhibitors of nitric oxide synthase and guanylyl cyclase. However, A769662 did not augment currents evoked by the K-ATP channel opener diazoxide. In the presence of NMDA, A769662 inhibited depolarizing plateau potentials and burst firing, both of which could be antagonized by tolbutamide or dorsomorphin. These studies show that AMPK augments NMDA-induced K-ATP currents by a Ca(2+)-dependent process that involves nitric oxide and cGMP. By augmenting K-ATP currents, AMPK activation would be expected to dampen the excitatory effect of glutamate-mediated transmission in the STN.