RESUMO
High fat diets have been used as complementary treatments for seizure disorders for more than a century. Moreover, many fatty acids and derivatives, including the broad-spectrum antiseizure medication valproic acid, have been explored and used as pharmacological agents to treat epilepsy. In this work, we have explored the anticonvulsant potential of a large library of fatty acids and fatty acid derivatives, the LIPID MAPS Structure Database, using structure-based virtual screening to assess their ability to block the voltage-gated sodium channel 1.2 (NaV1.2), a validated target for antiseizure medications. Four of the resulting in silico hits were submitted for experimental confirmation using in vitro patch clamp experiments, and their protective role was evaluated in an acute mice seizure model, the Maximal Electroshock seizure model. These four compounds were found to protect mice against seizures. Two of them exhibited blocking effects on NaV1.2, CaV2.2, and CaV3.1.
RESUMO
The dopamine receptor type 1 (D1R) and the dopamine receptor type 5 (D5R), which are often grouped as D1R-like due to their sequence and signaling similarities, exhibit high levels of constitutive activity. The molecular basis for this agonist-independent activation has been well characterized through biochemical and mutagenesis in vitro studies. In this regard, it was reported that many antipsychotic drugs act as inverse agonists of D1R-like constitutive activity. On the other hand, D1R is highly expressed in the medial prefrontal cortex (mPFC), a brain area with important functions such as working memory. Here, we studied the impact of D1R-like constitutive activity and chlorpromazine (CPZ), an antipsychotic drug and D1R-like inverse agonist, on various neuronal CaV conductances, and we explored its effect on calcium-dependent neuronal functions in the mouse medial mPFC. Using ex vivo brain slices containing the mPFC and transfected HEK293T cells, we found that CPZ reduces CaV2.2 currents by occluding D1R-like constitutive activity, in agreement with a mechanism previously reported by our lab, whereas CPZ directly inhibits CaV1 currents in a D1R-like activity independent manner. In contrast, CPZ and D1R constitutive activity did not affect CaV2.1, CaV2.3, or CaV3 currents. Finally, we found that CPZ reduces excitatory postsynaptic responses in mPFC neurons. Our results contribute to understanding CPZ molecular targets in neurons and describe a novel physiological consequence of CPZ non-canonical action as a D1R-like inverse agonist in the mouse brain.
Assuntos
Clorpromazina , Receptores Dopaminérgicos , Camundongos , Humanos , Animais , Clorpromazina/farmacologia , Agonismo Inverso de Drogas , Células HEK293 , Neurônios/metabolismo , Canais de Cálcio , Córtex Pré-Frontal/metabolismo , Cálcio/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
AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system (RAS) recently identified as the membrane receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we aim to study whether two receptors from RAS, the angiotensin receptor type 1 (AT1R) and the bradykinin 2 receptor (B2R) modulate ACE2 internalization induced by a recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. Also, we investigated the impact of ACE2 coexpression on AT1R and B2R functionality. MATERIALS AND METHODS: To study ACE2 internalization, we assessed the distribution of green fluorescent protein (GFP) signal in HEK293T cells coexpressing GFP-tagged ACE2 and AT1R, or B2R, or AT1R plus B2R in presence of RBD alone or in combination with AT1R or B2R ligands. To estimate ACE2 internalization, we classified GFP signal distribution as plasma membrane uniform GFP (PMU-GFP), plasma membrane clustered GFP (PMC-GFP) or internalized GFP and calculated its relative frequency. Additionally, we investigated the effect of ACE2 coexpression on AT1R and B2R inhibitory action on voltage-gated calcium channels (CaV2.2) currents by patch-clamp technique. KEY FINDINGS: RBD induced ACE2-GFP internalization in a time-dependent manner. RBD-induced ACE2-GFP internalization was increased by angiotensin II and reduced by telmisartan in cells coexpressing AT1R. RBD-induced ACE2-GFP internalization was strongly inhibited by B2R co-expression. This effect was mildly modified by bradykinin and rescued by angiotensin II in presence of AT1R. ACE2 coexpression impacted on B2R- and AT1R-mediated inhibition of CaV2.2 currents. SIGNIFICANCE: Our work contributes to understand the role of RAS modulators in the susceptibility to SARS-CoV-2 infection and severity of COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2/biossíntese , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor B2 da Bradicinina/biossíntese , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Enzima de Conversão de Angiotensina 2/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Células HEK293 , Humanos , Receptor Tipo 1 de Angiotensina/análise , Receptor B2 da Bradicinina/análise , Proteínas Recombinantes/administração & dosagemRESUMO
OBJECTIVE: Binding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. METHODS: We generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. RESULTS: In COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first â¼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. CONCLUSIONS: Our data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.
Assuntos
Alelos , Substituição de Aminoácidos , Metabolismo Energético/genética , Mutação , Receptores de Grelina/genética , Animais , Pesos e Medidas Corporais , Sinalização do Cálcio , Linhagem Celular , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Marcação de Genes , Estudos de Associação Genética , Células HEK293 , Hormônios/metabolismo , Humanos , Hipotálamo/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Técnicas de Patch-Clamp , Receptores de Grelina/metabolismoRESUMO
The mechanisms by which ghrelin controls electrical activity in the hypothalamus are not fully understood. One unexplored target of ghrelin is CaV3, responsible for transient calcium currents (T-currents) that control neuronal firing. We investigated the effect of ghrelin on CaV3 subtypes and how this modulation impacts on neuronal activity. We performed whole-cell patch-clamp recordings in primary mouse hypothalamic cultures to explore the effect of ghrelin on T-currents. We also recorded calcium currents from transiently transfected tsA201 cells to study the sensitivity of each CaV3 subtype to GHSR activation. Finally, we ran a computational model combining the well-known reduction of potassium current by ghrelin with the CaV3 biophysical parameter modifications induced by ghrelin to predict the impact on neuronal electrical behavior. We found that ghrelin inhibits native NiCl2 sensitive current currents in hypothalamic neurons. We determined that CaV3.3 is the only CaV3 subtype sensitive to ghrelin. The modulation of CaV3.3 by ghrelin comprises a reduction in maximum conductance, a shift to hyperpolarized voltages of the I-V and steady-state inactivation curves, and an acceleration of activation and inactivation kinetics. Our model-based prediction indicates that the inhibition of CaV3.3 would attenuate the stimulation of firing originating from the inhibition of potassium currents by ghrelin. In summary, we discovered a new target of ghrelin in neurons: the CaV3.3. This mechanism would imply a negative feed-forward regulation of the neuronal activation exerted by ghrelin. Our work expands the knowledge of the wide range of actions of GHSR, a receptor potentially targeted by therapeutics for several diseases.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Grelina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Hipotálamo/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Voltage-gated calcium channels type 2.2 (CaV2.2) are activated by action potentials at presynaptic terminals, and their calcium current induces neurotransmitter release. In this context, regulating CaV2.2 is critical, and one of the most important mechanisms for doing so is through its G protein-coupled receptor (GPCR) activity. Two such GPCRs are the ghrelin (GHSR) and the dopamine type 2 (D2R) receptors. We previously demonstrated that constitutive GHSR activity reduces CaV2.2 forward trafficking and that ghrelin-induced GHSR activity inhibits CaV2.2 currents. On the other hand, dopamine-induced D2R activity also inhibits CaV2.2 currents. It has been recently shown that D2R and GHSR form heteromers in hypothalamic neurons. This interaction profoundly changes the signaling cascades activated by dopamine and is necessary for dopamine-dependent anorexia. Here we explored how D2R-GHSR coexpression in HEK293T cells modulates the effect that each GPCR has on CaV2.2. We found that D2R-GHSR coexpression reduces the inhibition of CaV2.2 currents by agonist-induced D2R activation and added a new source of basal CaV2.2 current inhibition to the one produced by GHSR solely expression. We investigated the signaling cascades implicated and found that constitutive GHSR activity, Gq protein, and Gßγ subunit play a critical role in these altered effects. Moreover, we found that the effect of D2R agonist on native calcium currents in hypothalamic neurons is reduced when both D2R and GHSR are overexpressed. In summary, our results allow us to propose a novel mechanism for controlling CaV2.2 currents involving the coexpression of two physiologically relevant GPCRs.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Grelina/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G protein-coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the voltage-gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and agonist-dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Grelina/metabolismo , Hipotálamo/fisiologia , Neurônios/fisiologia , Receptores de Grelina/metabolismo , Animais , Sequência de Bases , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Camundongos , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Receptores de Grelina/genéticaRESUMO
Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood. Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons. However, the recent study from the Ikeda Laboratory revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.