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1.
Obes Rev ; : e13769, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38830619

RESUMEN

Given the high and growing prevalence of obesity among adults in the United States, obesity treatment and prevention are important topics in biomedical and public health research. Although researchers recognize the significance of this problem, much remains unknown about safe and effective prevention and treatment of obesity in adults. In response to the worsening obesity epidemic and the many unknowns regarding the disease, a group of key scientific and program staff members of the National Institutes of Health (NIH) and other federal and non-government agencies gathered virtually in September 2021 to discuss the current state of obesity research, research gaps, and opportunities for future research in adult obesity prevention and treatment. The current article synthesizes presentations given by attendees and shares their organizations' current initiatives and identified gaps and opportunities. By integrating the information discussed in the meeting and current initiatives, we identify potential targets and overlapping priorities for future research, including health equity and disparities in obesity, the heterogeneity of obesity, and the use of technological and innovative approaches in interventions.

2.
Circulation ; 144(23): e461-e471, 2021 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-34719260

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic has had worldwide repercussions for health care and research. In spring 2020, most non-COVID-19 research was halted, hindering research across the spectrum from laboratory-based experimental science to clinical research. Through the second half of 2020 and the first half of 2021, biomedical research, including cardiovascular science, only gradually restarted, with many restrictions on onsite activities, limited clinical research participation, and the challenges associated with working from home and caregiver responsibilities. Compounding these impediments, much of the global biomedical research infrastructure was redirected toward vaccine testing and deployment. This redirection of supply chains, personnel, and equipment has additionally hampered restoration of normal research activity. Transition to virtual interactions offset some of these limitations but did not adequately replace the need for scientific exchange and collaboration. Here, we outline key steps to reinvigorate biomedical research, including a call for increased support from the National Institutes of Health. We also call on academic institutions, publishers, reviewers, and supervisors to consider the impact of COVID-19 when assessing productivity, recognizing that the pandemic did not affect all equally. We identify trainees and junior investigators, especially those with caregiving roles, as most at risk of being lost from the biomedical workforce and identify steps to reduce the loss of these key investigators. Although the global pandemic highlighted the power of biomedical science to define, treat, and protect against threats to human health, significant investment in the biomedical workforce is required to maintain and promote well-being.


Asunto(s)
Investigación Biomédica/tendencias , COVID-19 , Cardiología/tendencias , Proyectos de Investigación/tendencias , Investigadores/tendencias , Comités Consultivos , American Heart Association , Investigación Biomédica/educación , Cardiología/educación , Difusión de Innovaciones , Educación Profesional/tendencias , Predicción , Humanos , Opinión Pública , Investigadores/educación , Factores de Tiempo , Estados Unidos
3.
J Pharmacol Exp Ther ; 362(3): 395-404, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28642232

RESUMEN

The muscle relaxant carisoprodol has recently been controlled at the federal level as a Schedule IV drug due to its high abuse potential and consequences of misuse, such as withdrawal syndrome, delusions, seizures, and even death. Recent work has shown that carisoprodol can directly gate and allosterically modulate the type A GABA (GABAA) receptor. These actions are subunit-dependent; compared with other GABAA receptors, carisoprodol has nominal direct gating effects in α3ß2γ2 receptors. Here, using site-directed mutagenesis and whole-cell patch-clamp electrophysiology in transiently transfected human embryonic kidney 293 cells, we examined the role of GABAA receptor α subunit transmembrane domain 4 (TM4) amino acids in direct gating and allosteric modulatory actions of carisoprodol. Mutation of α3 valine at position 440 to leucine (present in the equivalent position in the α1 subunit) significantly increased the direct gating effects of carisoprodol without affecting its allosteric modulatory effects. The corresponding reverse mutation, α1(L415V), decreased carisoprodol direct gating potency and efficacy. Analysis of a series of amino acid mutations at the 415 position demonstrated that amino acid volume correlated positively with carisoprodol efficacy, whereas polarity inversely correlated with carisoprodol efficacy. We conclude that α1(415) of TM4 is involved in the direct gating, but not allosteric modulatory, actions of carisoprodol. In addition, the orientation of alkyl or hydroxyl groups at this position influences direct gating effects. These findings support the likelihood that the direct gating and allosteric modulatory effects of carisoprodol are mediated via distinct binding sites.


Asunto(s)
Aminoácidos/metabolismo , Carisoprodol/farmacología , Relajantes Musculares Centrales/farmacología , Transporte de Proteínas/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Sustitución de Aminoácidos , Sitios de Unión/efectos de los fármacos , Agonistas del GABA/farmacología , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Pentobarbital/farmacología , Receptores de GABA-A/genética , Esteroides/farmacología
4.
Neuropharmacology ; 121: 167-178, 2017 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-28456686

RESUMEN

Efavirenz is a widely prescribed medicine used to treat type 1 human immunodeficiency virus (HIV-1), the most prevalent pathogenic strain of the virus responsible for the acquired immune deficiency syndrome (AIDS) pandemic. Under prescribed dosing conditions, either alone or in combination therapy, efavirenz-induced CNS disturbances are frequently reported. Efavirenz was recently reported to interact in a similar concentration range with a number of receptors, transporters and ion channels including recombinant rat α1ß2γ2 GABAA receptors whose actions were potentiated (Gatch et al., 2013; Dalwadi et al., 2016). Now we report on the molecular mechanism of efavirenz on GABAA receptors as a function of concentration and subunit composition via whole-cell recordings of GABA-activated currents from HEK293 cells expressing varying subunit configurations of GABAA receptors. Efavirenz elicited dual effects on the GABA response; it allosterically potentiated currents at low concentrations, whereas it inhibited currents at higher concentrations. The allosteric potentiating action on GABAA receptors was pronounced in the α1ß2γ2, α2ß2γ2 and α4ß2γ2 configurations, greatly diminished in the α6ß2γ2 configuration, and completely absent in the α3ß2γ2 or α5ß2γ2 configuration. In stark contrast, the inhibitory modulation of efavirenz at higher concentrations was evident in all subunit configurations examined. Moreover, efavirenz-induced modulatory effects were dependent on GABA concentration ([GABA]), with a pronounced impact on currents activated by low [GABA] but little effect at saturating [GABA]. Mutation of a highly-conserved threonine to phenylalanine in transmembrane domain 2 of the α1 subunit abolished the inhibitory effect of efavirenz in α1ß2 receptors. Finally, mutations of any of the three conserved extracellular residues in α1/2/4 subunits to the conserved residues at the corresponding positions in α3/5 subunits (i.e., R84P, M89L or I120L) completely eliminated the potentiating effect of efavirenz in α1ß2γ2 configuration. These findings demonstrate that efavirenz's positive allosteric modulation of the GABAA receptor is mediated via a novel allosteric site associated with the extracellular domain of the receptor.


Asunto(s)
Benzoxazinas/farmacología , Receptores de GABA-A/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Alquinos , Regulación Alostérica , Animales , Ciclopropanos , Diazepam/farmacología , Relación Dosis-Respuesta a Droga , Flumazenil/farmacología , Moduladores del GABA/farmacología , Células HEK293 , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Mutagénesis/genética , Técnicas de Placa-Clamp , Dominios Proteicos/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Receptores de GABA-A/genética , Transfección , Ácido gamma-Aminobutírico/farmacología
5.
Neuropharmacology ; 119: 100-110, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28390894

RESUMEN

Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAA receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAA receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 µM, and it had an IC50 of 31 µM in human α1ß2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the ß2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAA receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1ß2 receptors to MB was similar to that observed in α1ß2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and ß2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAA receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAA receptor function, which may underlie some of the effects of MB on the CNS.


Asunto(s)
Sitios de Unión/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Azul de Metileno/farmacología , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células HEK293 , Hipocampo/citología , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Unión Proteica/efectos de los fármacos , Ratas , Receptores de GABA-A/genética , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacología
6.
Eur J Pharmacol ; 775: 149-58, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26872987

RESUMEN

Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxßzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1ß1γ2 and α1ß2γ2 receptors, whereas allosteric effects were enhanced in α1ß2 compared to α1ß2γ2 receptors. In "extrasynaptic" (α1ß3δ and α4ß3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric ß3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted.


Asunto(s)
Ansiolíticos/farmacología , Moduladores del GABA/farmacología , Meprobamato/farmacología , Relajantes Musculares Centrales/farmacología , Subunidades de Proteína/fisiología , Receptores de GABA-A/fisiología , Bemegrida/farmacología , Carisoprodol/farmacología , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Pentobarbital/farmacología , Subunidades de Proteína/genética , Receptores de GABA-A/genética
7.
Neuropharmacology ; 97: 414-25, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25896767

RESUMEN

Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1ß2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1ß2, α1ß3 or αxßzγ2 (where x = 1-6 and z = 1-3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the ß1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the ß2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1ß3δ and α4ß3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects.


Asunto(s)
Carisoprodol/farmacología , GABAérgicos/farmacología , Relajantes Musculares Centrales/farmacología , Receptores de GABA-A/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Receptores de GABA-A/genética , Trastornos Relacionados con Sustancias/metabolismo , Transfección , Ácido gamma-Aminobutírico/metabolismo
8.
Pharmacology ; 92(1-2): 84-9, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23942137

RESUMEN

SV 293 [1-([5-methoxy-1H-indol-3-yl]methyl)-4-(4-[methylthio]​phenyl)piperidin-4-ol] binds with 100-fold higher affinity to human D2 receptors compared to the human D3 and D4 dopamine receptor subtypes. We investigated the intrinsic efficacy of this compound at the D2 dopamine receptor subtype using both: (1) a forskolin-dependent adenylyl cyclase inhibition assay and (2) an electrophysiological assay for evaluating coupling to G-protein-coupled inwardly rectifying potassium channels. In both assays SV 293 was found to be a neutral antagonist capable of blocking the effects of the full D2-like receptor agonist quinpirole. Based upon these results we propose that SV 293 is a useful pharmacological tool that can be used for both in vitro and in vivo studies to investigate the role of D2-like dopamine receptor subtypes in neurological, neuropsychiatric and movement disorders where dopaminergic pathways have been implicated.


Asunto(s)
Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Indoles/farmacología , Piperidinas/farmacología , Inhibidores de Adenilato Ciclasa , Animales , Línea Celular Tumoral , Colforsina/farmacología , Agonistas de Dopamina/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Humanos , Ratones , Quinpirol/farmacología , Receptores de Dopamina D2/fisiología
9.
Neurobiol Dis ; 59: 18-25, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23845275

RESUMEN

Vascular dementia ranks as the second leading cause of dementia in the United States. However, its underlying pathophysiological mechanism is not fully understood and no effective treatment is available. The purpose of the current study was to evaluate long-term cognitive deficits induced by transient middle cerebral artery occlusion (tMCAO) in rats and to investigate the underlying mechanism. Sprague-Dawley rats were subjected to tMCAO or sham surgery. Behavior tests for locomotor activity and cognitive function were conducted at 7 or 30days after stroke. Hippocampal long term potentiation (LTP) and involvement of GABAergic neurotransmission were evaluated at 30days after sham surgery or stroke. Immunohistochemistry and Western blot analyses were conducted to determine the effect of tMCAO on cell signaling in the hippocampus. Transient MCAO induced a progressive deficiency in spatial performance. At 30days after stroke, no neuron loss or synaptic marker change in the hippocampus were observed. LTP in both hippocampi was reduced at 30days after stroke. This LTP impairment was prevented by blocking GABAA receptors. In addition, ERK activity was significantly reduced in both hippocampi. In summary, we identified a progressive decline in spatial learning and memory after ischemic stroke that correlates with suppression of hippocampal LTP, elevation of GABAergic neurotransmission, and inhibition of ERK activation. Our results indicate that the attenuation of GABAergic activity or enhancement of ERK/MAPK activation in the hippocampus might be potential therapeutic approaches to prevent or attenuate cognitive impairment after ischemic stroke.


Asunto(s)
Trastornos del Conocimiento/etiología , Regulación de la Expresión Génica/fisiología , Infarto de la Arteria Cerebral Media/complicaciones , Transducción de Señal/fisiología , Animales , Estimulantes del Sistema Nervioso Central/farmacología , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Lateralidad Funcional , Hipocampo/fisiopatología , Técnicas In Vitro , Masculino , Aprendizaje por Laberinto/fisiología , Proteínas de la Membrana/metabolismo , Actividad Motora/fisiología , Proteínas del Tejido Nervioso/metabolismo , Picrotoxina/farmacología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Percepción Visual/fisiología
10.
Neuropsychopharmacology ; 38(12): 2373-84, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23702798

RESUMEN

Anecdotal reports have surfaced concerning misuse of the HIV antiretroviral medication efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) by HIV patients and non-infected teens who crush the pills and smoke the powder for its psychoactive effects. Molecular profiling of the receptor pharmacology of efavirenz pinpointed interactions with multiple established sites of action for other known drugs of abuse including catecholamine and indolamine transporters, and GABAA and 5-HT(2A) receptors. In rodents, interaction with the 5-HT(2A) receptor, a primary site of action of lysergic acid diethylamine (LSD), appears to dominate efavirenz's behavioral profile. Both LSD and efavirenz reduce ambulation in a novel open-field environment. Efavirenz occasions drug-lever responding in rats discriminating LSD from saline, and this effect is abolished by selective blockade of the 5-HT(2A) receptor. Similar to LSD, efavirenz induces head-twitch responses in wild-type, but not in 5-HT(2A)-knockout, mice. Despite having GABAA-potentiating effects (like benzodiazepines and barbiturates), and interactions with dopamine transporter, serotonin transporter, and vesicular monoamine transporter 2 (like cocaine and methamphetamine), efavirenz fails to maintain responding in rats that self-administer cocaine, and it fails to produce a conditioned place preference. Although its molecular pharmacology is multifarious, efavirenz's prevailing behavioral effect in rodents is consistent with LSD-like activity mediated via the 5-HT(2A) receptor. This finding correlates, in part, with the subjective experiences in humans who abuse efavirenz and with specific dose-dependent adverse neuropsychiatric events, such as hallucinations and night terrors, reported by HIV patients taking it as a medication.


Asunto(s)
Fármacos Anti-VIH/toxicidad , Benzoxazinas/toxicidad , Alucinógenos/toxicidad , Dietilamida del Ácido Lisérgico/toxicidad , Receptor de Serotonina 5-HT2A/metabolismo , Agonistas del Receptor de Serotonina 5-HT2/metabolismo , Alquinos , Animales , Conducta Animal/efectos de los fármacos , Condicionamiento Psicológico/efectos de los fármacos , Ciclopropanos , Discriminación en Psicología , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/metabolismo
11.
ACS Chem Neurosci ; 3(12): 1050-62, 2012 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-23259040

RESUMEN

We previously reported on the synthesis of substituted phenyl-4-hydroxy-1-piperidyl indole analogues with nanomolar affinity at D2 dopamine receptors, ranging from 10- to 100-fold selective for D2 compared to the D3 dopamine receptor subtype. More recently, we evaluated a panel of aripiprazole analogues, identifying several analogues that also exhibit D2 vs D3 dopamine receptor binding selectivity. These studies further characterize the intrinsic efficacy of the compound with the greatest binding selectivity from each chemical class, 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol (SV 293) and 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (SV-III-130s), using an adenylyl cyclase inhibition assay, a G-protein-coupled inward-rectifying potassium (GIRK) channel activation assay, and a cell based phospho-MAPK (pERK1/2) assay. SV 293 was found to be a neutral antagonist at D2 dopamine receptors using all three assays. SV-III-130s is a partial agonist using an adenylyl cyclase inhibition assay but an antagonist in the GIRK and phospho ERK1/2 assays. To define the molecular basis for the binding selectivity, the affinity of these two compounds was evaluated using (a) wild type human D2 and D3 receptors and (b) a panel of chimeric D2/D3 dopamine receptors. Computer-assisted modeling techniques were used to dock these compounds to the human D2 and D3 dopamine receptor subtypes. It is hoped that these studies on D2 receptor selective ligands will be useful in the future design of (a) receptor selective ligands used to define the function of D2-like receptor subtypes, (b) novel pharmacotherapeutic agents, and/or (c) in vitro and in vivo imaging agents.


Asunto(s)
Antagonistas de Dopamina/síntesis química , Antagonistas de los Receptores de Dopamina D2 , Receptores de Dopamina D3/antagonistas & inhibidores , Antagonistas de Dopamina/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Transfección
12.
Neuropharmacology ; 58(8): 1246-51, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20303997

RESUMEN

The protein tyrosine kinase (PTK) inhibitor genistein has been widely used to examine potential effects of tyrosine phosphorylation on neurotransmitter function. We report here that genistein inhibits N-methyl-d-aspartate (NMDA) receptors through a direct effect. Whole-cell NMDA-activated current was recorded in native receptors from mouse hippocampal slice culture and rat recombinant NR1aNR2A and NR1aNR2B receptors transiently expressed in HEK293 cells. Extracellular application of genistein and NMDA reversibly inhibited NMDA-activated current. The inhibition of NMDA-activated current by genistein applied externally was not affected when genistein was also pre-equilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited NMDA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the NMDA response. Moreover, genistein-induced inhibition of NMDA-activated current displayed concentration- and voltage-dependence. Our results demonstrate that genistein has a direct inhibitory effect on NMDA receptors that is not mediated via inhibition of tyrosine kinase. Thus, other PTK inhibitors may be more suitable for studying involvement of PTKs in NMDA receptor-mediated events.


Asunto(s)
Genisteína/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Técnicas In Vitro , Activación del Canal Iónico , Isoflavonas/farmacología , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Fenoles/farmacología , Ratas , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética
13.
J Pharmacol Exp Ther ; 329(2): 827-37, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19244096

RESUMEN

Carisoprodol is a frequently prescribed muscle relaxant. In recent years, this drug has been increasingly abused. The effects of carisoprodol have been attributed to its metabolite, meprobamate, a controlled substance that produces sedation via GABA(A) receptors (GABA(A)Rs). Given the structural similarities between carisoprodol and meprobamate, we used electrophysiological and behavioral approaches to investigate whether carisoprodol directly affects GABA(A)R function. In whole-cell patch-clamp studies, carisoprodol allosterically modulated and directly activated human alpha1beta2gamma2 GABA(A)R function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric rho1 GABA or glycine alpha1 receptors. In the absence of GABA, carisoprodol produced picrotoxin-sensitive, inward currents that were significantly larger than those produced by meprobamate, suggesting carisoprodol may directly produce GABAergic effects in vivo. When administered to mice via intraperitoneal or oral routes, carisoprodol elicited locomotor depression within 8 to 12 min after injection. Intraperitoneal administration of meprobamate depressed locomotor activity in the same time frame. In drug discrimination studies with carisoprodol-trained rats, the GABAergic ligands pentobarbital, chlordiazepoxide, and meprobamate each substituted for carisoprodol in a dose-dependent manner. In accordance with findings in vitro, the discriminative stimulus effects of carisoprodol were antagonized by a barbiturate antagonist, bemegride, but not by the benzodiazepine site antagonist, flumazenil. The results of our studies in vivo and in vitro collectively suggest the barbiturate-like effects of carisoprodol may not be due solely to its metabolite, meprobamate. Furthermore, the functional traits we have identified probably contribute to the abuse potential of carisoprodol.


Asunto(s)
Conducta Animal/efectos de los fármacos , Carisoprodol/farmacología , Moduladores del GABA/farmacología , Receptores de GABA-A/metabolismo , Regulación Alostérica , Sitio Alostérico , Animales , Carisoprodol/química , Línea Celular , Aprendizaje Discriminativo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Moduladores del GABA/química , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Meprobamato/química , Meprobamato/farmacología , Ratones , Actividad Motora/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Relación Estructura-Actividad , Transfección
14.
Mol Cell Pharmacol ; 1(4): 180-186, 2009 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-20419052

RESUMEN

Soma(®) (carisoprodol) is an increasingly abused, centrally-acting muscle relaxant. Despite the prevalence of carisoprodol abuse, its mechanism of action remains unclear. Its sedative effects, which contribute to its therapeutic and recreational use, are generally attributed to the actions of its primary metabolite, meprobamate, at GABA(A) receptors (GABA(A)R). Meprobamate is a controlled substance at the federal level; ironically, carisoprodol is not currently classified as such. Using behavioral and molecular pharmacological approaches, we recently demonstrated carisoprodol, itself, is capable of modulating GABA(A)R function in a manner similar to central nervous system depressants. Its functional similarities with this highly addictive class of drugs may contribute to the abuse potential of carisoprodol. The site of action of carisoprodol has not been identified; based on our studies, interaction with benzodiazepine or barbiturate sites is unlikely. These recent findings, when coupled with numerous reports in the literature, support the contention that the non-controlled status of carisoprodol should be reevaluated.

15.
Proc Natl Acad Sci U S A ; 105(39): 15148-53, 2008 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-18815371

RESUMEN

L-type voltage-gated Ca(2+)channels (VGCC) play an important role in dendritic development, neuronal survival, and synaptic plasticity. Recent studies have demonstrated that the gonadal steroid estrogen rapidly induces Ca(2+) influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this Ca(2+) influx is not clearly understood. We show by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate VGCC in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, in a manner that was estrogen receptor (ER)-independent. Equilibrium, competitive, and whole-cell binding assays indicate that estrogen directly interacts with the VGCC. Furthermore, a L-type VGCC antagonist to the dihydropyridine site displaced estrogen binding to neuronal membranes, and the effects of estrogen were markedly attenuated in a mutant, dihydropyridine-insensitive L-type VGCC, demonstrating a direct interaction of estrogens with L-type VGCC. Thus, estrogen-induced potentiation of calcium influx via L-type VGCC may link electrical events with rapid intracellular signaling seen with estrogen exposure leading to modulation of synaptic plasticity, neuroprotection, and memory formation.


Asunto(s)
Canales de Calcio Tipo L/metabolismo , Estrógenos/metabolismo , Neuronas/metabolismo , Animales , Canales de Calcio Tipo L/genética , Línea Celular , Estradiol/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Humanos , Mutación , Neuronas/efectos de los fármacos , Ratas , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Invest Ophthalmol Vis Sci ; 49(11): 4993-5002, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18641291

RESUMEN

PURPOSE: The sigma-1 receptor belongs to a recently discovered family of transmembrane proteins expressed in the central nervous system, including the eye, and mediates the regulation of ion channels. The exact function of sigma receptors remains to be elucidated. The purpose of this study was to investigate the effect of sigma-1 receptor ligands on calcium homeostasis in a retinal ganglion cell line (RGC)-5 and rat primary RGCs. METHODS: Calcium imaging was used to assess the effect of sigma-1 receptor agonist (+)-N-allylnormetazocine ((+)-SKF10047) on potassium chloride (KCl)-induced calcium influx in RGC-5. The whole-cell patch clamp technique was used to analyze the effect of (+)-SKF10047 on calcium currents in primary RGCs. Coimmunoprecipitation assessed the interaction between the sigma-1 receptor and the L-type voltage-gated calcium channel. RESULTS: The sigma-1 receptor agonist (+)-SKF10047 inhibited potassium chloride (KCl)-induced calcium influx. The sigma-1 receptor antagonist, BD1047, reversed the inhibitory effect of (+)-SKF10047. Whole-cell patch clamp recordings of rat cultured primary RGCs demonstrated that (+)-SKF10047 inhibited calcium currents. Coimmunoprecipitation studies demonstrated an association between L-type calcium channels and the sigma-1 receptors. CONCLUSIONS: These results suggest that sigma-1 receptor activation can regulate calcium homeostasis and signaling in RGCs, likely by directly influencing the activity of L-type voltage-gated calcium channels. Regulation of calcium influx in RGCs by sigma-1 receptor ligands may represent in part the neuroprotective effect of sigma-1 receptors.


Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Receptores sigma/biosíntesis , Células Ganglionares de la Retina/metabolismo , Animales , Western Blotting , Canales de Calcio Tipo L/efectos de los fármacos , Células Cultivadas , ADN/genética , Etilenodiaminas/farmacología , Expresión Génica , Líquido Intracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Microscopía Fluorescente , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Fenazocina/análogos & derivados , Fenazocina/farmacología , Cloruro de Potasio/farmacología , Ratas , Receptores sigma/antagonistas & inhibidores , Receptores sigma/efectos de los fármacos , Receptores sigma/genética , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor Sigma-1
17.
Neurosci Lett ; 431(2): 184-9, 2008 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-18162311

RESUMEN

The presence of phenylalanine (F) at the 6' position of transmembrane domain 2 (TM2) in the alpha4 subunit of alpha4beta2 nicotinic receptors enhances desensitization. As the GABA A receptor affords the ability to study the influence of as few as one and as many as five Fs at this position, we have used it to investigate potential subunit- and stoichiometry-dependent effects of the TM2 6'F mutation on desensitization. Whereas the presence of one F at this position decreased extent of desensitization, desensitization was increased in all configurations that included two or more Fs at the TM2 6' position; desensitization was particularly rapid with 3 or 4 F residues present. Our results demonstrate the ability of F residues at the TM2 6' position to modulate desensitization is likely conserved in the cys-loop family of ligand-gated ion channels. Moreover, our findings demonstrate both stoichiometric- and subunit-dependent effects of the ability of this mutation to regulate desensitization in GABA A receptors.


Asunto(s)
Mutación/fisiología , Fenilalanina/genética , Receptores de GABA-A/fisiología , Procesos Estocásticos , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Estimulación Eléctrica/métodos , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Receptores de GABA-A/genética , Transfección/métodos , Ácido gamma-Aminobutírico/farmacología
18.
Biochemistry ; 46(41): 11484-93, 2007 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-17887775

RESUMEN

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.


Asunto(s)
Endocitosis/fisiología , Receptores de Glicina/fisiología , Línea Celular , Dinaminas/fisiología , Humanos , Cinética , Microscopía Confocal , Técnicas de Placa-Clamp , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología
19.
J Neurobiol ; 66(13): 1467-74, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17013930

RESUMEN

The GABAA receptor is a ligand-gated ion channel whose function and activity can be regulated by ligand binding or alternatively may be influenced indirectly through the phosphorylation of specific subunits that comprise the GABAA receptor pentamer. With respect to phosphorylation, most studies have focused on either beta or gamma subunits, whereas the role of the alpha subunit as a relevant target of signaling kinases is largely unknown. Interestingly, we found a putative phosphorylation site for extracellular-signal regulated kinase (ERK), a key effector of the MAPK pathway, in almost all known alpha subunits of the GABAA receptor, including the ubiquitously expressed alpha1 subunit. To determine whether this putative ERK phosphorylation site was functionally relevant, we evaluated if ERK inhibition (through pharmacological inhibition of its upstream kinase, MEK) altered GABA-gated currents. Using HEK293 cells stably transfected with the alpha1beta2gamma2 form of the GABAA receptor, we found that UO126 reduced basal ERK phosphorylation and resulted in an enhancement of GABA-induced peak current amplitudes. Further, the enhancement of GABA-gated currents required an intact intracellular environment as it was robust in perforated patch recordings (which preserves the intracellular milieu), but absent in conventional whole-cell recordings (which dialyzes the cytosolic contents), supporting the involvement of an intracellular signaling pathway. Finally, mutation of the ERK phosphorylation site (T375-->A) prevented the UO126-induced enhancement of GABA-gated currents. Collectively, our results implicate the MAPK pathway as a negative modulator of GABAA receptor function, whose influence on GABA-gated currents may be mediated by phosphorylation of the alpha subunit.


Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Receptores de GABA-A/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting/métodos , Línea Celular , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Mutagénesis/fisiología , Técnicas de Placa-Clamp/métodos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Receptores de GABA-A/genética , Transfección/métodos
20.
Neuropharmacology ; 51(4): 701-8, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16842826

RESUMEN

Endogenous divalent cations Cu2+ and Zn2+ suppress the activity of glycine receptors (glyRs). Whereas residues critical for the effects of Zn2+ on glyRs have been identified, little is known about the determinants of Cu2+-mediated inhibition. In the present studies, we have assessed the potential commonality of Zn2+ and Cu2+-mediated inhibition of glyRs. Cu2+ potently inhibited recombinant human glycine alpha1 receptors, with an IC50 of 4.1+/-0.7 microM. Systematic mutation of extracellular histidine residues revealed that mutation H215A greatly reduced the inhibitory modulation by Cu2+. Substitution of H215 with C produced receptors with Cu2+ sensitivity similar to the wild type. Furthermore, modification of H215C with a thio-specific reagent, [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), reduced Cu2+ sensitivity of H215C receptors. However, mutation of other extracellular histidine residues including H107 and H109, which are known inhibitory Zn2+coordination sites, failed to influence inhibition of glycine currents by Cu2+. Moreover, mutation to alanine of two threonine residues (T112, T133) critical for Zn2+ inhibition had no effect (T133A) or only partial inhibitory effects (T112A) on Cu2+-induced inhibition. The double mutation, T112A/H215A, caused greater effects on Cu2+-mediated inhibition than either mutation alone. In addition, the glycine currents recorded from T112A/H215A mutant receptors were significantly potentiated by low concentrations of Cu2+. Our results have identified critical determinants of Cu2+-mediated inhibition of glyRs. Moreover, we demonstrate for the first time a clear difference in residues responsible for Cu2+-mediated compared to Zn2+-mediated inhibition of glyRs.


Asunto(s)
Cobre/metabolismo , Receptores de Glicina/química , Receptores de Glicina/fisiología , Línea Celular Transformada , Clonación Molecular/métodos , Cobre/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Glicina/farmacología , Histidina/genética , Histidina/metabolismo , Humanos , Concentración 50 Inhibidora , Potenciales de la Membrana/efectos de los fármacos , Mesilatos/farmacología , Mutagénesis/fisiología , Técnicas de Placa-Clamp/métodos , Reactivos de Sulfhidrilo/farmacología , Transfección/métodos , Zinc/farmacología
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