RESUMO
The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gßγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gßγ induces the release of DA through DAT. Specifically, a Gßγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gßγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gßγ. A TAT peptide containing the Gßγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gßγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gßγ-dependent mechanism. Collectively, our data show that Gßγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Animais , Ligação Competitiva , Encéfalo/metabolismo , Células Cultivadas , Cricetulus , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/metabolismo , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Ratos Sprague-Dawley , Receptor Muscarínico M5/metabolismoRESUMO
The dopamine transporter (DAT) has been implicated in a variety of arousal-related processes including the regulation of motor activity, learning, motivated behavior, psychostimulant abuse, and, more recently, sleep/wake state. We previously demonstrated that DAT uptake regulates fluctuations in extracellular dopamine (DA) in the striatum across the light/dark cycle with DA levels at their highest during the dark phase and lowest during the light phase. Despite this evidence, whether fluctuations in DA uptake across the light/dark cycle are associated with changes in sleep/wake has not been tested. To address this, we employed a combination of sleep/wake recordings, fast scan cyclic voltammetry, and western blotting to examine whether sleep/wake state and/or light/dark phase impact DA terminal neurotransmission in male rats. Further, we assessed whether variations in plasma membrane DAT levels and/or phosphorylation of the threonine 53 site on the DAT accounts for fluctuations in DA neurotransmission. Given the extensive evidence indicating that psychostimulants increase DA through interactions with the DAT, we also examined to what degree the effects of cocaine at inhibiting the DAT vary across sleep/wake state. Results demonstrated a significant association between individual sleep/wake states and DA terminal neurotransmission, with higher DA uptake rate, increased phosphorylation of the DAT, and enhanced cocaine potency observed after periods of sleep. These findings suggest that sleep/wake state influences DA neurotransmission in a manner that is likely to impact a host of DA-dependent processes including a variety of neuropsychiatric disorders.
Assuntos
Cocaína , Proteínas da Membrana Plasmática de Transporte de Dopamina , Animais , Dopamina , Inibidores da Captação de Dopamina/farmacologia , Masculino , Ratos , SonoRESUMO
Abnormal levels of dopamine (DA) are thought to contribute to several neurological and psychiatric disorders including drug addiction. Extracellular DA levels are regulated primarily via reuptake by the DA transporter (DAT). Amphetamine, a potent psychostimulant, increases extracellular DA by inducing efflux through DAT. Recently, we discovered that G protein ßγ subunits (Gßγ) interact with DAT, and that in vitro activation of Gßγ promotes DAT-mediated efflux. Here, we investigated the role of Gßγ in the actions of amphetamine in DA neurons in culture, ex vivo nucleus accumbens (NAc), and freely moving rats. Activation of Gßγ with the peptide myr-Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (mSIRK) in the NAc potentiated amphetamine-induced hyperlocomotion, but not cocaine-induced hyperlocomotion, and systemic or intra-accumbal administration of the Gßγ inhibitor gallein attenuated amphetamine-induced, but not cocaine-induced hyperlocomotion. Infusion into the NAc of a TAT-fused peptide that targets the Gßγ-binding site on DAT (TAT-DATct1) also attenuated amphetamine-induced but not cocaine-induced hyperlocomotion. In DA neurons in culture, inhibition of Gßγ with gallein or blockade of the Gßγ-DAT interaction with the TAT-DATct1 peptide decreased amphetamine-induced DA efflux. Furthermore, activation of Gßγ with mSIRK potentiated and inhibition of Gßγ with gallein reduced amphetamine-induced increases of extracellular DA in the NAc in vitro and in freely moving rats. Finally, systemic or intra-accumbal inhibition of Gßγ with gallein blocked the development of amphetamine-induced, but not cocaine-induced place preference. Collectively, these results suggest that interaction between Gßγ and DAT plays a critical role in the actions of amphetamine and presents a novel target for modulating the actions of amphetamine in vivo.
Assuntos
Anfetamina/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Anfetamina/efeitos adversos , Animais , Estimulantes do Sistema Nervoso Central/efeitos adversos , Cocaína/administração & dosagem , Neurônios Dopaminérgicos/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
PDZ domain-containing proteins play an important role in the targeting and localization of synaptic membrane proteins. Here, we report an interaction between the PDZ domain-containing protein PICK1 and monoamine neurotransmitter transporters in vitro and in vivo. In dopaminergic neurons, PICK1 colocalizes with the dopamine transporter (DAT) and forms a stable protein complex. Coexpression of PICK1 with DAT in mammalian cells and neurons in culture results in colocalization of the two proteins in a cluster pattern and an enhancement of DAT uptake activity through an increase in the number of plasma membrane DAT. Deletion of the PDZ binding site at the carboxyl terminus of DAT abolishes its association with PICK1 and impairs the localization of the transporter in neurons. These findings indicate a role for PDZ-mediated protein interactions in the localization, expression, and function of monoamine transporters.
Assuntos
Proteínas de Transporte/metabolismo , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Simportadores , Membranas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Transformada/metabolismo , Células Cultivadas/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Feto , Imuno-Histoquímica , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Estrutura Terciária de Proteína/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Leveduras/metabolismoRESUMO
6-Hydroxydopamine (6-OHDA), a neurotoxic substrate of the dopamine transporter (DAT), is widely used in Parkinson's disease models. However, the molecular mechanisms underlying 6-OHDA's selectivity for dopamine neurons and the injurious sequelae that it triggers are not well understood. We tested whether ectopic expression of DAT induces sensitivity to 6-OHDA in non-dopaminergic rat cortical neurons and evaluated the contribution of voltage-dependent potassium channel (Kv)-dependent apoptosis to the toxicity of this compound in rat cortical and midbrain dopamine neurons. Cortical neurons expressing DAT accumulated dopamine and were highly vulnerable to 6-OHDA. Pharmacological inhibition of DAT completely blocked this toxicity. We also observed a p38-dependent Kv current surge in DAT-expressing cortical neurons exposed to 6-OHDA, and p38 antagonists and Kv channel blockers were neuroprotective in this model. Thus, DAT-mediated uptake of 6-OHDA recruited the oxidant-induced Kv channel dependent cell death pathway present in cortical neurons. Finally, we report that 6-OHDA also increased Kv currents in cultured midbrain dopamine neurons and this toxicity was blocked with Kv channel antagonists. We conclude that native DAT expression accounts for the dopamine neuron specific toxicity of 6-OHDA. Following uptake, 6-OHDA triggers the oxidant-associated Kv channel-dependent cell death pathway that is conserved in non-dopaminergic cortical neurons and midbrain dopamine neurons.
Assuntos
Adrenérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/fisiologia , Neurônios/efeitos dos fármacos , Oxidopamina/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Embrião de Mamíferos , Proteínas de Fluorescência Verde/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Tetraetilamônio/farmacologia , Transfecção/métodosRESUMO
Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA.
Assuntos
Ácido Araquidônico/metabolismo , Dopamina/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Núcleos Septais/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Endocanabinoides/metabolismo , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Glicerídeos/metabolismo , Homeostase , Incretinas/farmacologia , Camundongos , Microdiálise , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Núcleos Septais/efeitos dos fármacos , Peçonhas/farmacologiaRESUMO
Scanning cysteine mutagenesis was used to identify potential pore-forming residues in and around the first transmembrane domains of ionotropic P2X(2) receptor subunits. Twenty-eight unique cysteine-substituted mutants (R28C-Y55C) were individually expressed in HEK293 cells by lipofection. Twenty-three of these were functional as assayed by application of ATP to transfected voltage-clamped cells. Individual mutants varied in their sensitivity to ATP; otherwise, currents through functional mutant receptors resembled those of the homomeric wild-type (WT) receptor. In five (H33C, R34C, I50C, K53C, and S54C) of 23 functional mutants, coapplication of 30 microm ATP and 500 nm Ag(+) irreversibly inhibited inward current evoked by subsequent applications of ATP alone. These inhibitions did not result in a lateral shift in the agonist concentration-response curve and are unlikely to involve a modification of the agonist binding site. Two (K53C and S54C) of the five residues modified by Ag(+) applied in the presence of ATP when the channels were gating were also modified by 1 mm (2-aminoethyl)methanethiosulfonate applied in the absence of ATP when the channels were closed. These data suggest that domains near either end of the first transmembrane domain influence ion conduction through the pore of the P2X(2) receptor.
Assuntos
Trifosfato de Adenosina/metabolismo , Rim/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Substituição de Aminoácidos , Linhagem Celular , Cisteína/química , Cisteína/genética , Relação Dose-Resposta a Droga , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/química , Humanos , Ativação do Canal Iônico/fisiologia , Rim/citologia , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína/fisiologia , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Prata/farmacologia , Relação Estrutura-Atividade , TransfecçãoRESUMO
We investigated the transmembrane topology of the P2X2 receptor subunit expressed in HEK 293 cells. Initial studies using two P2X subunits expressed in tandem indicated that the amino- and carboxy-termini are on the same side of the membrane. Immunofluorescence studies showed the cytoplasmic orientation of the amino- and carboxy-termini. Finally, N-glycosylation scanning mutagenesis revealed that reporter sites inserted into the central loop, but not those in the amino- or carboxy-terminal regions, were glycosylated, thus suggesting an extracellular placement for that domain. Our results support a two-transmembrane arrangement for P2X receptors with intracellular amino- and carboxy-termini.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Ativação do Canal Iônico/efeitos dos fármacos , Proteínas de Membrana/química , Receptores Purinérgicos P2/química , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , DNA Complementar , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Conformação Proteica , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2X2RESUMO
P2X receptors are integral membrane proteins that belong to the growing family of transmitter-gated ion channels. The extracellular domain of these receptors contains several consensus sequences for N-linked glycosylation that may contribute to the functional expression of the channel. We have previously reported the extracellular orientation of asparagine residues 182, 239, and 298 of the P2X2 receptor subunit by showing that the protein is glycosylated at each site [Torres, G. E., et al. (1998) FEBS Lett. 425, 19-23 (1)]. In this study, we focused on the consequences of removing N-linked glycosylation from the P2X2 receptor by using two different approaches, tunicamycin treatment or site-directed mutagenesis. HEK-293 cells stably transfected with the P2X2 receptor subunit showed little or no response to ATP after tunicamycin treatment. In addition, loss of function was observed with the elimination of all three N-linked glycosylation sites from P2X2. Cell surface labeling with biotin or indirect immunofluorescence revealed that the expression of the nonglycosylated receptors produced by either tunicamycin or site-directed mutagenesis is greatly reduced at the cell surface, indicating that the nonglycosylated P2X2 receptors are retained inside the cell. These data provide the first direct evidence for a critical role of N-linked glycosylation in the cell surface expression of a P2X receptor subunit.
Assuntos
Asparagina/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Animais , Asparagina/genética , Biotinilação , Linhagem Celular , Sequência Consenso/genética , Eletrofisiologia , Técnica Direta de Fluorescência para Anticorpo , Glicosilação/efeitos dos fármacos , Humanos , Microscopia Confocal , Mutagênese Sítio-Dirigida , Oligopeptídeos , Peptídeos/genética , Testes de Precipitina , Ratos , Receptores Purinérgicos P2/fisiologia , Relação Estrutura-Atividade , Transfecção , Tunicamicina/farmacologiaRESUMO
In the CA1 region of the hippocampus, activation of serotonin receptors of the 5-hydroxytryptamine (5-HT)4 subtype increases membrane excitability by reducing the calcium-activated potassium current responsible for the slow afterhyperpolarization observed in these cells. In the present study, the signaling mechanism by which 5-HT4 receptors reduce the afterhyperpolarization in the CA1 region was examined using intracellular recording in brain slices. Administration of the membrane-permeable cAMP analog 8-bromo-cAMP mimicked the effect of serotonin on the afterhyperpolarization, whereas administration of the protein kinase inhibitor staurosporine inhibited the effects of serotonin. These observations suggested a role for protein kinase A in this response. This was confirmed by intracellular injection of the selective protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate ((Rp)-cAMPS), which noncompetitively inhibited the ability of serotonin to reduce the after-hyperpolarization. Additional evidence for the involvement of cAMP in the signaling by 5-HT4 receptors was obtained using the general phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. When this compound was bath administered at concentrations sufficient to enhance a known cAMP-mediated response, a significant enhancement of the ability of 5-HT4 receptors to reduce the afterhyperpolarization was observed. Together, these results indicate that serotonin reduces the afterhyperpolarization in the CA1 region by acting on 5-HT4 receptors that increase intracellular cAMP levels and activate protein kinase A.
Assuntos
Cálcio/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , Receptores de Serotonina/fisiologia , Alcaloides/farmacologia , Animais , AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Eletrofisiologia , Masculino , Ratos , Transdução de Sinais/fisiologia , EstaurosporinaRESUMO
The study of serotonin-4 (5-HT4) receptors in the central nervous system has been hindered by the lack of effective, selective antagonists. However, recently, several novel compounds have been synthesized and shown to act as antagonists at 5-HT4 receptors in smooth muscle and embryonic neurons in culture. In the present study, intracellular electrophysiological recordings were used to test the effects of three of these compounds: endo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl-2,3-dihydro-6-methoxy- 2-oxo-1H-benzimidazole-1-carboxylate (DAU 6285), [1-[2-(methylsulfonylamino)ethyl]-4-piperidinyl]methyl 1-methyl-1H-indole-3-carboxylate (GR 113808) and 2-diethylaminoethyl-(2-methoxy-4-amino-5-chloro) benzoate (SDZ 205-557) on the 5-HT4 reduction of the afterhyperpolarization seen in adult CA1 hippocampal neurons in brain slices. GR 113808, SDZ 205-557 and DAU 6285 all functioned as competitive antagonists at these 5-HT4 receptors. Although all three compounds tested acted as effective antagonists, they differed considerably in potency. When the potency of these antagonists at the 5-HT4 receptor that mediates the reduction of the afterhyperpolarization was compared with that observed for 5-HT4 receptors in biochemical and binding assays, an excellent correlation was observed. Among the antagonists tested, GR 113808 was the most potent (pA2 = GR 113808 > SDZ 205-507 > DAU 6285). It exhibited an apparent affinity for the 5-HT4 receptors in the low nanomolar range but did not antagonize 5-HT1A, beta-adrenergic or muscarinic receptor-mediated responses when applied at concentrations two orders of magnitude higher.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Hipocampo/efeitos dos fármacos , Receptores de Serotonina/fisiologia , Antagonistas da Serotonina/farmacologia , Ácido 4-Aminobenzoico/farmacologia , Animais , Benzimidazóis/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Hipocampo/fisiologia , Técnicas In Vitro , Indóis/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Ratos , Sulfonamidas/farmacologia , para-AminobenzoatosRESUMO
P2X receptors are ATP-gated ion channels found in a variety of tissues and cell types. Seven different subunits (P2X(1)-P2X(7)) have been molecularly cloned and are known to form homomeric, and in some cases heteromeric, channel complexes. However, the molecular determinants leading to the assembly of subunits into P2X receptors are unknown. To address this question we utilized a co-immunoprecipitation assay in which epitope-tagged deletion mutants and chimeric constructs were examined for their ability to co-associate with full-length P2X subunits. Deletion mutants of the P2X(2) receptor subunit were expressed individually and together with P2X(2) or P2X(3) receptor subunits in HEK 293 cells. Deletion of the amino terminus up to the first transmembrane domain (amino acid 28) and beyond (to amino acid 51) did not prevent subunit assembly. Analysis of the carboxyl terminus demonstrated that mutants missing the portion of the protein downstream of the second transmembrane domain could also still co-assemble. However, a mutant terminating 25 amino acids before the second transmembrane domain could not assemble with other subunits or itself, implicating the missing region of the protein in assembly. This finding was supported and extended by data utilizing a chimera strategy that indicated TMD2 is a critical determinant of P2X subunit assembly.
Assuntos
Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Ativação do Canal Iônico , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Proteínas Recombinantes de Fusão/metabolismo , Deleção de SequênciaRESUMO
Steroids and neuropeptides interact in the central nervous system (CNS) to regulate reproductive function and behavior. The preoptic regulatory factors, PORF-1 and PORF-2, are unique neuropeptides for which roles in gender-related brain development and function have been proposed. PORF-1 and PORF-2 expression in rat brain are age, region and gender dependent, and castration or hypophysectomy alter the metabolism of the PORF-1 and PORF-2 mRNAs in male rat brain and testes. If these two peptides have a role in gender-dependent brain function, then gonadal steroids might well affect their expression. The present study was designed to investigate the response of the PORF-1 and PORF-2 mRNAs to sex steroids in the female rat brain and to compare this response to that of two peptides whose roles in the neuroendocrinology of reproduction are well established, gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY). Rats were ovariectomized and treated with placebo, estradiol (E2), progesterone (P4) or a combination of the two (E2/P4) and NPY, PORF-2, GnRH and PORF-1 mRNAs were quantified by nuclease protection assays. PORF-1, PORF-2 and GnRH mRNAs were also measured in intact rats during estrus and proestrus. Responses were compared in the preoptic anterior hypothalamus (POA), medial basal hypothalamus (MBH), cerebral cortex (CC) and hippocampus (HIPP). Expression of PORF-1 and PORF-2 was also confirmed in the female rat hypothalamus by in situ hybridization analysis. PORF-1 and PORF-2 mRNAs were detected in the adult female rat brain by both in situ hybridization and ribonuclease protection analyses. In situ hybridization analysis demonstrated that PORF-1 and PORF-2 mRNAs are expressed in hypothalamic neurons. RNase protection analysis showed that PORF-1, PORF-2 and NPY mRNAs were present in all four brain regions examined while GnRH expression was detected only in the MBH and POA. Estradiol alone upregulated expression of the PORF-1 and PORF-2 mRNAs in the ovariectomized rat in the POA and HIPP, and of NPY mRNA in the MBH and HIPP. Progesterone alone had a stimulatory effect on NPY mRNA in the MBH and HIPP. Treatment with a combination of E2/P4 downregulated PORF-2 mRNA in the POA as well as PORF-1, PORF-2 and NPY mRNAs in the CC. In contrast, E2/P4 upregulated the PORF-2 and NPY mRNAs in the HIPP and NPY mRNA in the MBH. In the cycling rat, PORF-1 mRNA levels were higher during proestrus than estrus in both the MBH and POA, while PORF-2 mRNA levels did not change. In contrast GnRH mRNA was lower in the POA and higher in the MBH during proestrus compared with estrus. Thus, intrinsic factors, most likely both ovarian and neuroendocrine, regulate PORF-1 and GnRH expression in the intact cycling rat CNS in a region-dependent manner. In the ovariectomized rat, PORF-1, PORF-2, NPY and GnRH mRNAs all respond in a region-specific manner to sex steroid treatment. These data support the role of PORF-1 and PORF-2 in gender-dependent brain function in the adult female rat.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Estradiol/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Progesterona/farmacologia , Animais , Química Encefálica , Implantes de Medicamento , Estradiol/sangue , Estro/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/genética , Hipotálamo/química , Hibridização In Situ , Iodeto Peroxidase , Neuropeptídeo Y/genética , Ovariectomia , Progesterona/sangue , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Iodotironina Desiodinase Tipo IIRESUMO
P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X1 through P2X7) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homo-oligomeric complexes, the exception being P2X6. When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X7. Whereas P2X1, P2X2, P2X5, and P2X6 were able to assemble with most subunits, P2X3 and P2X4 presented a more restricted pattern of co-association. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP.
Assuntos
Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P2/química , Transporte Biológico , Dimerização , Humanos , Receptores Purinérgicos P2/metabolismo , Relação Estrutura-AtividadeRESUMO
Serotonin acting on 5-hydroxytryptamine4 receptors increases membrane excitability in CA1 hippocampal pyramidal cells by reducing the slow calcium-activated afterhyperpolarization. This effect is mediated through an increase in cAMP and activation of protein kinase A, although subsequent steps have not been elucidated. We now report that a significant portion of the calcium responsible for the generation of the afterhyperpolarization originates from the release of intracellular calcium through a calcium-induced calcium-release mechanism. Thus, the afterhyperpolarization is enhanced by caffeine, whereas it is inhibited by dantrolene and ruthenium red, two blockers of calcium-induced calcium release. The afterhyperpolarization is also inhibited by thapsigargin, which depletes intracellular calcium stores. These observations raised the possibility that serotonin might reduce the afterhyperpolarization by regulating calcium-induced calcium release. Consistent with this possibility, administration of calcium-induced calcium-release blockers, as well as of thapsigargin, occluded the ability of serotonin to inhibit the afterhyperpolarization. Similarly, administration of caffeine, which enhances the contribution of calcium-induced calcium release to the afterhyperpolarization, enhanced the effect of serotonin. These results indicate that serotonin inhibits the afterhyperpolarization in the CA1 region of hippocampus by reducing the ability of extracellular calcium to trigger calcium release from intracellular stores. As such, they identify a physiological role for the calcium-induced calcium release in hippocampus and provide evidence for its regulation by G protein-coupled receptors and, more specifically, 5-hydroxytryptamine4 receptors.
Assuntos
Cálcio/fisiologia , Hipocampo/fisiologia , Receptores de Serotonina/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Cafeína/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Dantroleno/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores 5-HT4 de Serotonina , Rutênio Vermelho/farmacologia , Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Tapsigargina/farmacologiaRESUMO
We recently reported that a novel hetero-oligomeric P2X receptor is formed from the P2X(1) and P2X(5) isoforms when coexpressed in human embryonic kidney 293 cells (). A more complete description of the pharmacology of this novel receptor is presented here. A brief application of ATP to a voltage-clamped cell transiently expressing P2X(1/5) receptors resulted in a biphasic current that rapidly reached a peak and then decayed to a sustained plateau. Washout of ATP was accompanied by generation and fade of a pronounced tail of inward current. EC(50) values were determined from concentration-response curves for a range of agonists. The rank order of agonist potency was ATP >/= 2 methylthio ATP > adenosine 5'-O-(3-thiotriphosphate) > alpha,beta-methylene ATP > ADP > CTP. alpha,beta-methylene ADP, UTP, GTP, and AMP were ineffective. Only ATP and 2 methylthio ATP were full agonists. IC(50) values were determined from concentration-response curves for three commonly used purinergic antagonists. Suramin and pyridoxal phosphate-6-azophenyl-2', 4'-disulfonic acid were equipotent at P2X(1) and P2X(1/5) receptors; however, the P2X(1/5) receptor was much less sensitive to TNP-ATP than was the P2X(1) receptor. The amplitude of peak ATP-gated current was relatively insensitive to changes in [Ca(2+)](O) (1-30 mM). Finally, plateau currents were potentiated by low concentrations of pyridoxal phosphate-6-azophenyl-2', 4'-disulfonic acid and by raising [Ca(2+)](O). These results provide additional information on the pharmacological profile of the recombinant P2X(1/5) receptor channel and provide a basis to further evaluate ATP-induced currents in native tissues.
Assuntos
Isoformas de Proteínas/fisiologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Células COS , Cálcio/farmacologia , Células Cultivadas , Cricetinae , Cricetulus , Eletrofisiologia , Humanos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inibidores , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X5RESUMO
P2X receptors are a family of ion channels gated by extracellular ATP. Each member of the family can form functional homomeric channels, but only P2X2 and P2X3 have been shown to combine to form a unique heteromeric channel. Data from in situ hybridization studies suggest that P2X1 and P2X5 may also co-assemble. In this study, we tested this hypothesis by expressing recombinant P2X1 and P2X5 receptor subunits either individually or together in human embryonic kidney 293 cells. In cells expressing the homomeric P2X1 receptor, 30 microM alpha,beta-methylene ATP (alpha,beta-me-ATP) evoked robust currents that completely desensitized in less than 1 sec, whereas alpha,beta-me-ATP failed to evoke current in cells expressing the homomeric P2X5 receptor. By contrast, alpha, beta-me-ATP evoked biphasic currents with a pronounced nondesensitizing plateau phase in cells that co-expressed both subunits. Further, the EC50 for alpha,beta-me-ATP was greater in cells expressing both P2X1 and P2X5 than in cells expressing P2X1 alone (5 and 1.6 microM, respectively). Heteromeric assembly was confirmed using a co-immunoprecipitation assay of epitope-tagged P2X1 and P2X5 subunits. In summary, this study provides biochemical and functional evidence of a novel channel formed by P2X subunit heteropolymerization. This finding suggests that heteromeric subunit assembly constitutes an important mechanism for generating functional diversity of ATP-mediated responses.