RESUMEN
BACKGROUND: Dual regimen dolutegravir (DTG) plus lamivudine (3TC) has demonstrated non-inferior efficacy compared to DTG-based three-drug regimens (3DRs), yet directly comparative data regarding the efficacy and safety of DTG + 3TC and bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) for therapy-naïve people with human immunodeficiency virus (HIV)-1 (PWH) are still limited. We aimed to assess the antiviral potency and safety profiles of DTG + 3TC vs. B/F/TAF based on antiretroviral therapy (ART)-naïve PWH in China. METHODS: This retrospective multicenter study enrolled PWH initiating ART with DTG + 3TC or B/F/TAF from 2020 to 2022 in Guangdong and Guangxi. We analyzed response rates based on target not detected (TND) status using intention-to-treat (ITT) analysis. Subgroups were formed based on baseline viral load (VL) (<100,000 vs . ≥100,000 copies/mL) and CD4 + cell count (<200 vs . ≥200 cell/µL). Median time to TND VL was assessed by Kaplan-Meier method. We also measured changes from baseline in CD4 + cell counts, CD4/CD8 ratio, lipid parameters, weight, creatinine (Cr), estimated glomerular filtration rate (eGFR), and drug-related adverse effects (DRAEs). RESULTS: We enrolled 280 participants, including 137 (48.9%) on DTG + 3TC and 143 (51.1%) on B/F/TAF. At week 48, 96.4% (132/137) on DTG+3TC and 100% (143/143) on B/F/TAF achieved TND ( P = 0.064). At week 12, TND responses were higher with B/F/TAF (78.3% [112/143]) than DTG+3TC (30.7% [42/137]) ( P <0.001). This trend held across subgroups. B/F/TAF achieved TND faster (12 weeks) than DTG+3TC (24 weeks) ( P <0.001). No differences were seen in CD4 + cell count and CD4/CD8 ratio, except in the high-VL subgroup, where B/F/TAF showed better recovery. DRAEs were significantly lower with B/F/TAF (4.9% [7/143]) than with DTG + 3TC (13.1% [18/137]) ( P = 0.016). Lipid parameters, body weight, and Cr increased in both groups over 48 weeks, with DTG+3TC showing a more favorable effect on triglycerides, high-density lipoprotein (HDL) cholesterol, and weight gain. CONCLUSIONS: In this real-life study, B/F/TAF led to a faster viral decline and fewer DRAEs compared to DTG+3TC. No significant difference was observed in the TND rate at week 48, regardless of baseline VL and CD4 + cell count. CD4 + recovery was superior for B/F/TAF in participants with high VL. The DTG + 3TC regimen had less impact on metabolic changes than B/F/TAF.
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Fármacos Anti-VIH , Infecciones por VIH , VIH-1 , Adulto , Humanos , Fármacos Anti-VIH/uso terapéutico , China , Emtricitabina/uso terapéutico , Emtricitabina/farmacología , Infecciones por VIH/tratamiento farmacológico , Lamivudine/uso terapéutico , Lamivudine/farmacología , Lípidos , Estudios RetrospectivosRESUMEN
Limited real-world data on dolutegravir (DTG) plus lamivudine (3TC) for HIV-1-infected individuals have been reported. This study aimed to evaluated the real-world efficacy and safety of DTG + 3TC in ART-naïve HIV-1-infected adults in China. This real-world prospective observational cohort study enrolled HIV-1-infected adults receiving ART initiation with DTG + 3TC (D3 group) or tenofovir plus lamivudine and efavirenz (TDF + 3TC + EFV, TLE group) with subgroups of low viral load (LVL, ≤500,000 copies/mL) and high viral load (HVL, >500,000 copies/mL) according to baseline HIV-1 RNA. Efficacy were assessed by proportion of virologic suppression, changes of CD4+ cell count and CD4/CD8 ratio, HIV-1 DNA decay, and safety by symptoms and changes of laboratory indicators at week 4, 12, 24, 36, and 48. Totally 45 participants in D3 group and 95 in TLE group were enrolled. The proportion of HIV RNA < 50 copies/mL were 48.7% (19/39), 84.6% (33/39), 100% (39/39), 100% (39/39) in D3-LVL subgroup at week 4, 12, 24, 48, compared with 1.3% (1/75), 14.7% (11/75), 86.7% (65/75), 96.0% (72/75) in TLE-LVL subgroup, with P < .05 at week 4, 12, and 36. The proportion were 0.0% (0/6), 66.7% (4/6), 83.3% (5/6), 100% (6/6) in D3-HVL subgroup compared with 0.0% (0/20), 5.0% (1/20), 85.0% (17/20), 100% (20/20) in TLE-HVL subgroup, with P < .05 at week 12. No virologic rebound was observed in D3 group. Mean change of CD4/CD8 ratio were higher in D3-LVL versus TLE-LVL subgroup at each scheduled visit (P < .05), while CD4+ cell counts increased significantly in D3-HVL versus TLE-HVL subgroup at week 4 and 12 (P < .05). Less complaint of dizziness, insomnia, dreaminess and amnesia, lower elevated level of triglyceride and higher elevated level of creatinine from baseline to week 48 were documented in D3 group (P < .05). Total HIV-1 DNA decayed along with HIV-1 RNA after DTG + 3TC initiation in both D3-LVL and D3-HVL subgroups. DTG + 3TC achieved virological suppression more rapidly and stably versus TDF + 3TC + EFV in ART-naïve HIV-1-infected adults, with better immunological response and less adverse drug effect, and reduced total HIV-1 DNA effectively. DTG + 3TC is a potent regimen for ART-naïve individuals with HIV-1 infection.
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Fármacos Anti-VIH , Infecciones por VIH , VIH-1 , Adulto , Humanos , Lamivudine/efectos adversos , VIH-1/genética , Tenofovir/uso terapéutico , Fármacos Anti-VIH/efectos adversos , Creatinina , Estudios Prospectivos , Didesoxinucleósidos/uso terapéutico , Benzoxazinas/efectos adversos , Piridonas/uso terapéutico , ARN , Triglicéridos/farmacologíaRESUMEN
Background: Heroin use disorder (HUD) is a complex brain disease that includes multiple phenotypes. Heroin acts primarily as a mu-opioid receptor (OPRM1) agonist. The κ opioid receptor 1 (OPRK1) is critically involved in abstinence and remission. Multiple studies confirm that the OPRM1 and OPRK1 genes are associated with HUD. However, their relationship with the addictive phenotype is still unclear. This study was designed to identify the genetic polymorphisms within OPRM1 and OPRK1 with six HUD phenotypes. Methods: A total of 801 patients with HUD were recruited from the Methadone Maintenance Treatment Program in Xi'an. We identified eight potential functional single nucleotide polymorphisms (SNPs) in the two genes that were genotyped using SNaPshot SNP technology. We then performed a case-control association analysis, investigated particular disease phenotypes, and assessed the extent of epistasis among the variants of the two genes. Results: The OPRK1 rs3802279, rs3802281, and rs963549 genotypes were significantly associated with methadone dosage analyzed by Pearson's chi-square test or binary logistic regression to correct for covariates. The rs3802279 CC, rs3802281 TT, and rs963549 CC genotype carriers required a lower methadone maintenance dose per day. Multifactor dimensionality reduction analysis indicated strong interactions between sex and OPRK1 rs963549. The results of the OPRM1 genotyping did not reveal any associations with the various HUD phenotypes. Conclusion: These findings support an important role of the OPRK1 polymorphism in determining the daily methadone dose and may guide future studies in identifying additional genetic risk factors for HUD.
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Dependencia de Heroína/genética , Receptores Opioides kappa/genética , Receptores Opioides mu/genética , Adulto , Pueblo Asiatico/genética , Biomarcadores Farmacológicos , Estudios de Casos y Controles , China/epidemiología , Relación Dosis-Respuesta a Droga , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos , Humanos , Masculino , Metadona/farmacología , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Trastornos Relacionados con Sustancias/genéticaRESUMEN
Heroin abuse is linked to a deleterious effect on cognitive functioning in the individual. Recent evidences suggest that the serotonin7 receptor (5-HT7R) is engaged in the regulation of cognitive control and the drug use-associated behaviors. However, the role of 5-HT7R in the cognitive control after acute heroin administration has not been studied. The present study aims to investigate whether the knockdown of the 5-HT7R by virus-mediated gene silencing in the medial prefrontal cortex (mPFC) could ameliorate the acute heroin-induced cognitive impairments. The attentional function, impulsivity and compulsivity were assessed by the 5-choice serial reaction time task (5-CSRTT) in mice. The memory ability and locomotor activity were examined by the novel objects recognition (NOR), Y-maze and open-field test (OFT). Acute heroin administration at 5â¯mg/kg produced robust disruptions in attention, impulsivity and motivation in mice. 5-HT7R knockdown in the mPFC did not affect the 5-CSRTT baseline performance, spatial working memory, visual episodic memory and locomotion. However, mPFC 5-HT7R knockdown selectively ameliorated acute heroin-induced increase in omissions and premature responses under conditions of increased perceptual load. In addition, mPFC 5-HT7R knockdown induced increases in perseverative responding observed across both saline and heroin-treated animals. Moreover, 5-HT7R knockdown prevented the heroin-induced decrease in NR1/CaMKII phosphorylation in mPFC, thus suggesting that 5-HT7R and N-methyl-d-aspartic acid (NMDA) receptor signaling may be involved in the cognitive outcomes of acute heroin administration. Altogether, these observations suggest modest and restricted effects of mPFC 5-HT7R knockdown on cognitive behaviors, both in the presence or absence of acute heroin treatment.
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Analgésicos Opioides/administración & dosificación , Cognición/efectos de los fármacos , Heroína/administración & dosificación , Corteza Prefrontal/efectos de los fármacos , Receptores de Serotonina/deficiencia , Animales , Conducta de Elección/efectos de los fármacos , Conducta de Elección/fisiología , Conducta Exploratoria/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Locomoción/efectos de los fármacos , Locomoción/genética , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microinyecciones , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Tiempo de Reacción/efectos de los fármacos , Receptores de Serotonina/genéticaRESUMEN
Recurrent spontaneous abortion (RSA) is a common health problem that affects women of reproductive age. Recent studies have indicated that microRNAs are important factors in miscarriage. This study investigated the role of miR-16 in regulating vascular endothelial growth factor (VEGF) expression and the pathogenesis of RSA. In this report, clinical samples revealed that miR-16 expression was significantly elevated in the villi and decidua of RSA patients. In vitro, miR-16 upregulation inhibited human umbilical vein endothelial cell proliferation, migration and tube formation. Conversely, the downregulation of miR-16 reversed these effects. In vivo, we demonstrated that abnormal miR-16 levels affect the weights of the placenta and embryo and the number of progeny and microvascular density, as well as cause recurrent abortions by controlling VEGF expression in pregnant mice. VEGF, a potential target gene of miR-16, was inversely correlated with miR-16 expression in the decidua of clinical samples. Furthermore, the luciferase reporter system demonstrated that miR-16 was found to directly downregulate the expression of VEGF by binding a specific sequence of its 3'-untranslated region (3'UTR). Collectively, these data strongly suggest that miR-16 regulates placental angiogenesis and development by targeting VEGF expression and is involved in the pathogenesis of RSA.
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Aborto Habitual/genética , MicroARNs/metabolismo , Neovascularización Fisiológica/genética , Factor A de Crecimiento Endotelial Vascular/genética , Regiones no Traducidas 3'/genética , Adulto , Animales , Secuencia de Bases , Movimiento Celular/genética , Proliferación Celular/genética , Decidua/metabolismo , Decidua/patología , Regulación hacia Abajo/genética , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos C57BL , MicroARNs/genética , Placentación/genética , Embarazo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To investigate the relationship between the -308G/A, -857C/T and -1031T/C of tumor necrosis factor alpha gene (TNF- alpha), -174G/C and -572C/G of interleukin-6 gene (IL-6) polymorphisms and schizophrenia. METHODS: Genomic DNA was isolated from the venous blood leukocytes of 346 unrelated patients with schizophrenia and 323 healthy unrelated individuals (control group). All of the polymorphisms were genotyped by PCR-restriction fragment length polymorphisms (PCR-RFLP). Genotype and allele frequencies were analyzed by SPSS13.0 software. RESULTS: There were significant differences in both allele and genotype frequencies of -857C/T of TNF-alpha gene between the schizophrenia and control groups (P< 0.05). The allele T of -857C/T in schizophrenia group was significantly higher than that in control group (chi-square was 9.414, P=0.002, OR=1.511, 95%CI:1.160-1.969). In addition, there were significant differences in the positive and negative syndrome scale (PANSS) total score and negative symptoms between the patients with different -857C/T genotypes, and the negative symptom score of TT genotype was significantly higher than that of CC genotype (P<0.05). CONCLUSION: There is an association between -857C/T of TNF-alpha gene and schizophrenia, individuals with T allele of -857C/T are susceptible to schizophrenia, and there is an association between -857C/T and negative symptom score.
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Interleucina-6/genética , Polimorfismo Genético/genética , Regiones Promotoras Genéticas/genética , Esquizofrenia/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Adulto JovenRESUMEN
GABA is expressed in carotid body (CB) chemoreceptor type I cells and has previously been reported to modulate sensory transmission via presynaptic GABA(B) receptors. Because low doses of clinically important GABA(A) receptor (GABA(A)R) agonists, e.g. benzodiazepines, have been reported to depress afferent CB responses to hypoxia, we investigated the potential contribution of GABA(A)R in co-cultures of rat type I cells and sensory petrosal neurones (PNs). During gramicidin perforated-patch recordings (to preserve intracellular Cl-), GABA and/or the GABA(A) agonist muscimol (50 microm) induced a bicuculline-sensitive membrane depolarization in isolated PNs. GABA-induced whole-cell currents reversed at approximately -38 mV and had an EC50 of approximately 10 microm (Hill coefficient = approximately 1) at -60 mV. During simultaneous PN and type I cell recordings at functional chemosensory units in co-culture, bicuculline reversibly potentiated the PN, but not type I cell, depolarizing response to hypoxia. Application of the CB excitatory neurotransmitter ATP (1 microm) over the soma of functional PN induced a spike discharge that was markedly suppressed during co-application with GABA (2 microm), even though GABA alone was excitatory. RT-PCR analysis detected expression of GABAergic markers including mRNA for alpha1, alpha2, beta2, gamma2S, gamma2L and gamma3 GABA(A)R subunits in petrosal ganglia extracts. Also, CB extracts contained mRNAs for GABA biosynthetic markers, i.e. glutamate decarboxylase (GAD) isoforms GAD 67A,E, and GABA transporter isoforms GAT 2,3 and BGT-1. In CB sections, sensory nerve endings apposed to type I cells were immunopositive for the GABA(A)R beta subunit. These data suggest that GABA, released from the CB during hypoxia, inhibits sensory discharge postsynaptically via a shunting mechanism involving GABA(A) receptors.
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Cuerpo Carotídeo/citología , Células Quimiorreceptoras/fisiología , Receptores de GABA-A/fisiología , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adenosina Trifosfato/farmacología , Animales , Cuerpo Carotídeo/metabolismo , Hipoxia de la Célula/fisiología , Células Cultivadas , Células Quimiorreceptoras/efectos de los fármacos , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Agonistas de Receptores GABA-B , Antagonistas de Receptores de GABA-B , Ganglios/citología , Ganglios/metabolismo , Expresión Génica/efectos de los fármacos , Glutamato Descarboxilasa/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Ratas , Ratas Wistar , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-B/fisiología , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
BACKGROUND: The general anesthetic etomidate acts via gamma-aminobutyric acid type A (GABA(A)) receptors, enhancing activation at low GABA and prolonging deactivation. Azi-etomidate is a photo-reactive etomidate derivative with similar pharmacological actions, which has been used to identify putative binding sites. The authors examine the irreversible effects of azi-etomidate photo-modification on functional GABA(A) receptors in cell membranes. METHODS: GABA(A) receptors (alpha1beta2gamma2L) were expressed in both Xenopus oocytes and human embryonic kidney cells exposed to 365 nm light-activated azi-etomidate with or without GABA, then extensively washed. Receptor-mediated chloride currents were measured using voltage clamp electrophysiology to assess the ratio of peak responses at 10 microm and 1 mm GABA (I10/I1000) and deactivation time course. RESULTS: After azi-etomidate photo-modification, I10/I1000 ratios were persistently enhanced and deactivation was prolonged, mimicking reversible azi-etomidate actions. Azi-etomidate and ultraviolet light were required to produce irreversible receptor modulation. Adding GABA during photo-modification greatly enhanced irreversible modulation. Azi-etomidate modification also dose-dependently reduced maximal GABA-activated currents, consistent with accumulation of permanently desensitized receptors. Excess etomidate during azi-etomidate photo-modification competitively reduced permanent desensitization. Persistent channel modulation was blocked by 320-fold excess etomidate but enhanced when 32-fold excess etomidate was present. CONCLUSIONS: Azi-etomidate efficiently photo-modifies etomidate sites on GABA(A) receptors in intact cells, producing persistent functional changes that mimic its reversible effects. The results demonstrate sequential modification at more than one etomidate site per receptor. The sites display reciprocal positive cooperativity. In combination with focal photo-activation, azi-etomidate may prove useful for studies of anesthetic actions in neural circuits.
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Anestésicos Generales , Etomidato/análogos & derivados , Activación del Canal Iónico/efectos de los fármacos , Estimulación Luminosa , Etiquetas de Fotoafinidad/farmacología , Receptores de GABA-A/metabolismo , Animales , Línea Celular , Femenino , Humanos , Activación del Canal Iónico/fisiología , Estimulación Luminosa/métodos , Xenopus laevisRESUMEN
Presynaptic Ca(2+) channels are inhibited by neurotransmitters acting through G protein-coupled receptors via a membrane-delimited pathway. Inhibition is reversed by strong depolarization, resulting in prepulse facilitation. Activated G protein betagamma subunits (Gbetagamma) are required for maximal prepulse facilitation. Gbetagamma binds to multiple sites on Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 alpha1 subunits. Here we examine the functional relevance of a C-terminal binding site for Gbetagamma on Ca(v)2.2b channels, which mediate N-type Ca(2+) currents. In vitro binding studies showed that Gbetagamma subunits bind to the intracellular loop connecting domains I and II and the C-terminal domain of Ca(v)2.2b but not the intracellular loops connecting domains II and III or III and IV. Deletion analysis revealed that the binding site is located near the C terminus, within amino acid residues 2257 to 2336. Directed yeast two-hybrid analysis confirmed this specific binding interaction in vivo in yeast cells. Ca(v)2.2b channels with this site deleted had normal function properties, and they were inhibited essentially normally by strong activation of G proteins with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) and were facilitated nearly normally by depolarizing prepulses. Similarly deletion of this site had small, statistically insignificant effects on inhibition of Ca(2+) current and on prepulse facilitation in the presence of somatostatin to stimulate receptor-mediated activation of G proteins. In contrast, deletion of the C-terminal Gbetagamma site substantially reduced the low level of intrinsic prepulse facilitation present at the basal level of G protein activation in tsA-201 cells. Thus, this C-terminal Gbetagamma binding site contributes to the affinity or efficacy of Gbetagamma regulation at basal levels of G protein activation. The simplest interpretation of our results is that the C-terminal binding site increases the affinity of Gbetagamma for the channel but is not required for Gbetagamma action. C-terminal binding of Gbetagamma may influence the physiological responsiveness of Ca(2+) channels to low-level G protein activation.
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Canales de Calcio Tipo N/fisiología , Proteínas de Unión al GTP/metabolismo , Estructura Terciaria de Proteína/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Aminoácido , Somatostatina/farmacologíaRESUMEN
At clinical concentrations, the potent intravenous general anesthetic etomidate enhances gamma-aminobutyric acid, type A (GABA(A)) receptor activity elicited with low gamma-aminobutyric acid (GABA) concentrations, whereas much higher etomidate concentrations activate receptors in the absence of GABA. Therefore, GABA(A) receptors may possess two types of etomidate sites: high affinity GABA-modulating sites and low affinity channel-activating sites. However, GABA modulation and direct activation share stereoselectivity for the (R)(+)-etomidate isomer and display parallel dependence on GABA(A) beta subunit isoforms, suggesting that these two actions may be mediated by a single class of etomidate site(s) that exert one or more molecular effects. In this study, we assessed GABA modulation by etomidate using leftward shifts of electrophysiological GABA concentration responses in cells expressing human alpha1beta2gamma2L receptors. Etomidate at up to 100 microm reduced GABA EC(50) values by over 100-fold but without apparent saturation, indicating the absence of high affinity etomidate sites. In experiments using a partial agonist, P4S, etomidate both reduced EC(50) and increased maximal efficacy, demonstrating that etomidate shifts the GABA(A) receptor gating equilibrium toward open states. Results were quantitatively analyzed using equilibrium receptor gating models, wherein a postulated class of equivalent etomidate sites both directly activates receptors and enhances agonist gating. A Monod-Wyman-Changeux co-agonist mechanism with two equivalent etomidate sites that allosterically enhance GABA(A) receptor gating independently of agonist binding most simply accounts for direct activation and agonist modulation. This model also correctly predicts the actions of etomidate on GABA(A) receptors containing a point mutation that increases constitutive gating activity.
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Etomidato/farmacología , Receptores de GABA-A/fisiología , Sitio Alostérico , Animales , Línea Celular , ADN Complementario , Femenino , Agonistas del GABA/farmacología , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Oocitos/fisiología , Receptores de GABA-A/química , Receptores de GABA-A/efectos de los fármacos , Transfección , Xenopus laevis , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The peripheral control of breathing is mediated by O2-sensitive carotid body (CB) type 1 cells, which express multiple neurotransmitters including the monoamines, dopamine and serotonin (5-HT). Whereas dopamine has been extensively studied, 5-HT has received little attention. Here, to elucidate the role of 5-HT in CB chemotransmission, we used perforated-patch recording from rat type 1 cell clusters and co-cultured petrosal (afferent) neurones. 5-HT induced action potentials and/or membrane depolarization associated with a conductance decrease in approximately 40% of recordings from type 1 cells (n = 78/192). These responses were markedly inhibited by the 5-HT2 receptor antagonist ketanserin (10-50 microM) and by the protein kinase C (PKC) inhibitor chelerythrine (50 microM). The PKC activator 1-oleoyl-2-acetylglycerol (OAG; 50 microM) mimicked the 5-HT-induced depolarization, and the combined effects of 5-HT and OAG were non-additive. The 5-HT-induced responses reversed near the potassium (K+) equilibrium potential (at approximately -82 mV; EK = -83 mV), suggesting inhibition of a resting K+ conductance. In type 1 cells (n = 7), voltage-activated outward K+ current was also inhibited by 1-50 microM 5-HT, an effect that was prevented by PKC inhibitors (chelerythrine and NPC 15437) and mimicked by OAG; the outward K+ current inhibited by 5-HT appeared to be predominantly a Ca(2+)-dependent K+ current. The 5-HT2 receptor blockers ketanserin and ritanserin reversibly inhibited spontaneous action potentials and the hypoxia-induced receptor potential recorded from clustered type 1 cells. Moreover, these blockers reversibly inhibited the hypoxic chemosensory response recorded postsynaptically in petrosal neurones that functionally innervated type 1 clusters in co-culture. RT-PCR and confocal immunofluorescence techniques revealed 5-HT2a receptor expression in rat CB type 1 cells. These results suggest that release of endogenous 5-HT regulates CB chemoreceptor function presynaptically, by a positive feedback mechanism involving autocrine-paracrine stimulation of 5-HT2a receptors and PKC modulation of resting and Ca(2+)-dependent K+ conductances.
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Cuerpo Carotídeo/fisiología , Células Quimiorreceptoras/fisiología , Canales de Potasio/metabolismo , Proteína Quinasa C/metabolismo , Serotonina/farmacología , Animales , Comunicación Autocrina/fisiología , Calcio/metabolismo , Cuerpo Carotídeo/citología , Células Cultivadas , Células Quimiorreceptoras/citología , Técnicas de Cocultivo , Retroalimentación Fisiológica/fisiología , Expresión Génica , Hipoxia/metabolismo , Hipoxia/fisiopatología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Comunicación Paracrina/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Terminales Presinápticos/fisiología , Ratas , Receptor de Serotonina 5-HT2A/genética , Receptor de Serotonina 5-HT2A/metabolismoRESUMEN
Ca(v)2.1 channels, which conduct P/Q-type Ca(2+) currents, were expressed in superior cervical ganglion neurons in cell culture, and neurotransmission initiated by these exogenously expressed Ca(2+) channels was measured. Deletions in the synaptic protein interaction (synprint) site in the intracellular loop between domains II and III of Ca(v)2.1 channels reduced their effectiveness in synaptic transmission. Surprisingly, this effect was correlated with loss of presynaptic localization of the exogenously expressed channels. Ca(v)1.2 channels, which conduct L-type Ca(2+) currents, are ineffective in supporting synaptic transmission, but substitution of the synprint site from Ca(v)2.1 channels in Ca(v)1.2 was sufficient to establish synaptic transmission initiated by L-type Ca(2+) currents through the exogenous Ca(v)1.2 channels. Substitution of the synprint site from Ca(v)2.2 channels, which conduct N-type Ca(2+) currents, was even more effective than Ca(v)2.1. Our results show that localization and function of exogenous Ca(2+) channels in nerve terminals of superior cervical ganglion neurons require a functional synprint site and suggest that binding of soluble NSF attachment protein receptor (SNARE) proteins to the synprint site is a necessary permissive event for nerve terminal localization of presynaptic Ca(2+) channels.