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
Loss-of-function mutations in melanocortin-4 receptor (MC4R) are the most common cause of monogenic obesity, a severe type of early-onset obesity. Our aim was to determine the prevalence of MC4R mutations in a cohort of 97 Argentinian children with early-onset obesity. We found two novel mutations (p.V52E and p.G233S) and estimated a prevalence of 2.1%. We investigated the pathogenicity of mutations in HEK293T cells expressing wild-type or mutant MC4R and found that both mutants exhibited reduced plasma membrane expression and altered agonist-induced cAMP responses, with no changes in basal activity. Besides, MC4R G233S mutant demonstrated an altered agonist-dependent inhibition of voltage-gated calcium channels type 2.2. Results using a Gαs protein inhibitor suggest that the G233S mutation could be recruiting a different G-protein signaling pathway. The identification of new mutations in MC4R and characterization of their functional impact provide tools for the diagnosis and treatment of monogenic obesity.
Assuntos
Obesidade Infantil , Receptor Tipo 4 de Melanocortina , Criança , Humanos , Estudos de Coortes , Células HEK293 , Mutação , Receptor Tipo 4 de Melanocortina/genética , Obesidade Infantil/genética , ArgentinaRESUMO
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
The growth hormone secretagogue receptor (GHSR) signals in response to ghrelin, but also acts via ligand-independent mechanisms that include either constitutive activation or interaction with other G protein-coupled receptors, such as the dopamine 2 receptor (D2R). A key target of GHSR in neurons is voltage-gated calcium channels type 2.2 (CaV2.2). Recently, the liver-expressed antimicrobial peptide 2 (LEAP2) was recognized as a novel GHSR ligand, but the mechanism of action of LEAP2 on GHSR is not well understood. Here, we investigated the role of LEAP2 on the canonical and non-canonical modes of action of GHSR on CaV2.2 function. Using a heterologous expression system and patch-clamp recordings, we found that LEAP2 impairs the reduction of CaV2.2 currents induced by ghrelin-evoked and constitutive GHSR activities, acting as a GHSR antagonist and inverse agonist, respectively. We also found that LEAP2 prevents GHSR from modulating the effects of D2R signaling on CaV2.2 currents, and that the GHSR-binding N-terminal region LEAP2 underlies these effects. Using purified labeled receptors assembled into lipid nanodiscs and Forster Resonance Energy Transfer (FRET) assessments, we found that the N-terminal region of LEAP2 stabilizes an inactive conformation of GHSR that is dissociated from Gq protein and, consequently, reverses the effect of GHSR on D2R-dependent Gi activation. Thus, our results provide critical molecular insights into the mechanism mediating LEAP2 modulation of GHSR.
RESUMO
The growth hormone secretagogue receptor (GHSR) has emerged as one of the most fascinating molecules from the perspective of neuroendocrine control. GHSR is mainly expressed in the pituitary and the brain, and plays key roles regulating not only growth hormone secretion but also food intake, adiposity, body weight, glucose homeostasis and other complex functions. Quite atypically, GHSR signaling displays a basal constitutive activity that can be up- or downregulated by two digestive system-derived hormones: the octanoylated-peptide ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2), which was recently recognized as an endogenous GHSR ligand. The existence of two ligands with contrary actions indicates that GHSR activity can be tightly regulated and that the receptor displays the capability to integrate such opposing inputs in order to provide a balanced intracellular signal. This article provides a summary of the current understanding of the biology of ghrelin, LEAP2 and GHSR and discusses the reconceptualization of the cellular and physiological implications of the ligand-regulated GHSR signaling, based on the latest findings.
Assuntos
Receptores de Grelina/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
The growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor (GPCR) highly expressed in the brain, and also in some peripheral tissues. GHSR activity is evoked by the stomach-derived peptide hormone ghrelin and abrogated by the intestine-derived liver-expressed antimicrobial peptide 2 (LEAP2). In vitro, GHSR displays ligand-independent actions, including a high constitutive activity and an allosteric modulation of other GPCRs. Beyond its neuroendocrine and metabolic effects, cumulative evidence shows that GHSR regulates the activity of the mesocorticolimbic pathway and modulates complex reward-related behaviors towards different stimuli. Here, we review current evidence indicating that ligand-dependent and ligand-independent actions of GHSR enhance reward-related behaviors towards appetitive stimuli and drugs of abuse. We discuss putative neuronal networks and molecular mechanisms that GHSR would engage to modulate such reward-related behaviors. Finally, we briefly discuss imaging studies showing that ghrelin would also regulate reward processing in humans. Overall, we conclude that GHSR is a key regulator of the mesocorticolimbic pathway that influences its activity and, consequently, modulates reward-related behaviors via ligand-dependent and ligand-independent actions.
Assuntos
Grelina , Receptores de Grelina , Humanos , Ligantes , Recompensa , Transdução de SinaisRESUMO
OBJECTIVE: The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor that plays major roles in the central control of energy balance. Loss-of-function mutations of MC4R constitute the most common monogenic cause of early-onset extreme obesity in humans, whereas gain-of-function mutations appear to be protective. In particular, two relatively frequent alleles carrying the non-synonymous coding mutations V103I or I251L are associated with lower risks of obesity and type-2 diabetes. Although V103I and I251L MC4Rs showed more efficient signalling in transfected cells, their specific effects in live animals remain unexplored. Here, we investigated whether the introduction of V103I and I251L mutations into the mouse MC4R leads to a lean phenotype and provides protection against an obesogenic diet. METHODS: Using CRISPR/Cas9, we generated two novel strains of mice carrying single-nucleotide mutations into the mouse Mc4r which are identical to those present in V103I and I251L MCR4 human alleles, and studied their phenotypic outcomes in mice fed with normal chow or a high-fat diet. In particular, we measured body weight progression, food intake and adiposity. In addition, we analysed glucose homeostasis through glucose and insulin tolerance tests. RESULTS: We found that homozygous V103I females displayed shorter longitudinal length and decreased abdominal white fat, whereas homozygous I251L females were also shorter and leaner due to decreased weight in all white fat pads examined. Homozygous Mc4rV103I/V103I and Mc4rI251L/I251L mice of both sexes showed improved glucose homeostasis when challenged in a glucose tolerance test, whereas Mc4rI251L/I251L females showed improved responses to insulin. Despite being leaner and metabolically more efficient, V103I and I251L mutants fed with a hypercaloric diet increased their fasting glucose levels and adiposity similar to their wild-type littermates. CONCLUSIONS: Our results demonstrate that mice carrying V103I and I251L MC4R mutations displayed gain-of-function phenotypes that were more evident in females. However, hypermorphic MC4R mutants were as susceptible as their control littermates to the obesogenic and diabetogenic effects elicited by a long-term hypercaloric diet, highlighting the importance of healthy feeding habits even under favourable genetic conditions.
Assuntos
Adiposidade/genética , Receptor Tipo 4 de Melanocortina/metabolismo , Aumento de Peso/genética , Adiposidade/fisiologia , Animais , Dieta Hiperlipídica , Metabolismo Energético , Feminino , Mutação com Ganho de Função/genética , Glucose/metabolismo , Homeostase/genética , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/genética , Receptor Tipo 4 de Melanocortina/genéticaRESUMO
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
Alterations in dopamine receptor type 1 (D1R) density are associated with cognitive deficits of aging and schizophrenia. In the prefrontal cortex (PFC), D1R plays a critical role in the regulation of working memory, which is impaired in these cognitive deficit states, but the cellular events triggered by changes in D1R expression remain unknown. A previous report demonstrated that interaction between voltage-gated calcium channel type 2.2 (CaV2.2) and D1R stimulates CaV2.2 postsynaptic surface location in medial PFC pyramidal neurons. Here, we show that in addition to the occurrence of the physical receptor-channel interaction, constitutive D1R activity mediates up-regulation of functional CaV2.2 surface density. We performed patch-clamp experiments on transfected HEK293T cells and wild-type C57BL/6 mouse brain slices, as well as imaging experiments and cAMP measurements. We found that D1R coexpression led to â¼60% increase in CaV2.2 currents in HEK293T cells. This effect was occluded by preincubation with a D1/D5R inverse agonist, chlorpromazine, and by replacing D1R with a D1R mutant lacking constitutive activity. Moreover, D1R-induced increase in CaV2.2 currents required basally active Gs protein, as well as D1R-CaV2.2 interaction. In mice, intraperitoneal administration of chlorpromazine reduced native CaV currents' sensitivity to ω-conotoxin-GVIA and their size by â¼49% in layer V/VI pyramidal neurons from medial PFC, indicating a selective effect on CaV2.2. Additionally, we found that reducing D1/D5R constitutive activity correlates with a decrease in the agonist-induced D1/D5R inhibitory effect on native CaV currents. Our results could be interpreted as a stimulatory effect of D1R constitutive activity on the number of CaV2.2 channels available for dopamine-mediated modulation. Our results contribute to the understanding of the physiological role of D1R constitutive activity and may explain the noncanonical postsynaptic distribution of functional CaV2.2 in PFC neurons.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Córtex Pré-Frontal/metabolismo , Células Piramidais/metabolismo , Receptores Dopaminérgicos/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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 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
Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Encéfalo/metabolismo , Corantes Fluorescentes/química , Grelina/metabolismo , Rim/metabolismo , Animais , Células Cultivadas , Ingestão de Alimentos , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Transdução de SinaisRESUMO
KEY POINTS: Presynaptic CaV 2 voltage-gated calcium channels link action potentials arriving at the presynaptic terminal to neurotransmitter release. Hence, their regulation is essential to fine tune brain circuitry. CaV 2 channels are highly sensitive to G protein-coupled receptor (GPCR) modulation. Our previous data indicated that growth hormone secretagogue receptor (GHSR) constitutive activity impairs CaV 2 channels by decreasing their surface density. We present compelling support for the impact of CaV 2.2 channel inhibition by agonist-independent GHSR activity exclusively on GABA release in hippocampal cultures. We found that this selectivity arises from a high reliance of GABA release on CaV 2.2 rather than on CaV 2.1 channels. Our data provide new information on the effects of the ghrelin-GHSR system on synaptic transmission, suggesting a putative physiological role of the constitutive signalling of a GPCR that is expressed at high levels in brain areas with restricted access to its natural agonist. ABSTRACT: Growth hormone secretagogue receptor (GHSR) displays high constitutive activity, independent of its endogenous ligand, ghrelin. Unlike ghrelin-induced GHSR activity, the physiological role of GHSR constitutive activity and the mechanisms that underlie GHSR neuronal modulation remain elusive. We previously demonstrated that GHSR constitutive activity modulates presynaptic CaV 2 voltage-gated calcium channels. Here we postulate that GHSR constitutive activity-mediated modulation of CaV 2 channels could be relevant in the hippocampus since this brain area has high GHSR expression but restricted access to ghrelin. We performed whole-cell patch-clamp in hippocampal primary cultures from E16- to E18-day-old C57BL6 wild-type and GHSR-deficient mice after manipulating GHSR expression with lentiviral transduction. We found that GHSR constitutive activity impairs CaV 2.1 and CaV 2.2 native calcium currents and that CaV 2.2 basal impairment leads to a decrease in GABA but not glutamate release. We postulated that this selective effect is related to a higher CaV 2.2 over CaV 2.1 contribution to GABA release (â¼40% for CaV 2.2 in wild-type vs. â¼20% in wild-type GHSR-overexpressing cultures). This effect of GHSR constitutive activity is conserved in hippocampal brain slices, where GHSR constitutive activity reduces local GABAergic transmission of the granule cell layer (intra-granule cell inhibitory postsynaptic current (IPSC) size â¼-67 pA in wild-type vs. â¼-100 pA in GHSR-deficient mice), whereas the glutamatergic output from the dentate gyrus to CA3 remains unchanged. In summary, we found that GHSR constitutive activity impairs IPSCs both in hippocampal primary cultures and in brain slices through a CaV 2-dependent mechanism without affecting glutamatergic transmission.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Hipocampo/citologia , Neurônios/fisiologia , Receptores de Grelina/metabolismo , Animais , Bário/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Voltage-gated Ca2+ (CaV) channels couple membrane depolarization to Ca2+ influx, triggering a range of Ca2+-dependent cellular processes. CaV channels are, therefore, crucial in shaping neuronal activity and function, depending on their individual temporal and spatial properties. Furthermore, many neurotransmitters and drugs that act through G protein coupled receptors (GPCRs), modulate neuronal activity by altering the expression, trafficking, or function of CaV channels. GPCR-dependent mechanisms that downregulate CaV channel expression levels are observed in many neurons but are, by comparison, less studied. Here we show that the growth hormone secretagogue receptor type 1a (GHSR), a GPCR, can inhibit the forwarding trafficking of several CaV subtypes, even in the absence of agonist. This constitutive form of GPCR inhibition of CaV channels depends on the presence of a CaVß subunit. CaVß subunits displace CaVα1 subunits from the endoplasmic reticulum. The actions of GHSR on CaV channels trafficking suggest a role for this signaling pathway in brain areas that control food intake, reward, and learning and memory.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio/metabolismo , Receptores de Grelina/metabolismo , Sinalização do Cálcio , Linhagem Celular , HumanosRESUMO
Hypothalamic tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle. Given their strategic location, tanycytes are believed to play several key functions including being a selective barrier and controlling the amount of hypothalamic-derived factors reaching the anterior pituitary. The in vitro culture of these cells has proved to be difficult. Here, we report an improved method for the generation of primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day-old rats and cultured in a chemically defined medium containing DMEM:F12, serum albumin, insulin, transferrin and the antibiotic gentamycin. After 7 days in vitro, â¼30% of the cultured cells exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. Tanycyte-like cells were strongly immuno-reactive for vimentin and dopamine-cAMP-regulated phospho-protein (DARPP-32) and weakly immune-reactive for glial fibrillary acidic protein. Tanycyte-like cells displayed a stable negative resting plasma membrane potential and failed to show spiking properties in response to current injections. When exposed to fluorescent beads in the culture medium, tanycyte-like cells exhibited a robust endocytosis. Thus, the present method effectively yields cultures containing tanycyte-like cells that resemble in vivo tanycytes in terms of morphologic features and molecular markers as well as electrical and endocytic activity. To our knowledge, this is the first protocol that allows the culturing of tanycyte-like cells that can be individually identified and that conserve the morphology of tanycytes in their natural physiological environment.
Assuntos
Técnicas de Cultura de Células/métodos , Forma Celular , Células Ependimogliais/citologia , Hipotálamo/citologia , Animais , Células Cultivadas , Fenômenos Eletrofisiológicos , Endocitose , Imuno-Histoquímica , Ratos Sprague-DawleyRESUMO
Ghrelin is an octanoylated peptide hormone that plays a key role in the regulation of the body weight and glucose homeostasis. In plasma, ghrelin circulates bound to larger proteins whose identities are partially established. Here, we used size exclusion chromatography, mass spectrometry and isothermal titration microcalorimetry to show that ghrelin interacts with serum albumin. Furthermore, we found that such interaction displays an estimated dissociation constant (KD) in the micromolar range and involves albumin fatty-acid binding sites as well as the octanoyl moiety of ghrelin. Notably, albumin-ghrelin interaction reduces the spontaneous deacylation of the hormone. Both in vitro experiments-assessing ghrelin ability to inhibit calcium channels-and in vivo studies-evaluating ghrelin orexigenic effects-indicate that the binding to albumin affects the bioactivity of the hormone. In conclusion, our results suggest that ghrelin binds to serum albumin and that this interaction impacts on the biological activity of the hormone.
Assuntos
Grelina/metabolismo , Albumina Sérica/metabolismo , Sequência de Aminoácidos , Animais , Calorimetria , Cromatografia em Gel , Grelina/química , Humanos , Camundongos , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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
The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.
Assuntos
Tonsila do Cerebelo/fisiologia , Canais de Cálcio Tipo N/metabolismo , Terminações Pré-Sinápticas/fisiologia , Receptor Tipo 4 de Melanocortina/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Fármacos do Sistema Nervoso Central/farmacologia , Toxina da Cólera/farmacologia , Células HEK293 , Humanos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Peptídeos/farmacologia , Peptídeos Cíclicos/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor Tipo 4 de Melanocortina/agonistas , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , ômega-Conotoxina GVIA/farmacologiaRESUMO
Growth hormone secretagogue receptor (GHSR) 1a is the only molecularly identified receptor for ghrelin, mediating ghrelin-related effects on eating, body weight, and blood glucose control, among others. The expression pattern of GHSR within the brain has been assessed previously by several neuroanatomical techniques. However, inherent limitations to these techniques and the lack of reliable anti-GHSR antibodies and reporter rodent models that identify GHSR-containing neurons have prevented a more comprehensive functional characterization of ghrelin-responsive neurons. Here we have systematically characterized the brain expression of an enhanced green fluorescence protein (eGFP) transgene controlled by the Ghsr promoter in a recently reported GHSR reporter mouse. Expression of eGFP in coronal brain sections was compared with GHSR mRNA expression detected in the same sections by in situ hybridization histochemistry. eGFP immunoreactivity was detected in several areas, including the prefrontal cortex, insular cortex, olfactory bulb, amygdala, and hippocampus, which showed no or low GHSR mRNA expression. In contrast, eGFP expression was low in several midbrain regions and in several hypothalamic nuclei, particularly the arcuate nucleus, where robust GHSR mRNA expression has been well-characterized. eGFP expression in several brainstem nuclei showed high to moderate degrees of colocalization with GHSR mRNA labeling. Further quantitative PCR and electrophysiological analyses of eGFP-labeled hippocampal cells confirmed faithful expression of eGFP within GHSR-containing, ghrelin-responsive neurons. In summary, the GHSR-eGFP reporter mouse model may be a useful tool for studying GHSR function, particularly within the brainstem and hippocampus; however, it underrepresents GHSR expression in nuclei within the hypothalamus and midbrain.