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PURPOSE: This document describes the consensus process and intervention for a National Institutes of Health (NIH)-funded multi-site feasibility study utilizing acupuncture for ACUte paIn in The EmergencY Department (ACUITY). The acupuncture intervention is designed to be flexible and responsive to the most common Emergency Department (ED) scenarios, including trauma, acute pain of the low back, abdomen and/or musculoskeletal system, renal colic and headache. BACKGROUND: Opioids remain a primary treatment for acute ED pain with attendant risk of adverse effects, addiction liability, diversion and death. Effective/safer options for acute pain are needed. Although acupuncture therapy has shown promise for acute pain in the ED alone or in conjunction with usual care, pragmatic trials are needed to obtain definitive and generalizable evidence. METHODS: An Acupuncture Advisory Panel was convened that included nine acupuncture experts with 5-44 years of experience in practice and 2-16 years of experience in the acute pain care setting. A modified Delphi process was used with provision of a literature review, surveys of our panel members, three online discussions and email discussion as needed. The STandards for Reporting Interventions in Controlled Trials (STRICTA) checklist was used as a guide. RESULTS: A responsive acupuncture intervention was agreed on for ACUITY. Session forms were fashioned in REDCap (Research Electronic Data Capture program to capture essential treatment data, assess fidelity and inform our design for a future pragmatic multi-site randomized controlled trial (RCT) of acupuncture in the ED, and for use by other future researchers. CONCLUSION: Development of a responsive manualization intervention provides the appropriate framework for conducting a future, pragmatic, multi-site, definitive RCT of acupuncture in the ED. TRIAL REGISTRATION NUMBER: NCT04880733 (ClinicalTrials.gov).
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Terapia por Acupuntura , Dolor Agudo , Dolor Agudo/terapia , Consenso , Servicio de Urgencia en Hospital , Estudios de Factibilidad , Humanos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
The cytokines IL-1α and IL-1ß exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1). Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP), with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV)-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development.
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Alelos , Receptores Tipo I de Interleucina-1/genética , Transducción de Señal/genética , Animales , Encéfalo/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Transgénicos , Receptores Tipo I de Interleucina-1/metabolismoRESUMEN
BACKGROUND: Rare genetic variation is an important class of autism spectrum disorder (ASD) risk factors and can implicate biological networks for investigation. Altered serotonin (5-HT) signaling has been implicated in ASD, and we and others have discovered multiple, rare, ASD-associated variants in the 5-HT transporter (SERT) gene leading to elevated 5-HT re-uptake and perturbed regulation. We hypothesized that loci encoding SERT regulators harbor variants that impact SERT function and/or regulation and therefore could contribute to ASD risk. The adenosine A3 receptor (A3AR) regulates SERT via protein kinase G (PKG) and other signaling pathways leading to enhanced SERT surface expression and catalytic activity. METHODS: To test our hypothesis, we asked whether rare variants in the A3AR gene (ADORA3) were increased in ASD cases vs. controls. Discovery sequencing in a case-control sample and subsequent analysis of comparison exome sequence data were conducted. We evaluated the functional impact of two variants from the discovery sample on A3AR signaling and SERT activity. RESULTS: Sequencing discovery showed an increase of rare coding variants in cases vs. controls (P=0.013). While comparison exome sequence data did not show a significant enrichment (P=0.071), combined analysis strengthened evidence for association (P=0.0025). Two variants discovered in ASD cases (Leu90Val and Val171Ile) lie in or near the ligand-binding pocket, and Leu90Val was enriched individually in cases (P=0.040). In vitro analysis of cells expressing Val90-A3AR revealed elevated basal cGMP levels compared with the wildtype receptor. Additionally, a specific A3AR agonist increased cGMP levels across the full time course studied in Val90-A3AR cells, compared to wildtype receptor. In Val90-A3AR/SERT co-transfections, agonist stimulation elevated SERT activity over the wildtype receptor with delayed 5-HT uptake activity recovery. In contrast, Ile171-A3AR was unable to support agonist stimulation of SERT. Although both Val90 and Ile171 were present in greater numbers in these ASD cases, segregation analysis in families showed incomplete penetrance, consistent with other rare ASD risk alleles. CONCLUSIONS: Our results validate the hypothesis that the SERT regulatory network harbors rare, functional variants that impact SERT activity and regulation in ASD, and encourages further investigation of this network for other variation that may impact ASD risk.
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Activation of A3 adenosine receptors (A3ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A3AR agonist stimulation of SERT activity is lost in A3AR knockout mice. A3AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A3ARs target SERTs among other potential effectors is unknown. Here we present evidence that A3ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A3AR/hSERT cotransfected cells. Treatment of A3AR/SERT-cotransfected Chinese hamster ovary cells with the A3AR agonist N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (1 µM, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A3AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A3AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A3AR activation. Because PKGI and nitric-oxide synthetase are required for A3AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A3AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.
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Receptor de Adenosina A3/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Inmunohistoquímica , Ratones , Ratones Noqueados , Unión ProteicaRESUMEN
Serotonin (5-hydroxytryptamine, 5-HT) has long been implicated in regulation of mood. Medications that block the neuronal 5-HT transporter (SERT) are used as major pharmacological treatment for mood disorders. Conversely, stimuli that enhance SERT activity might be predicted to diminish synaptic 5-HT availability and increase the risk for 5-HT-related CNS disorders. We have shown that the inflammatory cytokines enhance brain SERT activity in cultured serotonergic cells and nerve terminal preparations in vitro. In this study, we establish that intraperitoneal injection of the cytokine-inducer lipopolysaccharide (LPS) stimulates brain SERT activity, acting at doses below those required to induce overt motor suppression. SERT stimulation by LPS is paralleled by increased immobility in both the tail suspension test (TST) and the forced swim test (FST); antidepressant-sensitive alterations are thought to model aspects of behavioral despair. Both the stimulation of SERT activity and induced immobility are absent when LPS is administered to interleukin-1 receptor (IL-1R)-deficient mice and in the presence of SB203580, an inhibitor of IL-1R-stimulated p38 MAPK. Moreover, the ability of LPS to enhance immobility in TST is lost in SERT knockout mice. These findings reveal an ability of peripheral inflammatory stimuli to enhance brain SERT activity through IL-1R and p38 MAPK pathways in vivo and identify a requirement for SERT expression in immune-system-modulated despair behaviors. Our studies identify IL-1R- and p38 MAPK-dependent regulation of SERT as one of the mechanisms by which environmentally driven immune system activation can trigger despair-like behavior in an animal model, encouraging future analysis of the pathway for risk factors in neuropsychiatric disorders.
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Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Depresión/inducido químicamente , Lipopolisacáridos/farmacología , Receptores de Interleucina-1/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Encéfalo/efectos de los fármacos , Depresión/prevención & control , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Piridinas/farmacología , Receptores de Interleucina-1/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Sinaptosomas/metabolismoRESUMEN
The human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT, SLC6A4) figures prominently in the etiology and treatment of many prevalent neurobehavioral disorders including anxiety, alcoholism, depression, autism, and obsessive-compulsive disorder (OCD). Here, we use naturally occurring polymorphisms in recombinant inbred (RI) lines to identify multiple phenotypes associated with altered SERT function. The widely used mouse strain C57BL/6J, harbors a SERT haplotype defined by 2 nonsynonymous coding variants [Gly-39 and Lys-152 (GK)]. At these positions, many other mouse lines, including DBA/2J, encode, respectively, Glu-39 and Arg-152 (ER haplotype), amino acids found also in hSERT. Ex vivo synaptosomal 5-HT transport studies revealed reduced uptake associated with the GK variant, a finding confirmed by in vitro heterologous expression studies. Experimental and in silico approaches using RI lines (C57BL/6J x DBA/2J = BXD) identify multiple anatomical, biochemical, and behavioral phenotypes specifically impacted by GK/ER variation. Among our findings are several traits associated with alcohol consumption and multiple traits associated with dopamine signaling. Further bioinformatic analysis of BXD phenotypes, combined with biochemical evaluation of SERT knockout mice, nominates SERT-dependent 5-HT signaling as a major determinant of midbrain iron homeostasis that, in turn, dictates iron-regulated DA phenotypes. Our studies provide an example of the power of coordinated in vitro, in vivo, and in silico approaches using mouse RI lines to elucidate and quantify the system-level impact of gene variation.
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Conducta Animal , Polimorfismo Genético , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Consumo de Bebidas Alcohólicas , Animales , Biología Computacional , Dopamina/metabolismo , Humanos , Hierro/metabolismo , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos , Fenotipo , Serotonina/metabolismoRESUMEN
The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A(3) adenosine receptors (A(3)AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A(3)ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A(3)AR agonist N(6)-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A(3)AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A(3)AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport V(max) with no significant change in K(m). As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK(5)H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A(3)ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A(3)ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.
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Receptor de Adenosina A3/fisiología , Serotonina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Transporte Biológico , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiología , Sinaptosomas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Proinflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of major psychiatric disorders. We have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) induces a catalytic activation of the serotonin transporter (SERT) arising from a reduction in the SERT Km for 5-hydroxytryptamine (5-HT). As inflammatory cytokines can activate p38 MAPK, we hypothesized that they might also activate neuronal SERT. Indeed, Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulated serotonin uptake in both the rat embryonic raphe cell line, RN46A, and in mouse midbrain and striatal synaptosomes. In RN46A cells, IL-1beta stimulated 5-HT uptake in a dose- and time-dependent manner, peaking in 20 min at 100 ng/ml. This was abolished by IL-1ra (20 ng/ml), an antagonist of the IL-1 receptor, and by SB203580 (5 microM), a p38 MAPK inhibitor. TNF-alpha also dose- and time-dependently stimulated 5-HT uptake that was only partially blocked by SB203580. Western blots showed that IL-1beta and TNF-alpha activated p38 MAPK, in an SB203580-sensitive manner. IL-1beta induced an SB203580-sensitive decrease in 5-HT Km with no significant change in Vmax. In contrast, TNF-alpha stimulation decreased 5-HT Km and increased SERT Vmax. SB203580 selectively blocked the TNF-alpha-induced change in SERT Km. In mouse midbrain and striatal synaptosomes, maximal stimulatory effects on 5-HT uptake occurred at lower concentrations (IL-1beta, 10 ng/ml; TNF-alpha, 20 ng/ml), and over shorter incubation times (10 min). As with RN46A cells, the effects of IL-1beta and TNF-alpha were completely (IL-1beta) or partially (TNF-alpha) blocked by SB203580. These results provide the first evidence that proinflammatory cytokines can acutely regulate neuronal SERT activity via p38 MAPK-linked pathways.
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Interleucina-1/farmacología , Neuronas/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Análisis de Varianza , Animales , Línea Celular , Cuerpo Estriado/ultraestructura , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mesencéfalo/ultraestructura , Ratones , Núcleos del Rafe/citología , Ratas , Serotonina/metabolismo , Serotonina/farmacología , Sinaptosomas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.
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Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Expresión Génica , Variación Genética , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Células HeLa , Herpesvirus Humano 4 , Humanos , Linfocitos/metabolismo , Fosforilación , Transporte de Proteínas/fisiología , Serotonina/metabolismo , TransfecciónRESUMEN
Presynaptic, plasma membrane serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) clear 5-HT following vesicular release and are regulated through trafficking-dependent pathways. Recently, we provided evidence for a trafficking-independent mode of SERT regulation downstream of adenosine receptor (AR) activation that is sensitive to p38 MAPK inhibitors. Here, we probe this pathway in greater detail, demonstrating elevation of 5-HT transport by multiple p38 MAPK activators (anisomycin, H(2)O(2), and UV radiation), in parallel with p38 MAPK phosphorylation, as well as suppression of anisomycin stimulation by p38 MAPK siRNA treatments. Studies with transporter-transfected Chinese hamster ovary cells reveal that SERT stimulation is shared with the human norepinephrine transporter but not the human dopamine transporter. Saturation kinetic analyses of anisomycin-SERT activity reveal a selective reduction in 5-HT K(m) supported by a commensurate increase in 5-HT potency (K(i)) for displacing surface antagonist binding. Anisomycin treatments that stimulate SERT activity do not elevate surface SERT surface density whereas stimulation is lost with preexposure of cells to the surface-SERT inactivating reagent, 2-(trimethylammonium)ethyl methane thiosulfonate. Guanylyl cyclase (1H-(1,2,4)-oxadiazolo[4,3-a]-quinoxalin-1-one) and protein kinase G inhibitors (H8, DT-2) block AR stimulation of SERT yet fail to antagonize SERT stimulation by anisomycin. We thus place p38 MAPK activation downstream of protein kinase G in a SERT-catalytic regulatory pathway, distinct from events controlling SERT surface density. In contrast, the activity of protein phosphatase 2A inhibitors (fostriecin and calyculin A) to attenuate anisomycin stimulation of 5-HT transport suggests that protein phosphatase 2A is a critical component of the pathway responsible for p38 MAPK up-regulation of SERT catalytic activity.
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Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Serotonina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Membrana Celular/metabolismo , Cricetinae , Activadores de Enzimas/farmacología , Humanos , Proteína Fosfatasa 2 , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacosRESUMEN
The serotonin (5-hydroxtryptamine, 5-HT) transporter (SERT) plays a critical role in the inactivation of synaptic 5-HT and has been implicated in multiple psychiatric and peripheral disorders. SERT regulation studies demonstrate that activation of cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-linked pathways can increase SERT activity. As cGMP actions are limited by cGMP-specific phosphodiesterase (PDEs), we investigated whether the cGMP-specific PDE5 inhibitor sildenafil (Viagra) can stimulate 5-HT uptake and potentiate cGMP-mediated regulation. In RBL-2H3 cells, SERT activity was stimulated by sildenafil in a concentration- and time-dependent manner. Sildenafil also enhanced the stimulation of SERT triggered by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), effects blocked by the PKG inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8). Sildenafil stimulation of 5-HT uptake arises from an increase in 5-HT transport Vmax and is paralleled by elevated SERT surface antagonist binding, also H8-sensitive. These findings implicate cGMP-targeted PDEs in limiting the regulation of antidepressant-sensitive 5-HT transport.
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3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Piperazinas/farmacología , Serotonina/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Animales , Células CHO , Cricetinae , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Purinas , Ratas , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Citrato de Sildenafil , SulfonasRESUMEN
Serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) are critical determinants of synaptic 5-HT inactivation and the targets for multiple drugs used to treat psychiatric disorders. In support of prior studies, we found that short-term (5-30 min) application of the adenosine receptor (AR) agonist 5'-N-ethylcarboxamidoadenosine (NECA) induces an increase in 5-HT uptake Vmax in rat basophilic leukemia 2H3 cells that is enhanced by pretreatment with the cGMP phosphodiesterase inhibitor sildenafil. NECA stimulation is blocked by the A3 AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191), by the phospholipase C inhibitor 1-(6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl)-1H-pyrrole-2,5-dione (U73122), by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, and by the guanyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Hydroxylamine, a nitric-oxide donor, and 8-bromo-cGMP, a membrane-permeant analog of cGMP, mimic the effects of NECA on 5-HT uptake, whereas the protein kinase G (PKG) inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) blocks NECA, hydroxylamine, and 8-bromo-cGMP effects. NECA stimulation activates p38 mitogen-activated protein kinase (MAPK), whereas p38 MAPK inhibitors block NECA stimulation of SERT activity, as does the protein phosphatase 2A (PP2A) inhibitor calyculin A. 5-HT-displaceable [125I]3beta-(4-iodophenyl)-tropane-2beta-carboxylic acid methylester tartrate (RTI-55) whole-cell binding is increased by NECA or sildenafil, and both surface binding and cell surface SERT protein are elevated after NECA or sildenafil stimulation of AR/SERT-cotransfected Chinese hamster ovary cells. Whereas p38 MAPK inhibition blocks NECA stimulation of 5-HT activity, it fails to blunt stimulation of SERT surface density. Moreover, inactivation of existing surface SERTs fails to eliminate NECA stimulation of SERT. Together, these results reveal two PKG-dependent pathways supporting rapid SERT regulation by A3 ARs, one leading to enhanced SERT surface trafficking, and a separate, p38 MAPK-dependent process augmenting SERT intrinsic activity.
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Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas del Tejido Nervioso/metabolismo , Receptores Purinérgicos P1/fisiología , Serotonina/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Transporte Biológico , Células CHO , Calcio/metabolismo , Células Cultivadas , Cricetinae , GMP Cíclico/metabolismo , Femenino , Guanilato Ciclasa/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Piperazinas/farmacología , Purinas , Ratas , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Citrato de Sildenafil , Sulfonas , Fosfolipasas de Tipo C/metabolismo , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
To study HIV-1 primary isolate neutralization, we have used DH012 as a model to study the immunogenicity of several DH012 immunogens and determine the potential neutralization epitopes in the virus envelope glycoprotein. Previously, we identified that DH012 infected animals mount potent neutralizing activity against a conformational epitope (CEV) that involves multiple variable regions. In this study, we show that the conformational epitope can be reconstituted with one gp120 recombinant fragment containing sequences from the V1/V2 loop and the bridging sheet of gp120 and a V3 peptide. In contrast to DH012 infection, we previously demonstrated that animals immunized with DH012 gp120 induced potent neutralizing antibodies directed at the V3 domain of gp120. In this study, a second neutralizing activity against the V1/V2 region of gp120 was identified from the same guinea pig sera. In summary, several neutralization epitopes are identified on DH012, including the CEV, V1/V2, V3, 17b, IgG1b12, and 2G12 epitopes. Infectious DH012 virus carrying oligomeric envelope appears to raise primarily neutralizing antibodies that recognize a discontinuous conformationally dependent epitope whereas the monomeric gp120 induces antibodies that are primarily directed at epitopes in the V3 and V1/V2 domains. The DH012 neutralizing epitopes, such as V1/V2 and V3, are either cryptic or poorly immunogenic in chimpanzees. However, immunogens, such as gp120, could be designed to induce neutralizing activity against epitopes that are poorly immunogenic, such as V1/V2 of DH012, in the native envelope glycoproteins.