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1.
Science ; 341(6152): 1394-9, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-24052307

RESUMO

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced µ-opioid receptor (MOR) constitutive activity (MOR(CA)) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3',5'-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MOR(CA) initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MOR(CA) suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.


Assuntos
Dor Crônica/metabolismo , Hiperalgesia/metabolismo , Dor Nociceptiva/metabolismo , Receptores Opioides mu/metabolismo , Dor Aguda/metabolismo , Monofosfato de Adenosina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Modelos Animais de Doenças , Adjuvante de Freund/farmacologia , Hiperalgesia/induzido quimicamente , Isoflurano/farmacologia , Masculino , Camundongos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo
2.
Genes Brain Behav ; 11(7): 837-47, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22925203

RESUMO

Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor-coordination, and have an attenuation of age-induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , Memória , Atividade Motora/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Fatores Etários , Animais , Ansiedade/genética , Encéfalo/enzimologia , Encéfalo/metabolismo , Condicionamento Psicológico , Medo , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia
3.
Mol Endocrinol ; 19(5): 1277-90, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15705663

RESUMO

Mammalian spermatids and spermatozoa express functional G protein-coupled receptors. However, bicarbonate-regulated soluble adenylyl cyclase (AC), the major AC present in these cells, is not directly coupled to G proteins. To understand how G protein-coupled receptors signal in spermatozoa, we investigated whether a conventional transmembrane cyclase is present and biologically active in these cells. Here, we provide evidence for expression of type 3 AC (AC3) in male germ cells and describe the effects of disruption of the AC3 gene on fertility and function of mouse spermatozoa. As previously reported in rat, AC3 mRNA is expressed in mouse testes and localized, together with soluble AC mRNA, mainly in postmeiotic germ cells. AC3 protein was detected by immunolocalization in round and elongating spermatids in a region corresponding to the developing acrosome and was retained in the mature spermatozoa of the epididymis. Forskolin caused a small increase in cAMP production in mouse spermatozoa, but this increase could not be detected in the AC3(-/-) mice. Inactivation of the AC3 gene did not have overt effects on spermatogenesis; however, AC3(-/-) males were subfertile with only three litters generated by 11 males over a period of 6 months. When used in in vitro fertilization, spermatozoa from these AC3(-/-) mice produced few embryos, but their fertilizing ability was restored after removal of the zona pellucida. Despite an apparently normal structure, these spermatozoa had decreased motility and showed an increase in spontaneous acrosome reactions. These data support the hypothesis that AC3 is required for normal spermatid or spermatozoa function and male fertility.


Assuntos
Adenilil Ciclases/genética , Infertilidade Masculina/genética , Isoenzimas/genética , Espermatozoides/metabolismo , Adenilil Ciclases/biossíntese , Animais , AMP Cíclico/metabolismo , Epididimo/anormalidades , Fertilização/genética , Imuno-Histoquímica , Infertilidade Masculina/metabolismo , Isoenzimas/biossíntese , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Motilidade dos Espermatozoides/genética , Testículo/anormalidades , Testículo/metabolismo
4.
Neuron ; 31(3): 409-20, 2001 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-11516398

RESUMO

The development of precise connections in the mammalian brain proceeds through refinement of initially diffuse patterns, a process that occurs largely within critical developmental windows. To elucidate the molecular pathways that orchestrate these early periods of circuit remodeling, we have examined the role of a calcium- and cAMP-regulated transcriptional pathway. We show that there is a window of CRE/CREB-mediated gene expression in the developing thalamus, which precedes neocortical expression. In the LGN, this wave of gene expression occurs prior to visual experience, but requires retinal function. Mutant mice with reduced CREB expression show loss of refinement of retinogeniculate projections. These results suggest an important role of the CRE/CREB transcriptional pathway in the coordination of experience-independent circuit remodeling during forebrain development.


Assuntos
Axônios/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Corpos Geniculados/fisiologia , Integrases/metabolismo , Retina/fisiologia , Tálamo/fisiologia , Transcrição Gênica , Proteínas Virais/metabolismo , Vias Visuais/fisiologia , Envelhecimento , Animais , Cruzamentos Genéticos , Enucleação Ocular , Feminino , Heterozigoto , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tálamo/crescimento & desenvolvimento , Proteínas Virais/genética , beta-Galactosidase/genética
5.
J Biol Chem ; 276(36): 34206-12, 2001 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-11432866

RESUMO

Arterial smooth muscle cell (SMC) proliferation contributes to a number of vascular pathologies. Prostaglandin E(2) (PGE(2)), produced by the endothelium and by SMCs themselves, acts as a potent SMC growth inhibitor. The growth-inhibitory effects of PGE(2) are mediated through activation of G-protein-coupled membrane receptors, activation of adenylyl cyclases (ACs), formation of cAMP, and subsequent inhibition of mitogenic signal transduction pathways in SMCs. Of the 10 different mammalian AC isoforms known today, seven isoforms (AC2-7 and AC9) are expressed in SMCs from various species. We show that, despite the presence of several different AC isoforms, the principal AC isoform activated by PGE(2) in human arterial SMCs is a calmodulin kinase II-inhibited AC with characteristics similar to those of AC3. AC3 is expressed in isolated human arterial SMCs and in intact aorta. We further show that arterial SMCs isolated from AC3-deficient mice are resistant to PGE(2)-induced growth inhibition. In summary, AC3 is the principal AC isoform activated by PGE(2) in arterial SMCs, and AC3 mediates the growth-inhibitory effects of PGE(2). Because AC3 activity is inhibited by intracellular calcium through calmodulin kinase II, AC3 may serve as an important integrator of growth-inhibitory signals that stimulate cAMP formation and growth factors that increase intracellular calcium.


Assuntos
Adenilil Ciclases/fisiologia , Artérias/enzimologia , Dinoprostona/metabolismo , Isoenzimas/fisiologia , Músculo Liso Vascular/enzimologia , Animais , Aorta/embriologia , Aorta Torácica/metabolismo , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Divisão Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Recém-Nascido , Camundongos , Modelos Biológicos , Músculo Liso/citologia , Testes de Precipitina , Isoformas de Proteínas
6.
Eur J Neurosci ; 13(11): 2054-66, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11422446

RESUMO

The Ca2+-stimulated type 1 adenylyl cyclase (AC1) contributes to several forms of synaptic plasticity and is the only known neurospecific adenylyl cyclase. Furthermore, the protein and mRNA levels of AC1 undergo a circadian oscillation in the pineal gland, and AC1 may play a pivotal role in regulating nocturnal melatonin synthesis. To better understand the expression of AC1, we isolated mouse genomic DNA clones of AC1. The transcription and translation start regions of mouse AC1 share extensive homologies with the bovine counterpart. The upstream proximal region has potential binding sites for transcription factors, including the steroid receptor family, the E-box factors, and Sp1. A 280-bp fragment that contains the transcription start site directed reporter gene expression in cultured cortical neurons and pinealocytes functioning as a basal neuro- and pineal-directed promoter. Interestingly, pinealocyte expression of the reporter gene was inhibited by increases in cAMP. This cAMP sensitivity may explain why AC1 mRNA in the pineal is low at night when cAMP is elevated and high during the day when cAMP signals drop. An adjacent 330-bp fragment interacted specifically with nuclear factor(s) that we designate binary E-box factor (BEF). Methylation interference and DNase I footprinting identified the BEF-binding site sequence as 5'-CCAAGGTCACGTGGC-3'. When linked to the basal tissue-directed promoter, this 15-bp sequence further enhanced reporter expression in neurons and pinealocytes. We propose that this 15-bp sequence may contribute to increased expression of AC1 in neurons and pinealocytes relative to other cells.


Assuntos
Adenilil Ciclases/genética , Encéfalo/enzimologia , DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Reporter/fisiologia , Neurônios/enzimologia , Glândula Pineal/enzimologia , Animais , Animais Recém-Nascidos , Sequência de Bases/fisiologia , Sítios de Ligação/genética , Ligação Competitiva/genética , Encéfalo/citologia , Células Cultivadas/citologia , Células Cultivadas/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , DNA/química , Proteínas de Ligação a DNA/metabolismo , Nucleotídeos de Guanina/metabolismo , Dados de Sequência Molecular , Neurônios/citologia , Oligonucleotídeos/metabolismo , Glândula Pineal/citologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Elementos de Resposta/fisiologia , Transdução de Sinais/genética , TATA Box/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia
7.
Int J Dev Neurosci ; 19(4): 387-94, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11378299

RESUMO

Studies with invertebrates and vertebrates have strongly implicated the CREB/CRE transcriptional pathway in long-term memory (LTM) and transcriptionally-dependent L-LTP. It is hypothesized that LTM and L-LTP are both dependent upon a Ca2+ signal generated through activation of NMDA receptors. This review discusses evidence that Ca2+ signals generated through activation of NMDA receptors coactivate the Erk/MAP kinase and cAMP signal transduction pathways. It is hypothesized that activation of these two regulatory pathways increases the transcription of a family of genes through the CREB/CRE transcriptional pathway. Gene disruption studies have shown that Ca2+ activated adenylyl cyclases play a critical role in generating the cAMP signal required for LTM and L-LTP. Although cAMP may be required for several events in this complex signal transduction cascade, one of the major roles of cAMP may be to support nuclear translocation of Erk/MAP kinase in hippocampal neurons.


Assuntos
Adenilil Ciclases/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/fisiologia , Potenciação de Longa Duração/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Memória/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fator 2 Ativador da Transcrição , Transporte Ativo do Núcleo Celular , Animais , Ritmo Circadiano/fisiologia , AMP Cíclico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Fibras Musgosas Hipocampais/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/fisiologia , Transcrição Gênica
8.
Neurosci Lett ; 299(3): 181-4, 2001 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-11165765

RESUMO

The mossy fiber pathway of the hippocampal formation and type 1 adenylyl cyclase (AC1) have been implicated in long-term potentiation and memory function. Using immunohistochemical labeling and light microscopy we demonstrated intense labeling of AC1 in the mossy fibers and less intense labeling in the molecular layers of both the dentate gyrus and fields CA1, CA2 and CA3 of the hippocampus, i.e. in terminal fields of the perforant pathway. These findings indicate that, in the non-human primate, AC1 is found in the mossy fibers and in terminal fields of the perforant pathway where it may play a role in long term potentiation similar to that demonstrated in the rodent.


Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/biossíntese , Fibras Musgosas Hipocampais/enzimologia , Vias Neurais/enzimologia , Animais , Imuno-Histoquímica , Aprendizagem/fisiologia , Potenciação de Longa Duração/fisiologia , Macaca nemestrina/anatomia & histologia , Macaca nemestrina/metabolismo , Fibras Musgosas Hipocampais/ultraestrutura , Vias Neurais/citologia
9.
J Biol Chem ; 276(3): 2047-52, 2001 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-11042208

RESUMO

Olfactory sensory neurons (OSNs) respond acutely to volatile molecules and exhibit adaptive responses including desensitization to odorant exposure. Although mechanisms for short term adaptation have been described, there is little evidence that odorants cause long lasting, transcription-dependent changes in OSNs. Here we report that odorants stimulate cAMP-response element (CRE)-mediated transcription in OSNs through Ca2+ activation of the ERK/MAPK/p90rsk pathway. Odorant stimulation of ERK phosphorylation was ablated by inhibition of calmodulin-dependent protein kinase II suggesting that odorant activation of ERK is mediated through this kinase. Moreover, a brief exposure in vivo to an odorant in vapor phase stimulated CRE-mediated gene transcription in discrete populations of OSNs. These data suggest that like central nervous system neurons, OSNs may undergo long term adaptive changes mediated through CRE-mediated transcription.


Assuntos
Sistema de Sinalização das MAP Quinases , Neurônios Aferentes/metabolismo , Odorantes , Condutos Olfatórios/metabolismo , Transcrição Gênica , Animais , AMP Cíclico/metabolismo , Cinética , Neurônios Aferentes/enzimologia , Condutos Olfatórios/citologia , Condutos Olfatórios/enzimologia , Ratos
10.
Curr Protoc Neurosci ; Appendix 4: Appendix 4A, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18428449

RESUMO

This unit provides protocols for cannulation and site-specific central microinjection of mice using a recently developed high-precision stereotaxic frame. The construction of cannulae, wire plugs and injection needles are also described.


Assuntos
Camundongos/cirurgia , Técnicas Estereotáxicas/instrumentação , Animais , Barreira Hematoencefálica , Encéfalo , Cateterismo , Desenho de Equipamento , Microinjeções/métodos , Agulhas , Crânio/anatomia & histologia
11.
Neuron ; 27(3): 487-97, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11055432

RESUMO

Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.


Assuntos
Adenilil Ciclases/genética , Marcação de Genes , Transtornos do Olfato/enzimologia , Transtornos do Olfato/genética , Adenilil Ciclases/metabolismo , Animais , Aprendizagem da Esquiva , Comportamento Animal , Cílios/metabolismo , AMP Cíclico/metabolismo , Eletrofisiologia , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/genética , Camundongos , Camundongos Transgênicos/genética , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , Transdução de Sinais , Estimulação Química
12.
EMBO J ; 19(18): 4955-66, 2000 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-10990459

RESUMO

Recent evidence indicates that phosphatidylinositol 3-kinase (PI3K) is a central regulator of mitosis, apoptosis and oncogenesis. Nevertheless, the mechanisms by which PI3K regulates proliferation are not well characterized. Mitogens stimulate entry into the cell cycle by inducing the expression of immediate early genes (IEGs) that in turn trigger the expression of G(1) cyclins. Here we describe a novel PI3K- regulated transcriptional cascade that is critical for mitogen regulation of the IEG, c-fos. We show that PI3K activates gene expression by transactivating SRF-dependent transcription independently of the previously described Rho and ETS TCF pathways. PI3K-stimulated cell cycle progression requires transactivation of SRF and expression of dominant- negative PI3K blocks mitogen-stimulated cell cycle progression. Furthermore, dominant-interfering SRF mutants attenuate mitogen-stimulated cell cycle progression, but are without effect on MEK-stimulated cell cycle entry. Moreover, expression of constitutively active SRF is sufficient for cell cycle entry. Thus, we delineate a novel SRF-dependent mitogenic cascade that is critical for PI3K- and growth factor-mediated cell cycle progression.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Células 3T3 , Androstadienos/farmacologia , Animais , Western Blotting , Ciclo Celular , Divisão Celular , Separação Celular , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Proteínas de Ligação ao GTP/metabolismo , Genes Dominantes , Células HeLa , Humanos , Imuno-Histoquímica , Óperon Lac , Luciferases/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno , Morfolinas/farmacologia , Mutagênese Sítio-Dirigida , Fator de Crescimento Neural/farmacologia , Células PC12 , Fosforilação , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Resposta Sérica , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Wortmanina , Proteínas Quinases p38 Ativadas por Mitógeno , Proteínas rho de Ligação ao GTP/metabolismo
13.
J Neurosci ; 20(13): 4809-20, 2000 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10864938

RESUMO

Stress results in alterations in behavior and physiology that can be either adaptive or maladaptive. To define the molecular pathways involved in the response to stress further, we generated mice deficient (KO) in the calcium-stimulated adenylyl cyclase type VIII (AC8) by homologous recombination in embryonic stem cells. AC8 KO mice demonstrate a compromise in calcium-stimulated AC activity in the hippocampus, hypothalamus, thalamus, and brainstem. Hippocampal slices derived from AC8 KO mice fail to demonstrate CA1-region long-term depression after low-frequency stimulation, and AC8 KO mice also fail to activate CRE-binding protein in the CA1 region after restraint stress. To define the behavioral consequences of AC8 deficiency, we evaluated AC8 KO mice in the elevated plus-maze and open field. Although naive AC8 KO mice exhibit indices of anxiety comparable with that of wild-type mice, AC8 KO mice do not show normal increases in behavioral markers of anxiety when subjected to repeated stress such as repetitive testing in the plus-maze or restraint preceding plus-maze testing. These results demonstrate a novel role for AC8 in the modulation of anxiety.


Assuntos
Adenilil Ciclases/genética , Ansiedade , Encéfalo/fisiologia , Aprendizagem em Labirinto/fisiologia , Estresse Psicológico/fisiopatologia , Adenilil Ciclases/deficiência , Animais , Encéfalo/enzimologia , Cálcio/metabolismo , Quimera , Cruzamentos Genéticos , Potenciais Evocados , Feminino , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Atividade Motora , Especificidade de Órgãos , Fenótipo , Células Piramidais/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Olfato/fisiologia , Estresse Psicológico/genética
14.
J Biol Chem ; 275(19): 14691-9, 2000 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-10799557

RESUMO

Capacitative Ca(2+) entry stimulates cAMP synthesis in mouse parotid acini, suggesting that one of the Ca(2+)-sensitive adenylyl cyclases (AC1 or AC8) may play an important role in the regulation of parotid function (Watson, E. L., Wu, Z., Jacobson, K. L., Storm, D. R., Singh, J. C., and Ott, S. M. (1998) Am. J. Physiol. 274, C557-C565). To evaluate the role of AC1 and AC8 in Ca(2+) stimulation of cAMP synthesis in parotid cells, acini were isolated from AC1 mutant (AC1-KO) and AC8 mutant (AC8-KO) mice and analyzed for Ca(2+) stimulation of intracellular cAMP levels. Although Ca(2+) stimulation of intracellular cAMP levels in acini from AC1-KO mice was indistinguishable from wild type mice, acini from AC8-KO mice showed no Ca(2+)-stimulated cAMP accumulation. This indicates that AC8, but not AC1, plays a major role in coupling Ca(2+) signals to cAMP synthesis in parotid acini. Interestingly, treatment of acini from AC8-KO mice with agents, i.e. carbachol and thapsigargin that increase intracellular Ca(2+), lowered cAMP levels. This decrease was dependent upon Ca(2+) influx and independent of phosphodiesterase activation. Immunoblot analysis revealed that AC5/6 and AC3 are expressed in parotid glands. Inhibition of calmodulin (CaM) kinase II with KN-62, or inclusion of the CaM inhibitor, calmidazolium, did not prevent agonist-induced inhibition of stimulated cAMP accumulation. In vitro studies revealed that Ca(2+), independently of CaM, inhibited isoproterenol-stimulated AC. Data suggest that agonist augmentation of stimulated cAMP levels is due to activation of AC8 in mouse parotid acini, and strongly support a role for AC5/6 in the inhibition of stimulated cAMP levels.


Assuntos
Adenilil Ciclases/metabolismo , Cálcio/farmacologia , AMP Cíclico/metabolismo , Glândula Parótida/efeitos dos fármacos , Adenilil Ciclases/genética , Animais , AMP Cíclico/biossíntese , Ativação Enzimática , Isoenzimas/metabolismo , Isoproterenol/antagonistas & inibidores , Isoproterenol/farmacologia , Camundongos , Camundongos Knockout , Glândula Parótida/enzimologia , Glândula Parótida/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteína Quinase C/metabolismo , Tapsigargina/farmacologia
15.
Neuron ; 23(4): 787-98, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10482244

RESUMO

It is hypothesized that Ca2+ stimulation of calmodulin (CaM)-activated adenylyl cyclases (AC1 or AC8) generates cAMP signals critical for late phase LTP (L-LTP) and long-term memory (LTM). However, mice lacking either AC1 or AC8 exhibit normal L-LTP and LTM. Here, we report that mice lacking both enzymes (DKO) do not exhibit L-LTP or LTM. To determine if these defects are due to a loss of cAMP increases in the hippocampus, DKO mice were unilaterally cannulated to deliver forskolin. Administration of forskolin to area CA1 before training restored normal LTM. We conclude that Ca2+-stimulated adenylyl cyclase activity is essential for L-LTP and LTM and that AC1 or AC8 can produce the necessary cAMP signal.


Assuntos
Adenilil Ciclases/metabolismo , Cálcio/fisiologia , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Animais , Aprendizagem da Esquiva/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Calmodulina/fisiologia , Colforsina/farmacologia , Sinais (Psicologia) , Eletrofisiologia , Medo/fisiologia , Medo/psicologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Imuno-Histoquímica , Potenciação de Longa Duração/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Camundongos Knockout , Microscopia Confocal
17.
J Biol Chem ; 274(25): 17748-56, 1999 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-10364217

RESUMO

A program of stringently-regulated gene expression is thought to be a fundamental component of the circadian clock. Although recent work has implicated a role for E-box-dependent transcription in circadian rhythmicity, the contribution of other enhancer elements has yet to be assessed. Here, we report that cells of the suprachiasmatic nuclei (SCN) exhibit a prominent circadian oscillation in cAMP response element (CRE)-mediated gene expression. Maximal reporter gene expression occurred from late-subjective night to mid-subjective day. Cycling of CRE-dependent transcription was not observed in other brain regions, including the supraoptic nucleus and piriform cortex. Levels of the phospho-active form of the transcription factor CREB (P-CREB) varied as a function of circadian time. Peak P-CREB levels occurred during the mid- to late-subjective night. Furthermore, photic stimulation during the subjective night, but not during the subjective day, triggered a marked increase in CRE-mediated gene expression in the SCN. Reporter gene experiments showed that activation of the p44/42 mitogen-activated protein kinase signaling cascade is required for Ca2+-dependent stimulation of CRE-mediated transcription in the SCN. These findings reveal the CREB/CRE transcriptional pathway to be circadian-regulated within the SCN, and raise the possibility that this pathway provides signaling information essential for normal clock function.


Assuntos
Ritmo Circadiano , AMP Cíclico/genética , Proteínas Quinases Ativadas por Mitógeno , Núcleo Supraquiasmático/metabolismo , Animais , Cálcio/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Elementos Facilitadores Genéticos , Ativação Enzimática , Regulação da Expressão Gênica , Genes Reporter , Imuno-Histoquímica , Luz , Camundongos , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno , Fosfoproteínas/genética , Fosforilação , Transdução de Sinais
18.
Nature ; 399(6732): 155-9, 1999 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-10335845

RESUMO

Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. Here we report a calcium-dependent interaction between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. Our results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity.


Assuntos
Canais de Cálcio Tipo N , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Quelantes , Clonagem Molecular , Ácido Egtázico , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Leveduras
19.
J Biol Chem ; 274(12): 7689-94, 1999 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-10075657

RESUMO

Neurogranin is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) within its IQ domain at serine 36. Since CaM binds to neurogranin through the IQ domain, PKC phosphorylation and CaM binding are mutually exclusive. Consequently, we hypothesize that neurogranin may function to concentrate CaM at specific sites in neurons and release free CaM in response to increased Ca2+ and PKC activation. However, it has not been established that neurogranin interacts with CaM in vivo. In this study, we examined this question using yeast two-hybrid methodology. We also searched for additional proteins that might interact with neurogranin by screening brain cDNA libraries. Our data illustrate that CaM binds to neurogranin in vivo and that CaM is the only neurogranin-interacting protein isolated from brain cDNA libraries. Single amino acid mutagenesis indicated that residues within the IQ domain are important for CaM binding to neurogranin in vivo. The Ile-33 --> Gln point mutant completely inhibited and Arg-38 --> Gln and Ser-36 --> Asp point mutants reduced neurogranin/CaM interactions. These data demonstrate that CaM is the major protein that interacts with neurogranin in vivo and support the hypothesis that phosphorylation of neurogranin at Ser-36 regulates its binding to CaM.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Inibidores de Adenilil Ciclases , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Células Cultivadas , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Neurogranina , Fosforilação , Mutação Puntual , Proteína Quinase C/metabolismo , Ratos , Serina/metabolismo
20.
Neuron ; 22(1): 63-72, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10027290

RESUMO

Neuronal activity-dependent processes are believed to mediate the formation of synaptic connections during neocortical development, but the underlying intracellular mechanisms are not known. In the visual system, altering the pattern of visually driven neuronal activity by monocular deprivation induces cortical synaptic rearrangement during a postnatal developmental window, the critical period. Here, using transgenic mice carrying a CRE-lacZ reporter, we demonstrate that a calcium- and cAMP-regulated signaling pathway is activated following monocular deprivation. We find that monocular deprivation leads to an induction of CRE-mediated lacZ expression in the visual cortex preceding the onset of physiologic plasticity, and this induction is dramatically downregulated following the end of the critical period. These results suggest that CRE-dependent coordinate regulation of a network of genes may control physiologic plasticity during postnatal neocortical development.


Assuntos
Envelhecimento/fisiologia , Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , AMP Cíclico/metabolismo , Plasticidade Neuronal/fisiologia , Transcrição Gênica , Córtex Visual/fisiologia , Animais , Corpos Geniculados/fisiologia , Camundongos , Camundongos Transgênicos , Privação Sensorial/fisiologia , Transcrição Gênica/fisiologia , Visão Monocular/fisiologia
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