RESUMO
Endothelial dysfunction may contribute to pathogenesis of Takotsubo cardiomyopathy, but mechanism underlying endothelial dysfunction in the setting of catecholamine excess has not been clarified. The study reports that D1/D5 dopamine receptor signaling and small conductance calcium-activated potassium channels contribute to high concentration catecholamine induced endothelial cell dysfunction. For mimicking catecholamine excess, 100 µM epinephrine (Epi) was used to treat human cardiac microvascular endothelial cells. Patch clamp, FACS, ELISA, PCR, western blot and immunostaining analyses were performed in the study. Epi enhanced small conductance calcium-activated potassium channel current (ISK1-3) without influencing the channel expression and the effect was attenuated by D1/D5 receptor blocker. D1/D5 agonists mimicked the Epi effect, suggesting involvement of D1/D5 receptors in Epi effects. The enhancement of ISK1-3 caused by D1/D5 activation involved roles of PKA, ROS and NADPH oxidases. Activation of D1/D5 and SK1-3 channels caused a hyperpolarization, reduced NO production and increased ROS production. The NO reduction was membrane potential independent, while ROS production was increased by the hyperpolarization. ROS (H2O2) suppressed NO production. The study demonstrates that high concentration catecholamine can activate D1/D5 and SK1-3 channels through NADPH-ROS and PKA signaling and reduce NO production, which may facilitate vasoconstriction in the setting of catecholamine excess.
Assuntos
Células Endoteliais , Epinefrina , Espécies Reativas de Oxigênio , Transdução de Sinais , Humanos , Transdução de Sinais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Óxido Nítrico/metabolismo , Catecolaminas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , NADPH Oxidases/metabolismo , Receptores de Dopamina D5/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores Dopaminérgicos/metabolismoRESUMO
Recent work demonstrated that activation of spinal D1 and D5 dopamine receptors (D1/D5Rs) facilitates non-Hebbian long-term potentiation (LTP) at primary afferent synapses onto spinal projection neurons. However, the cellular localization of the D1/D5Rs driving non-Hebbian LTP in spinal nociceptive circuits remains unknown, and it is also unclear whether D1/D5R signaling must occur concurrently with sensory input in order to promote non-Hebbian LTP at these synapses. Here we investigate these issues using cell-type-selective knockdown of D1Rs or D5Rs from lamina I spinoparabrachial neurons, dorsal root ganglion (DRG) neurons, or astrocytes in adult mice of either sex using Cre recombinase-based genetic strategies. The LTP evoked by low-frequency stimulation of primary afferents in the presence of the selective D1/D5R agonist SKF82958 persisted following the knockdown of D1R or D5R in spinoparabrachial neurons, suggesting that postsynaptic D1/D5R signaling was dispensable for non-Hebbian plasticity at sensory synapses onto these key output neurons of the superficial dorsal horn (SDH). Similarly, the knockdown of D1Rs or D5Rs in DRG neurons failed to influence SKF82958-enabled LTP in lamina I projection neurons. In contrast, SKF82958-induced LTP was suppressed by the knockdown of D1R or D5R in spinal astrocytes. Furthermore, the data indicate that the activation of D1R/D5Rs in spinal astrocytes can either retroactively or proactively drive non-Hebbian LTP in spinoparabrachial neurons. Collectively, these results suggest that dopaminergic signaling in astrocytes can strongly promote activity-dependent LTP in the SDH, which is predicted to significantly enhance the amplification of ascending nociceptive transmission from the spinal cord to the brain.
Assuntos
Astrócitos , Potenciação de Longa Duração , Receptores de Dopamina D1 , Receptores de Dopamina D5 , Sinapses , Animais , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genética , Potenciação de Longa Duração/fisiologia , Astrócitos/metabolismo , Astrócitos/fisiologia , Camundongos , Masculino , Receptores de Dopamina D5/metabolismo , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/genética , Feminino , Sinapses/fisiologia , Sinapses/metabolismo , Gânglios Espinais/citologia , Corno Dorsal da Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/citologia , Camundongos Transgênicos , Camundongos Endogâmicos C57BLRESUMO
INTRODUCTION: The dopamine D5 receptor (D5R) shows high expression in cortical regions, yet the role of the receptor in learning and memory remains poorly understood. This study evaluated the impact of prefrontal cortical (PFC) D5R knockdown in rats on learning and memory and assessed the role of the D5R in the regulation of neuronal oscillatory activity and glycogen synthase kinase-3 (GSK-3ß), processes integral to cognitive function. MATERIALS AND METHODS: Using an adeno-associated viral (AAV) vector, male rats were infused with shRNA to the D5R bilaterally into the PFC. Local field potential recordings were taken from freely moving animals and spectral power and coherence were evaluated in, and between, the PFC, orbitofrontal cortex (OFC), hippocampus (HIP), and thalamus. Animals were then assessed in object recognition, object location, and object in place tasks. The activity of PFC GSK-3ß, a downstream effector of the D5R, was evaluated. RESULTS: AAV-mediated knockdown of the D5R in the PFC induced learning and memory deficits. These changes were accompanied by elevations in PFC, OFC, and HIP theta spectral power and PFC-OFC coherence, reduced PFC-thalamus gamma coherence, and increased PFC GSK-3ß activity. CONCLUSION: This work demonstrates a role for PFC D5Rs in the regulation of neuronal oscillatory activity and learning and memory. As elevated GSK-3ß activity has been implicated in numerous disorders of cognitive dysfunction, this work also highlights the potential of the D5R as a novel therapeutic target via suppression of GSK-3ß.
Assuntos
Neurônios , Receptores de Dopamina D5 , Ratos , Masculino , Animais , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/metabolismo , Glicogênio Sintase Quinase 3 beta , Neurônios/metabolismo , Hipocampo/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de Dopamina D1/genéticaRESUMO
Aims: Reactive oxygen species are highly reactive molecules generated in different subcellular compartments. Both the dopamine D5 receptor (D5R) and endoplasmic reticulum (ER)-resident peroxiredoxin-4 (PRDX4) play protective roles against oxidative stress. This study is aimed at investigating the interaction between PRDX4 and D5R in regulating oxidative stress in the kidney. Results: Fenoldopam (FEN), a D1R and D5R agonist, increased PRDX4 protein expression, mainly in non-lipid rafts, in D5R-HEK 293 cells. FEN increased the co-immunoprecipitation of D5R and PRDX4 and their colocalization, particularly in the ER. The efficiency of Förster resonance energy transfer was increased with FEN treatment measured with fluorescence lifetime imaging microscopy. Silencing of PRDX4 increased hydrogen peroxide production, impaired the inhibitory effect of FEN on hydrogen peroxide production, and increased the production of interleukin-1ß, tumor necrosis factor (TNF), and caspase-12 in renal cells. Furthermore, in Drd5-/- mice, which are in a state of oxidative stress, renal cortical PRDX4 was decreased whereas interleukin-1ß, TNF, and caspase-12 were increased, relative to their normotensive wild-type Drd5+/+ littermates. Innovation: Our findings demonstrate a novel relationship between D5R and PRDX4 and the consequent effects of this relationship in attenuating hydrogen peroxide production in the ER and the production of proinflammatory cytokines. This study provides the potential for the development of biomarkers and new therapeutics for renal inflammatory disorders, including hypertension. Conclusion: PRDX4 interacts with D5R to decrease oxidative stress and inflammation in renal cells that may have the potential for translational significance. Antioxid. Redox Signal. 38, 1150-1166.
Assuntos
Peróxido de Hidrogênio , Receptores de Dopamina D5 , Camundongos , Humanos , Animais , Receptores de Dopamina D5/metabolismo , Interleucina-1beta/metabolismo , Peróxido de Hidrogênio/metabolismo , Caspase 12/metabolismo , Células HEK293 , Rim/metabolismo , Fenoldopam/metabolismo , Fenoldopam/farmacologia , Estresse Oxidativo , Inflamação/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismoRESUMO
BACKGROUND AND PURPOSE: CaV 3.1-3 currents differentially contribute to neuronal firing patterns. CaV 3 are regulated by G protein-coupled receptors (GPCRs) activity, but information about CaV 3 as targets of the constitutive activity of GPCRs is scarce. We investigate the impact of D5 recpetor constitutive activity, a GPCR with high levels of basal activity, on CaV 3 functionality. D5 recpetor and CaV 3 are expressed in the hippocampus and have been independently linked to pathophysiological states associated with epilepsy. EXPERIMENTAL APPROACH: Our study models were HEK293T cells heterologously expressing D1 or D5 receptor and CaV 3.1-3, and mouse brain slices containing the hippocampus. We used chlorpromazine (D1 /D5 inverse agonist) and a D5 receptor mutant lacking constitutive activity as experimental tools. We measured CaV 3 currents and excitability parameters using the patch-clamp technique. We completed our study with computational modelling and imaging technique. KEY RESULTS: We found a higher sensitivity to TTA-P2 (CaV 3 blocker) in CA1 pyramidal neurons obtained from chlorpromazine-treated animals compared with vehicle-treated animals. We found that CaV 3.2 and CaV 3.3-but not CaV 3.1-are targets of D5 receptor constitutive activity in HEK293T cells. Finally, we found an increased firing rate in CA1 pyramidal neurons from chlorpromazine-treated animals in comparison with vehicle-treated animals. Similar changes in firing rate were observed on a neuronal model with controlled CaV 3 currents levels. CONCLUSIONS AND IMPLICATIONS: Native hippocampal CaV 3 and recombinant CaV 3.2-3 are sensitive to D5 receptor constitutive activity. Manipulation of D5 receptor constitutive activity could be a valuable strategy to control neuronal excitability, especially in exacerbated conditions such as epilepsy.
Assuntos
Dopamina , Receptores de Dopamina D1 , Animais , Humanos , Camundongos , Clorpromazina/farmacologia , Agonismo Inverso de Drogas , Células HEK293 , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismoRESUMO
Tissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer. SIGNIFICANCE: Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.
Assuntos
Neoplasias Colorretais , Memória Imunológica , Animais , Linfócitos T CD8-Positivos , Neoplasias Colorretais/metabolismo , Dopamina/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Receptores de Dopamina D5/metabolismoRESUMO
Even before the first synapses appear, neurotransmitters and their receptors are present in the developing brain, regulating the cell fate of neuronal progenitors in neurogenic niches, such as the lateral ventricle. In particular, dopamine appears to play a pivotal role in the neurogenesis of the subventricular zone by controlling the proliferation and differentiation of progenitors through activation of different receptors. Although dopamine receptor 5 (D5R) is expressed prenatally, there is little information regarding its role in either pre- or postnatal forebrain development. To examine the role of D5Rs in neurogenesis in the rat lateral ventricle subventricular zone (V-SVZ), we immunohistochemically defined D5R expression, as well as BrdU incorporation in progenitor cells of various post-weaning stages (Post-natal day (P) 20 until P80). We found that the level of proliferating cells is stable from postnatal day 20 until 50, and then declines sharply on P80. Concomitantly, D5R is expressed in all ages examined, but we detected a progressive decrease in the density of D5R+ cells from P40 until P80. Moreover, double immunostaining for BrdU and D5R revealed that proliferating cells in V-SVZ also express D5R. Collectively, our data suggest that D5R is expressed in the post-weaning V-SVZ of rat at least until P80, and its expression pattern coincides with that of proliferating cells in the V-SVZ, hinting at a possible role of D5Rs in the regulation of neuronal progenitor division/differentiation.
Assuntos
Ventrículos Laterais , Neurogênese , Receptores de Dopamina D5 , Animais , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Proliferação de Células , Dopamina , Ventrículos Laterais/metabolismo , Ratos , Receptores de Dopamina D5/metabolismo , DesmameRESUMO
Highly correlated firing of primary afferent inputs and lamina I projection neurons evokes synaptic long-term potentiation (LTP), a mechanism by which ascending nociceptive transmission can be amplified at the level of the spinal dorsal horn. However, the degree to which neuromodulatory signaling shapes the temporal window governing spike-timing-dependent plasticity (STDP) at sensory synapses onto projection neurons remains unclear. The present study demonstrates that activation of spinal D1/D5 dopamine receptors (D1/D5Rs) creates a highly permissive environment for the production of LTP in male and female adult mouse spinoparabrachial neurons by promoting non-Hebbian plasticity. Bath application of the mixed D1/D5R agonist SKF82958 unmasked LTP at STDP pairing intervals that normally fail to alter synaptic efficacy. Furthermore, during D1/D5R signaling, action potential discharge in projection neurons became dispensable for LTP generation, and primary afferent stimulation alone was sufficient to induce strengthening of sensory synapses. This non-Hebbian LTP was blocked by the D1/D5R antagonist SCH 39166 or genetic deletion of D5R, and required activation of mGluR5 and intracellular Ca2+ release but was independent of NMDAR activation. D1/D5R-enabled non-Hebbian plasticity was observed across multiple neuronal subpopulations in the superficial dorsal horn but was more prevalent in spinoparabrachial neurons than interneurons. Interestingly, the ability of neonatal tissue damage to promote non-Hebbian LTP in adult projection neurons was not observed in D5R knock-out mice. Collectively, these findings suggest that joint spinal D1/D5R and mGluR5 activation can allow unfettered potentiation of sensory synapses onto the output neurons responsible for conveying pain and itch information to the brain.SIGNIFICANCE STATEMENT Synaptic LTP in spinal projection neurons has been implicated in the generation of chronic pain. Under normal conditions, plasticity at sensory synapses onto adult mouse spinoparabrachial neurons follows strict Hebbian learning rules, requiring coincident presynaptic and postsynaptic firing. Here, we demonstrate that the activation of spinal D1/D5Rs promotes a switch from Hebbian to non-Hebbian LTP so that primary afferent stimulation alone is sufficient to evoke LTP in the absence of action potential discharge in projection neurons, which required joint activation of mGluR5 and intracellular Ca2+ release but not NMDARs. These results suggest that D1/D5Rs cooperate with mGluR5 receptors in the spinal dorsal horn to powerfully influence the amplification of ascending nociceptive transmission to the brain.
Assuntos
Potenciação de Longa Duração/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D5/agonistas , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Benzazepinas/farmacologia , Cálcio/metabolismo , Agonistas de Dopamina/farmacologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Sinapses/metabolismoRESUMO
BACKGROUND AND PURPOSE: Dysregulation of dopaminergic transmission combined with transient hypofunction of N-methyl-d-aspartate receptors (NMDARs) is a key mechanism that may underlie cognitive symptoms of schizophrenia. EXPERIMENTAL APPROACH: Therefore, we aimed to identify electrophysiologic alterations in animals neonatally treated with the NMDA receptor antagonist, MK-801, or with saline solution. KEY RESULTS: Patch-clamp whole-cell recordings from MK-801-treated animals revealed altered passive and active electrophysiologic properties compared with CA1 pyramidal cells from saline-treated animals, including up-regulation of the K+ inward-rectifier conductance and fast-inactivating and slow/non-inactivating K+ currents. Up-regulation of these membrane ionic currents reduced the overall excitability and altered the firing properties of CA1 pyramidal cells. We also explored the capability of cells treated with MK-801 to express intrinsic excitability potentiation, a non-synaptic form of hippocampal plasticity associated with cognition and memory formation. CA1 pyramidal cells from animals treated with MK-801 were unable to convey intrinsic excitability potentiation and had blunted synaptic potentiation. Furthermore, MK-801-treated animals also exhibited reduced cognitive performance in the Barnes maze task. Notably, activation of D1/D5 receptors with SKF-38,393 partially restored electrophysiologic alterations caused by neonatal treatment with MK-801. CONCLUSION AND IMPLICATIONS: Our results offer a molecular and mechanistic explanation based on dysregulation of glutamatergic transmission, in addition to dopaminergic transmission, that may contribute to the understanding of the cognitive deterioration associated with schizophrenia.
Assuntos
Maleato de Dizocilpina , Receptores de Dopamina D1 , Receptores de Dopamina D5 , Receptores de N-Metil-D-Aspartato , Animais , Maleato de Dizocilpina/farmacologia , Dopamina/farmacologia , Hipocampo/metabolismo , Neurônios/metabolismo , Células Piramidais/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão SinápticaRESUMO
Mental stress has been shown to activate sympathetic adrenergic system to produce dopamine and finally promote the progression of cancer. Dopamine can also regulate the immune system through secreting kinds of cytokines. However, what role does dopamine play in acute myeloid leukemia (AML) remains unclear. Here, we investigated the effects and mechanisms of dopamine in NLRP3 inflammasome activation and cellular viability of acute myeloid leukemia U937 cells. Our results showed that dopamine enhanced the viability of U937 cells and activated the NLRP3 inflammasome in U937 cells. To further explore the mechanism of dopamine on U937 cells, we examined the expression level of dopamine receptors (DRs). We found that the mRNA expression level of DR5 in U937 cells was significantly higher than other dopamine receptors. Furthermore, we treated U937 cells with DR1/2/3/5 antagonist before dopamine, and it manifestly reversed the NLRP3 inflammasome activation and the viability-enhancing effect in U937 cells induced by dopamine. Anti-IL-1ß antibody also could partly reversed the viability-enhancing effect by dopamine. We concluded that dopamine could enhance the viability of U937 cells through DR1/5 receptor pathway and activate NLRP3 inflammasome.
Assuntos
Dopamina/farmacologia , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-1beta/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Receptores de Dopamina D5/antagonistas & inibidores , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/metabolismoRESUMO
Consolidation and reconsolidation are independent memory processes. Consolidation stabilizes new memories, whereas reconsolidation restabilizes memories destabilized when reactivated during recall. However, the biological role of the destabilization/reconsolidation cycle is still unknown. It has been hypothesized that reconsolidation links new information with reactivated memories, but some reports suggest that new and old memories are associated through consolidation mechanisms instead. Object-recognition memory (ORM) serves to judge the familiarity of items and is essential for remembering previous events. We took advantage of the fact that ORM consolidation, destabilization, and reconsolidation can be pharmacologically dissociated to demonstrate that, depending on the activation state of hippocampal dopamine D1/D5 receptors, the memory of a novel object presented during recall of the memory of a familiar one can be formed via reconsolidation or consolidation, but only reconsolidation can link them. We also found that recognition memories formed through reconsolidation can be destabilized even if indirectly reactivated. Our results indicate that dopamine couples novelty detection with memory destabilization to determine whether a new recognition trace is associated with an active network and suggest that declarative reminders should be used with caution during reconsolidation-based psychotherapeutic interventions.
Assuntos
Dopamina/metabolismo , Hipocampo/metabolismo , Consolidação da Memória , Rememoração Mental , Animais , Masculino , Ratos , Ratos Wistar , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Reconhecimento PsicológicoRESUMO
The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR-D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.
Assuntos
Potenciação de Longa Duração/genética , Neurônios/metabolismo , Receptor Cross-Talk , Receptores de Dopamina D5/genética , Receptores de GABA-A/genética , Transmissão Sináptica/genética , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Peptídeos/síntese química , Peptídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D5/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/genética , Sinapses/metabolismoRESUMO
Acute stressors are recurrent in multiple species' lives and can facilitate or impair cognition. The use of zebrafish (Danio rerio) as a translational species to understand the mechanisms by which stress induces different behavioral phenotypes has been widely studied. Two acute stressors are recognized when using this species: (1) conspecific alarm substance (CAS); and (2) net chasing. Here, we tested if CAS or net chasing would affect working memory and cognitive flexibility by testing performance in the FMP Y-maze after exposure to stress. We observed that CAS altered zebrafish behavioral phenotypes by increasing repetitive behavior; meanwhile, animals showed different patterns of repetitive behavior when exposed to net chasing, depending on the chasing direction. Because D1 receptors were previously studied as a potential mechanism underlying stress responses in different species, here, we pretreated fish with a D1/D5 agonist (SKF-38393) to assess whether this system plays a role in repetitive behavior in the FMP Y-maze. The pretreatment with D1/D5 agonist significantly decreased repetitive behavior in CAS exposed animals, and cortisol levels for both stressed groups, suggesting that the dopaminergic system plays an important role in zebrafish stress-related responses.
Assuntos
Comportamento Animal/fisiologia , Cognição/fisiologia , Hidrocortisona/metabolismo , Memória de Curto Prazo/fisiologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Estresse Psicológico/metabolismo , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória de Curto Prazo/efeitos dos fármacos , Feromônios , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D5/agonistas , Comportamento Estereotipado/efeitos dos fármacos , Comportamento Estereotipado/fisiologia , Peixe-ZebraRESUMO
The decrease of neurotransmitter dopamine (DA) levels in the intestine is closely related to the development of inflammatory bowel disease (IBD). However, the functional relevance and underlying mechanistic basis of the effects of DA signaling on IBD remains unclear. Here, we observed that the DRD5 receptor is highly expressed in colonic macrophages, and the deficiency of DA-DRD5 signaling exacerbated experimental colitis. Moreover, DA-DRD5 signaling can inhibit M1 by negatively regulating NF-κB signaling but promote M2 macrophage polarization through activation of the CREB pathway, respectively. The deficiency of DRD5 signaling increased colonic M1 macrophages but reduced M2 cells during colitis. Additionally, the administration of a D1-like agonist that has a higher affinity to DRD5 can attenuate the colitogenic phenotype of mice. Collectively, these findings provide the first demonstration of DA-DRD5 signaling in colonic macrophages controlling the development of colitis by regulating M1/M2 macrophage polarization.
Assuntos
Colite/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Receptores de Dopamina D5/metabolismo , Animais , Masculino , CamundongosRESUMO
Recent findings from this laboratory demonstrate that ethanol reduces the intrinsic excitability of orbitofrontal cortex (OFC) neurons via activation of strychnine-sensitive glycine receptors. Although the mechanism linking ethanol to the release of glycine is currently unknown, astrocytes are a source of neurotransmitters including glycine and activation of dopamine D1-like receptors has been reported to enhance extracellular levels of glycine via a functional reversal of the astrocytic glycine transporter GlyT1. We recently reported that like ethanol, dopamine or a D1/D5 receptor agonist increases a tonic current in lateral OFC (lOFC) neurons. Therefore, in this study, we used whole-cell patch-clamp electrophysiology to examine whether ethanol inhibition of OFC spiking involves the release of glycine from astrocytes and whether this release is dopamine receptor dependent. Ethanol, applied acutely, decreased spiking of lOFC neurons and this effect was blocked by antagonists of GlyT1, the norepinephrine transporter or D1-like but not D2-like receptors. Ethanol enhanced the tonic current of OFC neurons and occluded the effect of dopamine suggesting that ethanol and dopamine may share a common pathway. Altering astrocyte function by suppressing intracellular astrocytic calcium signaling or blocking the astrocyte-specific Kir4.1 potassium channels reduced but did not completely abolish ethanol inhibition of OFC neuron firing. However, when both astrocytic calcium signaling and Kir4.1 channels were inhibited, ethanol had no effect on firing. Ethanol inhibition was also prevented by inhibitors of phospholipase C and conventional isoforms of protein kinase C (cPKC) previously shown to block D1R-induced GlyT1 reversal and PKC inhibition of Kir4.1 channels. Finally, the membrane potential of OFC astrocytes was depolarized by bath application of a Kir4.1 blocker, a D1 agonist or ethanol and ethanol effect was blocked by a D1 antagonist. Together, these findings suggest that acute ethanol inhibits OFC neuron excitability via a D1 receptor-mediated dysregulation of astrocytic glycine transport.
Assuntos
Astrócitos/metabolismo , Etanol/toxicidade , Glicina/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Dopaminérgicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Córtex Pré-Frontal/efeitos dos fármacos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/antagonistas & inibidoresRESUMO
The insular cortex (IC) has a crucial role in taste recognition memory, including conditioned taste aversion (CTA). CTA is a learning paradigm in which a novel taste stimulus (CS) is associated with gastric malaise (US), inducing aversion to the CS in future encounters. The role of the IC in CTA memory formation has been extensively studied. However, the functional significance of neurotransmitter release during the presentation of taste stimuli and gastric malaise-inducing agents remains unclear. Using microdialysis in free-moving animals, we evaluated simultaneous changes in glutamate, norepinephrine and dopamine release in response to the presentation of an innate appetitive or aversive gustatory novel stimulus, as well as after i.p. administration of isotonic or hypertonic gastric malaise-inducing solutions. Our results demonstrate that the presentation of novel stimuli, regardless of their innate valence, induces an elevation of norepinephrine and dopamine. Administration of a gastric malaise inducing agent (LiCl) promotes an elevation of glutamate regardless of its concentration. In comparison, norepinephrine release is related to the LiCl concentration and its equimolar NaCl control. Additionally, we evaluated their functional role on short and long-term taste aversion memory. Results indicate that the blockade of noradrenergic ß1,2 receptors in the IC spares CTA acquisition and memory consolidation. In contrast, blockade of dopamine D1/D5 receptors impaired CTA consolidation, whereas the NMDA receptor blockade impedes both acquisition and consolidation of CTA. These results suggest that dopaminergic and noradrenergic release are related to the salience of conditioned taste stimuli. However, only cortical D1/D5 dopaminergic activity, but not the noradrenergic ß1,2 activity, is involved in the acquisition and consolidation of taste memory formation. Additionally, glutamatergic activity signals visceral distress caused by LiCl administration and activates NMDA receptors necessary for the acquisition and consolidation of long-lasting taste aversion memory.
Assuntos
Aprendizagem da Esquiva/fisiologia , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Córtex Insular/metabolismo , Norepinefrina/metabolismo , Reconhecimento Psicológico/fisiologia , Animais , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Injeções Intraperitoneais , Interocepção/fisiologia , Cloreto de Lítio/efeitos adversos , Estimulação Física , Ratos , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , PaladarRESUMO
BACKGROUND AND AIMS: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4ß7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. METHODS: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. RESULTS: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. CONCLUSIONS: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/patologia , Inflamação/imunologia , Multimerização Proteica , Receptores CCR/metabolismo , Receptores de Dopamina D5/metabolismo , Sequência de Aminoácidos , Animais , Movimento Celular , Proliferação de Células , Colite/imunologia , Colite/patologia , Humanos , Inflamação/patologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Integrina beta1/metabolismo , Células Jurkat , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Modelos Biológicos , Peptídeos/química , Fosforilação , Receptores CCR/deficiência , Receptores de Dopamina D5/deficiência , Transdução de Sinais , TropismoRESUMO
Overproduction of reactive oxygen species (ROS) plays an important role in the pathogenesis of hypertension. The dopamine D5 receptor (D5R) is known to decrease ROS production, but the mechanism is not completely understood. In HEK293 cells overexpressing D5R, fenoldopam, an agonist of the two D1-like receptors, D1R and D5R, decreased the production of mitochondria-derived ROS (mito-ROS). The fenoldopam-mediated decrease in mito-ROS production was mimicked by Sp-cAMPS but blocked by Rp-cAMPS. In human renal proximal tubule cells with DRD1 gene silencing to eliminate the confounding effect of D1R, fenoldopam still decreased mito-ROS production. By contrast, Sch23390, a D1R and D5R antagonist, increased mito-ROS production in the absence of D1R, D5R is constitutively active. The fenoldopam-mediated inhibition of mito-ROS production may have been related to autophagy because fenoldopam increased the expression of the autophagy hallmark proteins, autophagy protein 5 (ATG5), and the microtubule-associated protein 1 light chain (LC)3-II. In the presence of chloroquine or spautin-1, inhibitors of autophagy, fenoldopam further increased ATG5 and LC3-II expression, indicating an important role of D5R in the positive regulation of autophagy. However, when autophagy was inhibited, fenoldopam was unable to inhibit ROS production. Indeed, the levels of these autophagy hallmark proteins were decreased in the kidney cortices of Drd5-/- mice. Moreover, ROS production was increased in mitochondria isolated from the kidney cortices of Drd5-/- mice, relative to Drd5+/+ littermates. In conclusion, D5R-mediated activation of autophagy plays a role in the D5R-mediated inhibition of mito-ROS production in the kidneys.
Assuntos
Mitocôndrias , Espécies Reativas de Oxigênio , Receptores de Dopamina D5 , Animais , Autofagia , AMP Cíclico/metabolismo , Fenoldopam , Células HEK293 , Humanos , Rim/metabolismo , Camundongos , Mitocôndrias/metabolismo , Receptores de Dopamina D5/metabolismoRESUMO
Tumor-associated macrophages facilitate tumor progression and resistance to therapy. Their capacity for metabolic and inflammatory reprogramming represents an attractive therapeutic target. ONC201/TIC10 is an anticancer molecule that antagonizes the dopamine receptor D2 and affects mitochondria integrity in tumor cells. We examined whether ONC201 induces a metabolic and pro-inflammatory switch in primary human monocyte-derived macrophages that reactivates their antitumor activities, thus enhancing the onco-toxicity of ONC201. Contrary to glioblastoma cells, macrophages exhibited a low ratio of dopamine receptors D2/D5 gene expression and were resistant to ONC201 cytotoxicity. Macrophages responded to ONC201 with a severe loss of mitochondria integrity, a switch to glycolytic ATP production, alterations in glutamate transport, and a shift towards a pro-inflammatory profile. Treatment of macrophages-glioblastoma cells co-cultures with ONC201 induced similar alterations in glutamatergic and inflammatory gene expression profiles of macrophages. It induced as well metabolic changes and a pro-inflammatory switch of the co-culture milieu. However, these changes did not translate into increased onco-toxicity. This study provides the first evidence that ONC201 affects macrophage immunometabolism and leads to a pro-inflammatory tumor environment. This speaks in favor of implementing ONC201 in combinatorial therapies and warrants further investigation of the mechanisms of action of ONC201 in macrophages and other immune cells.
Assuntos
Antineoplásicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Imidazóis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/imunologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Ácido Glutâmico/metabolismo , Humanos , Macrófagos/imunologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Background: Although previous studies suggest that dopamine receptor genes partially affect physical activity-related behaviours, all of these studies were cross-sectional studies that examined the effects of dopamine receptor genes on physical activity-related behaviours at some point in time. Therefore, the nature and extent of this relationship across the lifespan are even more uncertain.Aim: The purpose of this study is to examine the effects of dopamine receptor genes (i.e. DRD2, DRD4 and DRD5) on sport participation trajectories from adolescence to young adulthood.Subjects and methods: This study used the National Longitudinal Study of Adolescent Health data (wave 1-4). Group-based trajectory modelling was used to investigate the effect of dopamine receptor genes on the probability of being in each sport participation trajectory group.Results: A three-group model was the best fitting model for men whereas a two-group model was the best fitting model for women. The more participants possess the A1 allele of the DRD2, the less likely they are to be in the "high-decreasing group" rather than the "low-stable group" in both men and women. In male participants, the more participants carry the A1 allele of the DRD2, the more likely they are to be in the "high-stable group" rather than the "high-decreasing group" (coefficient = 0.206, p<.05).Conclusions: These results can contribute to the literature by providing important information on the effects of dopamine receptor genes on sport participation trajectories from adolescence through young adulthood.