RESUMO
Spine motility analysis has become the mainstay for investigating synaptic plasticity but is limited in its versatility requiring complex, non automatized instrumentations. We describe an entropy-based method for determining the spatial distribution of dendritic spines that allows successful estimation of spine motility from still images. This method has the potential to extend the applicability of spine motility analysis to ex vivo preparations.
Assuntos
Espinhas Dendríticas , Modelos Biológicos , Plasticidade Neuronal , Neurônios/citologia , Neurônios/fisiologia , Algoritmos , Animais , Dendritos , Camundongos , Camundongos KnockoutRESUMO
Current therapeutic strategies for Huntington's disease (HD) are focused on symptom management of disease progression. Transcriptional dysregulation is one of the major characteristics in HD. REST is a transcriptional repressor that silences gene expression through binding to RE1/NRSE sites found in the regulatory regions of numerous neuronal genes. Dysregulation of REST and its targeted genes has been reported in different cell and mouse HD models, as well as in biopsies from human patients. In this work, we characterized transcriptional dysregulation associated with REST in two different HD mouse models and assessed the therapeutic effect of interfering with REST function by overexpressing a dominant-negative form (DN:REST). We show that delivery of DN:REST in the motor cortex restores brain-derived neurotrophic factor (BDNF) mRNA and protein levels by reducing endogenous REST occupancy at the Bdnf locus. Similarly, expression of other REST-regulated genes such as Synapsin I (Syn1), Proenkephalin (Penk1) and Cholinergic receptor muscarinic 4 (Chrm4) were restored to normal levels while non-REST-regulated genes were unaffected. This is the first study conducted to investigate REST's role in vivo in a neurodegenerative disease. Our data show that DN:REST in motor cortex reversed RESTs repressive effects on target genes. However, the lack of therapeutic effect on motor function suggests that a more widespread rescue of REST-regulated sites in the affected brain regions may be necessary.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Terapia Genética , Doença de Huntington/genética , Proteínas Repressoras/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Técnicas de Transferência de Genes , Humanos , Doença de Huntington/patologia , Doença de Huntington/terapia , Camundongos , Córtex Motor/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteínas Repressoras/uso terapêuticoRESUMO
Repressor element-1 silencing transcription factor (REST) is a candidate modulator of gene expression during status epilepticus in the rodent. In such models, full-length REST and the truncated REST4 variant are induced and can potentially direct differential gene expression patterns. We have addressed the regulation of these REST variants in rodent hippocampal seizure models and correlated this with expression of the proconvulsant, substance P encoding, PPT-A gene. REST and REST4 were differentially regulated following kainic acid stimulus both in in vitro and in vivo models. REST4 was more tightly regulated than REST in both models and its transient expression correlated with that of the differential regulation of PPT-A. Consistent with this, overexpression of a truncated REST protein (HZ4, lacking the C-terminal repression domain) increased expression of the endogenous PPT-A gene. Similarly the proximal PPT-A promoter reporter gene construct was differentially regulated by the distinct REST isoforms in hippocampal cells with HZ4 being the major inducer of increased reporter expression. Furthermore, REST and REST4 proteins were differentially expressed and compartmentalized within rat hippocampal cells in vitro following noxious stimuli. This differential localization of the REST isoforms was confirmed in the CA1 region following perforant path and kainic acid induction of status epilepticus in vivo. We propose that the interplay between REST and REST4 alter the expression of proconvulsant genes, as exemplified by the PPT-A gene, and may therefore regulate the progression of epileptogenesis.
Assuntos
Epilepsia/genética , Regulação da Expressão Gênica/fisiologia , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Agonistas de Aminoácidos Excitatórios , Imunofluorescência , Genes Reporter/genética , Hipocampo/citologia , Hipocampo/fisiologia , Ácido Caínico , Masculino , Microscopia Confocal , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Técnicas de Cultura de Órgãos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/genética , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/genéticaRESUMO
Antisense approaches are increasingly used to dissect signaling pathways linking cell surface receptors to intracellular effectors. Here we used a recombinant adenovirus to deliver G-protein alpha(q) antisense into rat superior cervical ganglion (SCG) neurons and neuronal cell lines to dissect G(alpha)(q)-mediated signaling pathways in these cells. This approach was compared with other G(alpha)(q) gene knockdown strategies, namely, antisense plasmid and knockout mice. Infection with adenovirus expressing G(alpha)(q) antisense (G(alpha)(q)AS AdV) selectively decreased immunoreactivity for the G(alpha)(q) protein. Expression of other G(alpha) protein subunits, such as G(alpha)(oA/B,) was unaltered. Consistent with this, modulation of Ca(2+) currents by the G(alpha)(q)-coupled M(1) muscarinic receptor was severely impaired in neurons infected with G(alpha)(q)AS AdV whereas modulation via the G(alpha)(oA)-coupled M(4) muscarinic receptor was unchanged. In agreement, activation of phospholipase C and consequent mobilization of intracellular Ca(2+) by UTP receptors was lost in NG108-15 cells infected with G(alpha)(q)AS AdV but not in cells infected with the control GFP-expressing adenovirus. Results obtained with this recombinant AdV strategy qualitatively and quantitatively replicated results obtained using SCG neurons microinjected with G(alpha)(q) antisense plasmids or SCG neurons from G(alpha)(q) knockout mice. This combined antisense/recombinant adenoviral approach can therefore be useful for dissecting signal transduction mechanisms in SCG and other neurons.
Assuntos
Sinalização do Cálcio/fisiologia , Neurônios/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Gânglio Cervical Superior/fisiologia , Adenoviridae/genética , Animais , Sequência de Bases , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Vetores Genéticos , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Ratos , Ratos Mutantes , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologiaRESUMO
The ionotropic ATP receptor subunits P2X(1-6) receptors play important roles in synaptic transmission, yet the P2X(7) receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X(7) receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X(7) receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X(7) receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP; 30 microm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X(7) receptor antagonists oxidized ATP (100 microm) and Brilliant Blue G (2 microm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X(7) receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1-43 in isolated NMJ preparations destained after application of BzATP (30 microm). This BzATP evoked destaining is blocked by oxidized ATP (100 microm) and Brilliant Blue G (1 microm). This indicates that activation of the P2X(7) receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.
Assuntos
Sistema Nervoso Central/metabolismo , Neurônios/metabolismo , Sistema Nervoso Periférico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Sistema Nervoso Central/química , Sistema Nervoso Central/citologia , Ácido Glutâmico/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Bulbo/química , Bulbo/citologia , Bulbo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/inervação , Junção Neuromuscular/metabolismo , Neurônios/citologia , Neurotransmissores/metabolismo , Gânglio Nodoso/química , Gânglio Nodoso/citologia , Gânglio Nodoso/metabolismo , Técnicas de Patch-Clamp , Sistema Nervoso Periférico/química , Sistema Nervoso Periférico/citologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/química , Medula Espinal/citologia , Medula Espinal/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
PURPOSE: To examine the acute urinary toxicity following transperineal prostate implant using a modified Quimby loading method with regard to time course, severity, and factors that may be associated with a higher incidence of morbidity. METHODS AND MATERIALS: One hundred thirty-nine patients with prostate adenocarcinoma treated with brachytherapy from 1997 through 1999 had follow-up records available for review. Patients considered for definitive brachytherapy alone included those with prostate specific antigen (PSA) < or = 6, Gleason score (GS) < or = 6, clinical stage < T2b, and prostate volumes generally less than 40 cc. Patients with larger prostate volumes were given neoadjuvant antiandrogen therapy. Those with GS > 6, PSA > 6, or Stage > T2a were treated with external beam radiation therapy followed by brachytherapy boost. Sources were loaded according to a modified Quimby method. At each follow-up, toxicity was graded based on a modified RTOG urinary toxicity scale. RESULTS: Acute urinary toxicity occurred in 88%. Grade I toxicity was reported in 23%, grade II in 45%, and grade III in 20%, with 14% requiring prolonged (greater than 1 week) intermittent or indwelling catheterization. Overall median duration of symptoms was 12 months. There was no difference in duration of symptoms between patients treated with I-125 or Pd-103 sources (p = 0.71). After adjusting for GS and PSA, multivariate logistic regression analysis showed higher incidence of grade 3 toxicity in patients with larger prostate volumes (p = 0.002), and those with more seeds implanted (p < 0.001). Higher incidence of prolonged catheterization was found in patients receiving brachytherapy alone (p = 0.01), with larger prostate volumes (p = 0.01), and those with more seeds implanted (p < 0.001). CONCLUSION: Interstitial brachytherapy for prostate cancer leads to a high incidence of acute urinary toxicity, most of which is mild to moderate in severity. A prolonged need for catheterization can occur in some patients. Patients receiving brachytherapy alone, those with prostate volumes greater than 30 cc, and those implanted with a greater number of seeds have the highest incidence of significant toxicity.
Assuntos
Adenocarcinoma/radioterapia , Braquiterapia/efeitos adversos , Neoplasias da Próstata/radioterapia , Transtornos Urinários/etiologia , Doença Aguda , Adenocarcinoma/sangue , Adulto , Idoso , Análise de Variância , Braquiterapia/métodos , Seguimentos , Humanos , Radioisótopos do Iodo/uso terapêutico , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Paládio/uso terapêutico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Radioisótopos/uso terapêuticoRESUMO
Many aspects of neurogenesis and neuronal differentiation are controlled by basic helix-loop-helix (bHLH) proteins. One such factor is SHARP-1, initially identified on the basis of its sequence similarity to hairy. Unlike hairy, and atypically for bHLHs, SHARP-1 is expressed late in development, suggestive of a role in terminal aspects of differentiation. Nevertheless, the role of SHARP-1 and the identity of its target genes remain unknown. During the course of a one-hybrid screen for transcription factors that bind to regulatory domains of the M1 muscarinic acetylcholine receptor gene, we isolated the bHLH transcription factor SHARP-1. In this study, we investigated the functional role of SHARP-1 in regulating transcription. Fusion proteins of SHARP-1 tethered to the gal4 DNA binding domain repress both basal and activated transcription when recruited to either a TATA-containing or a TATAless promoter. Furthermore, we identified two independent repression domains that operate via distinct mechanisms. Repression by a domain in the C terminus is sensitive to the histone deacetylase inhibitor trichostatin A, whereas repression by the bHLH domain is insensitive to TSA. Furthermore, overexpression of SHARP-1 represses transcription from the M(1) promoter. This study represents the first report to assign a function to, and to identify a target gene for, the bHLH transcription factor SHARP-1.
Assuntos
Sequências Hélice-Alça-Hélice , Histona Desacetilases/metabolismo , Neuropeptídeos/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular , Primers do DNA , Dados de Sequência Molecular , Neuropeptídeos/química , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/químicaRESUMO
Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (I(K(M))), which can be inhibited by activation of M(1) muscarinic receptors (M(1) mAChR) and bradykinin (BK) B(2) receptors. Inhibition by the M(1) mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein Galpha(q) (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves Galpha(q) and/or Galpha(11) (Jones et al., 1995). Galpha(q) and Galpha(11) can stimulate phospholipase C-beta (PLC-beta), raising the possibility that PLC is involved in I(K(M)) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-beta1, -beta2, -beta3, and -beta4 in rat SCG. We have tested the role of two PLC isoforms (PLC-beta1 and PLC-beta4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-beta1 and PLC-beta4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-beta isoform when compared to uninjected cells 48 hr later. BK inhibition of I(K(M)) was significantly reduced 48 hr after injection of the PLC-beta4, but not the PLC-beta1, antisense-encoding plasmid. Neither PLC-beta antisense altered M(1) mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M(1) mAChR, inhibition of I(K(M)) involves PLC and extends this finding by indicating that PLC-beta4 is involved.
Assuntos
Bradicinina/metabolismo , Isoenzimas/metabolismo , Antagonistas Muscarínicos/farmacologia , Neurônios/enzimologia , Bloqueadores dos Canais de Potássio , Fosfolipases Tipo C/metabolismo , Animais , Bradicinina/farmacologia , Células Cultivadas , Isoenzimas/genética , Microinjeções , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Fosfolipase C beta , Plasmídeos , Canais de Potássio/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/efeitos dos fármacos , Gânglio Cervical Superior/enzimologia , Fosfolipases Tipo C/genéticaRESUMO
Voltage-gated Ca2+ channels participate in dendritic integration, yet functional properties of Ca2+ channels and mechanisms of their modulation by neurotransmitters in dendrites are unknown. Here we report how pharmacologically identified Ca2+ channels behave in different neural compartments. Whole-cell and cell-attached patch-clamp recordings were made on both cell bodies and electrically isolated dendrites of sympathetic neurons. We found not only that Ca2+ channel populations differentially contribute to somatic and dendritic currents but also that families of Ca2+ channels display gating properties and neurotransmitter modulation that depend on channel compartmentalization. By comparison with their somatic counterparts, dendritic N-type Ca2+ currents were hypersensitive to neurotransmitters and G proteins. Single-channel analysis showed that dendrites express a unique N-type channel that has enhanced interaction with Gbetagamma. Thus Ca2+ channels in dendrites seem to be specialized elements with unique regulatory mechanisms.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Neurônios/fisiologia , Neurotransmissores/fisiologia , Animais , Células Cultivadas , Dendritos/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Ativação do Canal Iônico/fisiologia , Cinética , Potenciais da Membrana , Neuritos/fisiologia , Técnicas de Patch-Clamp , Ratos , Gânglio Cervical Superior/fisiologiaRESUMO
Activation of M(1) muscarinic acetylcholine receptors (M(1) mAChR) inhibits M-type potassium currents (I(K(M))) and N-type calcium currents (I(Ca)) in mammalian sympathetic ganglia. Previous antisense experiments suggested that, in rat superior cervical ganglion (SCG) neurons, both effects were partly mediated by the G-protein Galpha(q) (Delmas et al., 1998a; Haley et al., 1998a), but did not eliminate a contribution by other pertussis toxin (PTX)-insensitive G-proteins. We have tested this further using mice deficient in the Galpha(q) gene. PTX-insensitive M(1) mAChR inhibition of I(Ca) was strongly reduced in Galpha(q) -/- mouse SCG neurons and was fully restored by acute overexpression of Galpha(q). In contrast, M(1) mAChR inhibition of I(K(M)) persisted in Galpha(q)-/- mouse SCG cells. However, unlike rat SCG neurons, muscarinic inhibition of I(K(M)) was partly PTX-sensitive. Residual (PTX-insensitive) I(K(M)) inhibition was slightly reduced in Galpha(q) -/- neurons, and the remaining response was then suppressed by anti-Galpha(q/11) antibodies. Bradykinin (BK) also inhibits I(K(M)) in rat SCG neurons via a PTX-insensitive G-protein (G(q) and/or G(11); Jones et al., 1995). In mouse SCG neurons, I(K(M)) inhibition by BK was fully PTX-resistant. It was unchanged in Galpha(q) -/- mice but was abolished by anti-Galpha(q/11) antibody. We conclude that, in mouse SCG neurons (1) M(1) mAChR inhibition of I(Ca) is mediated principally by G(q), (2) M(1) mAChR inhibition of I(K(M)) is mediated partly by G(q), more substantially by G(11), and partly by a PTX-sensitive G-protein(s), and (3) BK-induced inhibition of I(K(M)) is mediated wholly by G(11).
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/fisiologia , Proteínas de Ligação ao GTP/deficiência , Agonistas Muscarínicos/farmacologia , Bloqueadores dos Canais de Potássio , Animais , Bradicinina/farmacologia , Canais de Cálcio Tipo N/efeitos dos fármacos , Células Cultivadas , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas de Ligação ao GTP/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microinjeções , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Toxina Pertussis , Canais de Potássio/genética , Receptor B2 da Bradicinina , Receptor Muscarínico M1 , Receptores da Bradicinina/efeitos dos fármacos , Receptores da Bradicinina/genética , Receptores Muscarínicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
A large number of neuron-specific genes characterized to date are under the control of negative transcriptional regulation. Many promoter regions of neuron-specific genes possess the repressor element repressor element 1/neuron-restrictive silencing element (RE1/NRSE). Its cognate binding protein, REST/NRSF, is an essential transcription factor; its null mutations result in embryonic lethality, and its dominant negative mutants produce aberrant expression of neuron-specific genes. REST/NRSF acts as a regulator of neuron-specific gene expression in both nonneuronal tissue and developing neurons. Here, we shown that heterologous expression of REST/NRSF in Saccharomyces cerevisiae is able to repress transcription from yeast promoters engineered to contain RE1/NRSEs. Moreover, we have taken advantage of this observation to show that this repression requires both yeast Sin3p and Rpd3p and that REST/NRSF physically interacts with the product of the yeast SIN3 gene in vivo. Furthermore, we show that REST/NRSF binds mammalian SIN3A and HDAC-2 and requires histone deacetylase activity to repress neuronal gene transcription in both nonneuronal and neuronal cell lines. We show that REST/NRSF binding to RE1/NRSE is accompanied by a decrease in the acetylation of histones around RE1/NRSE and that this decrease requires the N-terminal Sin3p binding domain of REST/NRSF. Taken together, these data suggest that REST/NRSF represses neuronal gene transcription by recruiting the SIN3/HDAC complex.
Assuntos
Histona Desacetilases/genética , Neurônios/fisiologia , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transcrição Gênica , Linhagem Celular , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoAssuntos
Núcleo Celular/efeitos dos fármacos , Proteínas de Ligação ao GTP/biossíntese , Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Plasmídeos/administração & dosagem , RNA Antissenso/administração & dosagem , Células Cultivadas , Microinjeções , Neurônios/citologia , Gânglio Cervical Superior/citologiaRESUMO
The potassium M current was originally identified in sympathetic ganglion cells, and analogous currents have been reported in some central neurons and also in some neural cell lines. It has recently been suggested that the M channel in sympathetic neurons comprises a heteromultimer of KCNQ2 and KCNQ3 (Wang et al., 1998) but it is unclear whether all other M-like currents are generated by these channels. Here we report that the M-like current previously described in NG108-15 mouse neuroblastoma x rat glioma cells has two components, "fast" and "slow", that may be differentiated kinetically and pharmacologically. We provide evidence from PCR analysis and expression studies to indicate that these two components are mediated by two distinct molecular species of K(+) channel: the fast component resembles that in sympathetic ganglia and is probably carried by KCNQ2/3 channels, whereas the slow component appears to be carried by merg1a channels. Thus, the channels generating M-like currents in different cells may be heterogeneous in molecular composition.
Assuntos
Neurônios/fisiologia , Canais de Potássio/fisiologia , Gânglio Cervical Superior/fisiologia , Animais , Benzimidazóis/farmacologia , Células CHO , Diferenciação Celular , Cricetinae , Glioma , Humanos , Células Híbridas , Indóis/farmacologia , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma , Neurônios/citologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Piridinas/farmacologia , Ratos , Receptor Muscarínico M1 , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Sulfanilamidas/farmacologia , TransfecçãoRESUMO
1. Using perforated-patch recordings, we have examined the part played by endogenous G-protein subunits in the alpha2-adrenoceptor-mediated inhibition of N-type Ca2+ currents in sympathetic neurones. 2. Two components of ICa inhibition by noradrenaline were recorded: a prominent, high affinity and voltage-dependent pertussis toxin (PTX)-sensitive pathway and a minor, low affinity and mostly voltage-insensitive PTX-resistant pathway. 3. PTX-sensitive inhibition was reduced by microinjection of antibodies against either GalphaoA,B or Galphai1,2. The voltage-dependent fraction of inhibition was reduced by anti-Galphao but not by anti-Galphai antibody. 4. Antisense depletion of GalphaoA led to a marked reduction of noradrenaline-induced inhibition and voltage dependence. By contrast, Galphai depletion attenuated noradrenergic modulation without affecting the voltage dependence. 5. Expression of the betagamma-binding agents beta-adrenergic receptor kinase 1 (C-terminus, betaARK1C-ter) or Galphai1 with a Cys3 to Ser mutation partially prevented noradrenergic inhibition while alpha-transducin abolished it. Residual inhibition was mostly voltage independent in cells expressing betaARK1C-ter but was strongly reversed by depolarization in Galphai1 Cys3Ser-expressing cells. 6. Expression of the PTX-resistant Galphai1 Cys351Ile mutant in cells treated with PTX restored alpha2-adrenoceptor inhibition. This restored inhibition was weakly reversed by depolarization. Both the degree and voltage dependence of inhibition were correlated with the level of expression of the Galphai1 Cys351Ile subunit. 7. Our findings identify betagamma dimers associated with GalphaoA and Galphai as mediators of the PTX-sensitive alpha2-adrenoceptor-mediated inhibition of N-type Ca2+ channels. Different betagamma combinations may account for the differential voltage-dependent effects of Go and Gi on ICa.
Assuntos
Canais de Cálcio/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Neurônios/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Células Cultivadas , Estimulação Elétrica , Eletrofisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Imuno-Histoquímica , Potenciais da Membrana/fisiologia , Norepinefrina/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Técnicas de Patch-Clamp , Toxina Pertussis , Plasmídeos/genética , Ratos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Sistema Nervoso Simpático/citologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Muscarinic acetylcholine receptor genes are members of the G-protein coupled receptor superfamily. Each member of this family studied to date appears to have a distinct expression profile, however the mechanisms determining these expression patterns remain largely unknown. We have previously isolated a genomic clone containing the M1 muscarinic receptor gene and determined its gene structure [Pepitoni, Wood and Buckley (1997) J. Biol. Chem. 272, 17112-17117]. We have now identified DNA elements responsible for driving cell specific expression in transient transfection assays of immortalized cell lines. A region of the gene spanning 974 nucleotides and containing 602 nucleotides of the first exon is sufficient to drive specific expression in cell lines. Like the M4 and M2 gene promoters, the M1 promoter contains an Sp1 motif which can recruit transcription factor Sp1 and at least one other protein, although this site does not appear to be functionally important for M1 expression in our assay. We have identified a region within the first exon of the M1 gene that regulates expression in cell lines, contains several positive and negative acting elements and is able to drive expression of a heterologous promoter. A polypyrimidine/polypurine tract and a sequence conserved between M1 genes of various species act in concert to enhance M1 transcription and are able to activate a heterologous promoter. We show that DNA binding proteins interact in vitro with single-stranded DNA derived from these regions and suggest that topology of the DNA is important for regulation of M1 expression.
Assuntos
Éxons , Regulação da Expressão Gênica , Neurônios/metabolismo , Receptores Muscarínicos/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , DNA Complementar , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pirimidinas/metabolismo , Ratos , Receptor Muscarínico M1 , Receptores Muscarínicos/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The specific cellular response to muscarinic receptor activation is dependent upon appropriate expression of each of the five muscarinic receptor genes by individual cells. Here we summarise recent work describing some of the genomic regulatory elements and transcriptional mechanisms that control expression of the M1 and M4 genes.
Assuntos
Regulação da Expressão Gênica , Receptores Muscarínicos/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas/genética , Ratos , Receptor Muscarínico M1 , Receptor Muscarínico M4 , Proteínas Repressoras/metabolismoRESUMO
One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N-type Ca2+ currents: this reduces Ca2+-activated K+ currents and facilitates high-frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage-dependent inhibition through activation of M4 muscarinic acetylcholine receptors (mAChRs), and a slower, voltage-independent inhibition via M1 mAChRs [Hille (1994) Trends in Neurosci., 17, 531-536]. We have analysed the mechanistic basis for this divergence at the level of the individual G-proteins and their alpha and betagamma subunits, using a combination of site-directed antibody injection, plasmid-driven antisense RNA expression, overexpression of selected constitutively active subunits, and antagonism of endogenously liberated betagamma subunits by over-expression of Dy-binding P-adrenergic receptor kinase 1 (PARK1) peptide. The results indicate that: (i) M4 mAChR-induced inhibition is mediated by GoA; (ii) a and Py subunits released from the activated GoA heterotrimer produce separate voltage-insensitive and voltage-sensitive components of inhibition, respectively; and (iii) voltage-insensitive M1 mAChR-induced inhibition is likely to be mediated by the alpha subunit of Gq. Hence, Ca2+ current inhibition results from the concerted, but independent actions of three different G-protein subunits.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Neurônios/fisiologia , Fragmentos de Peptídeos/fisiologia , Receptores Muscarínicos/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação ao GTP/química , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/citologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Muscarina/metabolismo , Neurônios/fisiologia , Potássio/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Elementos Antissenso (Genética)/genética , Aurora Quinases , Sequência de Bases , GTP Fosfo-Hidrolases/deficiência , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Dados de Sequência Molecular , Agonistas Muscarínicos/farmacologia , Neurônios/metabolismo , Plasmídeos/genética , Plasmídeos/farmacologia , Potássio/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/fisiologia , Sistema Nervoso Simpático/citologiaRESUMO
1. Using whole-cell and perforated-patch recordings, we have examined the part played by endogenous G-protein beta gamma subunits in neurotransmitter-mediated inhibition of N-type Ca2+ channel current (ICa) in dissociated rat superior cervical sympathetic neurones. 2. Expression of the C-terminus domain of beta-adrenergic receptor kinase 1 (beta ARK1), which contains the consensus motif (QXXER) for binding G beta gamma, reduced the fast (pertussis toxin (PTX)-sensitive) and voltage-dependent inhibition of ICa by noradrenaline and somatostatin, but not the slow (PTX-insensitive) and voltage-independent inhibition induced by angiotensin II. beta ARK1 peptide reduced GTP-gamma-S-induced voltage-dependent and PTX-sensitive inhibition of ICa but not GTP-gamma-S-mediated voltage-independent inhibition. 3. Overexpression of G beta 1 gamma 2, which mimicked the voltage-dependent inhibition by reducing ICa density and enhancing basal facilitation, occluded the voltage-dependent noradrenaline- and somatostatin-mediated inhibitions but not the inhibition mediated by angiotensin II. 4. Co-expression of the C-terminus of beta ARK1 with beta 1 and gamma 2 subunits prevented the effects of G beta gamma dimers on basal Ca2+ channel behaviour in a manner consistent with the sequestering of G beta gamma. 5. The expression of the C-terminus of beta ARK1 slowed down reinhibition kinetics of ICa following conditioning depolarizations and induced long-lasting facilitation by cumulatively sequestering beta gamma subunits. 6. Our findings identify endogenous G beta gamma as the mediator of the voltage-dependent, PTX-sensitive inhibition of ICa induced by both noradrenaline and somatostatin but not the voltage-independent. PTX-insensitive inhibition by angiotensin II. They also support the view that voltage-dependent inhibition results from a direct G beta gamma-Ca2+ channel interaction.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Neurotransmissores/fisiologia , Gânglio Cervical Superior/citologia , Angiotensina II/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Eletrofisiologia , Proteínas de Ligação ao GTP/genética , Expressão Gênica/genética , Expressão Gênica/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Ativação do Canal Iônico , Cinética , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Norepinefrina/antagonistas & inibidores , Norepinefrina/farmacologia , Toxina Pertussis , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa/fisiologia , Receptores de Somatostatina/fisiologia , Proteínas Recombinantes/genética , Somatostatina/antagonistas & inibidores , Somatostatina/farmacologia , Gânglio Cervical Superior/química , Fatores de Tempo , Fatores de Virulência de Bordetella/farmacologia , Quinases de Receptores Adrenérgicos betaRESUMO
The m1 receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia. RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire m1 gene demonstrated that the rat m1 gene consists of a single 657-base pairs (bp) non-coding exon separated by a 13. 5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940 and Wood, I. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the m1 promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich. The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-1, AP-2, E-box, NFkappaB, and Oct-1. Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the m1 promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.