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1.
Proc Natl Acad Sci U S A ; 110(51): 20813-8, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24297890

RESUMO

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.


Assuntos
Compostos Azo/química , Ativação do Canal Iônico/efeitos da radiação , Luz , Neurônios/metabolismo , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Ratos
2.
Proc Natl Acad Sci U S A ; 110(4): 1512-7, 2013 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-23297228

RESUMO

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.


Assuntos
Regulação do Apetite/fisiologia , Grelina/sangue , Hipotálamo/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Permeabilidade Capilar , Ingestão de Alimentos/fisiologia , Jejum/fisiologia , Hipotálamo/irrigação sanguínea , Hipotálamo/citologia , Masculino , Eminência Mediana/irrigação sanguínea , Eminência Mediana/citologia , Eminência Mediana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Neurológicos , Neurônios/fisiologia
3.
Neuron ; 111(3): 328-344.e7, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36731429

RESUMO

The mammalian spinal cord functions as a community of cell types for sensory processing, autonomic control, and movement. While animal models have advanced our understanding of spinal cellular diversity, characterizing human biology directly is important to uncover specialized features of basic function and human pathology. Here, we present a cellular taxonomy of the adult human spinal cord using single-nucleus RNA sequencing with spatial transcriptomics and antibody validation. We identified 29 glial clusters and 35 neuronal clusters, organized principally by anatomical location. To demonstrate the relevance of this resource to human disease, we analyzed spinal motoneurons, which degenerate in amyotrophic lateral sclerosis (ALS) and other diseases. We found that compared with other spinal neurons, human motoneurons are defined by genes related to cell size, cytoskeletal structure, and ALS, suggesting a specialized molecular repertoire underlying their selective vulnerability. We include a web resource to facilitate further investigations into human spinal cord biology.


Assuntos
Esclerose Lateral Amiotrófica , Animais , Humanos , Adulto , Esclerose Lateral Amiotrófica/metabolismo , Medula Espinal/metabolismo , Neurônios Motores/metabolismo , Modelos Animais , Neuroglia/metabolismo , Mamíferos
4.
Sci Adv ; 8(26): eabo7566, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35767616

RESUMO

Affective touch is necessary for proper neurodevelopment and sociability. However, it remains unclear how the neurons innervating the skin detect affective and social behaviors. The C low-threshold mechanoreceptors (C-LTMRs), a specific population of somatosensory neurons in mice, appear particularly well suited, physiologically and anatomically, to perceive affective and social touch. However, their contribution to sociability has not been resolved yet. Our observations revealed that C-LTMR functional deficiency induced social isolation and reduced tactile interactions in adulthood. Conversely, transient increase in C-LTMR excitability in adults, using chemogenetics, was rewarding, promoted touch-seeking behaviors, and had prosocial influences on group dynamics. This work provides the first empirical evidence that specific peripheral inputs alone can drive complex social behaviors. It demonstrates the existence of a specialized neuronal circuit, originating in the skin, wired to promote interactions with other individuals.

5.
J Clin Invest ; 132(12)2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35608912

RESUMO

The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase known for its oncogenic potential that is involved in the development of the peripheral and central nervous system. ALK receptor ligands ALKAL1 and ALKAL2 were recently found to promote neuronal differentiation and survival. Here, we show that inflammation or injury enhanced ALKAL2 expression in a subset of TRPV1+ sensory neurons. Notably, ALKAL2 was particularly enriched in both mouse and human peptidergic nociceptors, yet weakly expressed in nonpeptidergic, large-diameter myelinated neurons or in the brain. Using a coculture expression system, we found that nociceptors exposed to ALKAL2 exhibited heightened excitability and neurite outgrowth. Intraplantar CFA or intrathecal infusion of recombinant ALKAL2 led to ALK phosphorylation in the lumbar dorsal horn of the spinal cord. Finally, depletion of ALKAL2 in dorsal root ganglia or blocking ALK with clinically available compounds crizotinib or lorlatinib reversed thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury, respectively. Overall, our work uncovers the ALKAL2/ALK signaling axis as a central regulator of nociceptor-induced sensitization. We propose that clinically approved ALK inhibitors used for non-small cell lung cancer and neuroblastomas could be repurposed to treat persistent pain conditions.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Citocinas/metabolismo , Neoplasias Pulmonares , Animais , Humanos , Hiperalgesia/metabolismo , Inflamação/patologia , Ligantes , Camundongos , Dor/tratamento farmacológico , Receptores Proteína Tirosina Quinases , Células Receptoras Sensoriais/metabolismo , Corno Dorsal da Medula Espinal/patologia
6.
Sci Rep ; 9(1): 3112, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816223

RESUMO

The T-type calcium channel, Cav3.2, is necessary for acute pain perception, as well as mechanical and cold allodynia in mice. Being found throughout sensory pathways, from excitatory primary afferent neurons up to pain matrix structures, it is a promising target for analgesics. In our study, Cav3.2 was detected in ~60% of the lamina II (LII) neurons of the spinal cord, a site for integration of sensory processing. It was co-expressed with Tlx3 and Pax2, markers of excitatory and inhibitory interneurons, as well as nNOS, calretinin, calbindin, PKCγ and not parvalbumin. Non-selective T-type channel blockers slowed the inhibitory but not the excitatory transmission in LII neurons. Furthermore, T-type channel blockers modified the intrinsic properties of LII neurons, abolishing low-threshold activated currents, rebound depolarizations, and blunting excitability. The recording of Cav3.2-positive LII neurons, after intraspinal injection of AAV-DJ-Cav3.2-mcherry, showed that their intrinsic properties resembled those of the global population. However, Cav3.2 ablation in the dorsal horn of Cav3.2GFP-Flox KI mice after intraspinal injection of AAV-DJ-Cav3.2-Cre-IRES-mcherry, had drastic effects. Indeed, it (1) blunted the likelihood of transient firing patterns; (2) blunted the likelihood and the amplitude of rebound depolarizations, (3) eliminated action potential pairing, and (4) remodeled the kinetics of the action potentials. In contrast, the properties of Cav3.2-positive neurons were only marginally modified in Cav3.1 knockout mice. Overall, in addition to their previously established roles in the superficial spinal cord and in primary afferent neurons, Cav3.2 channel appear to be necessary for specific, significant and multiple controls of LII neuron excitability.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Neurônios/citologia , Nervos Espinhais/citologia , Potenciais de Ação , Animais , Hiperalgesia/metabolismo , Camundongos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Nervos Espinhais/metabolismo , Transmissão Sináptica
7.
J Neurosci ; 27(7): 1631-41, 2007 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-17301171

RESUMO

The organization of the peptidergic neurons of the hypothalamic arcuate nucleus is not fully understood. These include growth hormone-releasing hormone (GHRH) neurons involved in growth and metabolism. We studied identified GHRH neurons of GHRH-green fluorescent protein transgenic mice using patch-clamp methods and focused on gender differences, which govern the physiological patterns of GHRH release. Both the spontaneous firing rates and the intrinsic properties of GHRH neurons were similar in males and females, although higher glutamatergic currents were noticed in females. Surprisingly, marked gender differences in GHRH neuronal activity were observed in response to the muscarinic agonist carbachol (CCh). In females, CCh enhanced action potential firing in all GHRH neurons. In males, CCh enhanced action potential firing in two-thirds of GHRH neurons, whereas it decreased firing in the remainders. M1 agonist McN-A343 (10 microM) mimicked, and M1 antagonist pirenzepine (3 microM) blocked the effects of CCh. In both genders, CCh did not change the intrinsic properties of GHRH neurons, although it strongly increased the frequency of glutamatergic currents, in the presence or absence of tetrodotoxin. In males only, CCh enhanced the frequency of GABAergic currents, and this modulation was antagonized by tetrodotoxin. Thus, the muscarinic regulation involved differential control of afferent inputs at short and long distances in male and female mice. The dual-level control could be a mechanism whereby the selective modulation of the GHRH system (short-distance control) is adjusted to the integrated regulation of arcuate nucleus activity (long-distance control).


Assuntos
Vias Aferentes/fisiologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Neurônios/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Análise de Variância , Animais , Núcleo Arqueado do Hipotálamo/citologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Imuno-Histoquímica/métodos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Fatores Sexuais , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Ácido gama-Aminobutírico/metabolismo
8.
Aging Cell ; 6(2): 197-207, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17328688

RESUMO

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.


Assuntos
Senescência Celular/fisiologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Potenciais de Ação , Vias Aferentes/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Hormônio do Crescimento/fisiologia , Hormônio Liberador de Hormônio do Crescimento/genética , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo
9.
Sci Rep ; 6: 24394, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-27072430

RESUMO

Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K(+) channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic, and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These "feed-forward loops" may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.


Assuntos
Potenciais de Ação/fisiologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hipotálamo/fisiologia , Somatostatina/fisiologia , Animais , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp
10.
Endocrinology ; 155(5): 1887-98, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24601879

RESUMO

Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.


Assuntos
Lesões Encefálicas/fisiopatologia , Modelos Animais de Doenças , Células Ependimogliais/patologia , Hipopituitarismo/etiologia , Hipotálamo/patologia , Neurônios/patologia , Junções Íntimas/patologia , Animais , Núcleo Arqueado do Hipotálamo/imunologia , Núcleo Arqueado do Hipotálamo/metabolismo , Núcleo Arqueado do Hipotálamo/patologia , Biomarcadores/metabolismo , Células Ependimogliais/imunologia , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hipopituitarismo/imunologia , Hipopituitarismo/metabolismo , Hipopituitarismo/patologia , Hipotálamo/imunologia , Hipotálamo/metabolismo , Imunoglobulina G/metabolismo , Masculino , Eminência Mediana/imunologia , Eminência Mediana/metabolismo , Eminência Mediana/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/imunologia , Neurônios/metabolismo , Permeabilidade , Proteínas Recombinantes de Fusão/metabolismo , Terceiro Ventrículo/imunologia , Terceiro Ventrículo/metabolismo , Terceiro Ventrículo/patologia , Junções Íntimas/imunologia , Junções Íntimas/metabolismo
11.
Endocrinology ; 151(12): 5762-74, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20926590

RESUMO

Growth hormone (GH) is the key hormone involved in the regulation of growth and metabolism, two functions that are highly modulated during infancy. GH secretion, controlled mainly by GH releasing hormone (GHRH), has a characteristic pattern during postnatal development that results in peaks of blood concentration at birth and puberty. A detailed knowledge of the electrophysiology of the GHRH neurons is necessary to understand the mechanisms regulating postnatal GH secretion. Here, we describe the unique postnatal development of the electrophysiological properties of GHRH neurons and their regulation by gonadal hormones. Using GHRH-eGFP mice, we demonstrate that already at birth, GHRH neurons receive numerous synaptic inputs and fire large and fast action potentials (APs), consistent with effective GH secretion. Concomitant with the GH secretion peak occurring at puberty, these neurons display modifications of synaptic input properties, decrease in AP duration, and increase in a transient voltage-dependant potassium current. Furthermore, the modulation of both the AP duration and voltage-dependent potassium current are specifically controlled by gonadal hormones because gonadectomy prevented the maturation of these active properties and hormonal treatment restored it. Thus, GHRH neurons undergo specific developmental modulations of their electrical properties over the first six postnatal weeks, in accordance with hormonal demand. Our results highlight the importance of the interaction between the somatotrope and gonadotrope axes during the establishment of adapted neuroendocrine functions.


Assuntos
Hormônio Liberador de Hormônio do Crescimento/metabolismo , Neurônios/metabolismo , Potenciais de Ação , Animais , Encéfalo/crescimento & desenvolvimento , Etinilestradiol/farmacologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Neurônios/citologia , Orquiectomia , Ovariectomia , Caracteres Sexuais , Maturidade Sexual/fisiologia , Potenciais Sinápticos/fisiologia , Propionato de Testosterona/farmacologia , Ácido gama-Aminobutírico/metabolismo
12.
PLoS One ; 5(2): e9159, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-20161791

RESUMO

BACKGROUND: Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin. PRINCIPAL FINDINGS: Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by gamma-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate. CONCLUSION: Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Grelina/farmacologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Neurônios/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/citologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Relação Dose-Resposta a Droga , Estrenos/farmacologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hormônio Liberador de Hormônio do Crescimento/genética , Indóis , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Oligopeptídeos/farmacologia , Técnicas de Patch-Clamp , Pirrolidinonas/farmacologia , Receptores de Grelina/agonistas , Triptofano/análogos & derivados , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
13.
Hum Mol Genet ; 14(13): 1727-43, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15888488

RESUMO

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.


Assuntos
Caveolinas/metabolismo , Músculo Esquelético/embriologia , Mioblastos Esqueléticos/metabolismo , Miofibrilas/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Caveolina 3 , Caveolinas/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Fusão Celular , Embrião não Mamífero/embriologia , Humanos , Dados de Sequência Molecular , Músculo Esquelético/ultraestrutura , Mioblastos Esqueléticos/ultraestrutura , Miofibrilas/genética , Miofibrilas/ultraestrutura , Peixe-Zebra/genética
14.
Mol Cell Biochem ; 258(1-2): 35-48, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15030168

RESUMO

Little is known about the subcellular distribution and the dynamics of tubulins in adult cardiac myocytes although both are modified during cardiac hypertrophy and heart failure. Using confocal microscopy, we examined post-translational modifications of tubulin in fully differentiated ventricular myocytes isolated from adult rat hearts, as well as in immortalized and dividing HL-1 cardiomyocytes. Detyrosinated Glu-alpha-tubulin was the most abundant post-translationally modified tubulin found in ventricular myocytes, while acetylated- and delta2-alpha-tubulins were found in lower amounts or absent. In contrast, dividing HL-1 cardiomyocytes exhibited high levels of tyrosinated or acetylated alpha-tubulins. A mild nocodazole treatment (0.1 microM, 1 h) disrupted microtubules in HL-1 myocytes, but not in adult ventricular myocytes. A stronger treatment (10 microM, 2 h) was required to disassemble tubulins in adult myocytes. Glu-alpha-tubulin containing microtubules were more resistant to nocodazole treatment in HL-1 cardiomyocytes than in ventricular myocytes. Endogenous activation of the cAMP pathway with the forskolin analog L858051 (20 microM) or the beta-adrenergic agonist isoprenaline (10 microM) disrupted the most labile microtubules in HL-1 cardiomyocytes. In contrast, isoprenaline (10 microM), cholera toxin (200 ng/ml, a G(S)-protein activator), L858051 (20 microM) or forskolin (10 microM) had no effect on the microtubule network in ventricular myocytes. In addition, intracellular Ca2+ accumulation induced either by thapsigargin (2 microM) or caffeine (10 mM) did not modify microtubule stability in ventricular myocytes. Our data demonstrate the unique stability of the microtubule network in adult cardiac myocytes. We speculate that microtubule stability is required to support cellular integrity during cardiac contraction.


Assuntos
Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Antineoplásicos/farmacologia , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Transformada , Toxina da Cólera/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Ventrículos do Coração/citologia , Isoproterenol/farmacologia , Masculino , Miócitos Cardíacos/citologia , Nocodazol/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Wistar , Tapsigargina/farmacologia
15.
J Physiol ; 540(Pt 2): 411-24, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11956332

RESUMO

The effects of nitric oxide (NO) donors on the L-type Ca(2+) current (I(Ca,L)) and the muscarinic activated K(+) current (I(K,ACh)) were studied in isolated rat cardiac myocytes. The nitrosothiol S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1 pM-1 microM) strongly potentiated the stimulation of the I(Ca,L) elicited by subthreshold concentrations of isoprenaline (Iso, 0.1-0.5 nM) in ventricular myocytes. The effect of SNAP was mimicked by 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO, 1 pM-1 nM), a NONOate that spontaneously releases NO in a pH-controlled manner, and was blunted by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 microM), a NO trap. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (10 microM), a guanylyl cyclase inhibitor, did not alter the effect of SNAP. SNAP (1 pM-1 microM) did not modify the effect of L858051 (0.1-0.3 microM), a forskolin analogue that activates adenylyl cyclase, on I(Ca,L) and did not enhance the basal I(Ca,L) in the presence of rolipram (1 microM), a phosphodiesterase type 4 inhibitor. Superfusion with Rp-CPT-cAMPS (500 microM), or internal dialysis with cAMP-dependent protein kinase (cA-PK) inhibitory peptide (PKI; 20 microM), inhibitors of the cA-PK, blunted the effect of SNAP (1 nM and 1 microM) on the Iso-stimulated (1-100 pM) I(Ca,L). SNAP (1 nM and 1 microM) potentiated the threshold stimulation of I(Ca,L) elicited by internal GTP-gammaS (10 microM), a non-hydrolysable analogue of GTP. SNAP (1 pM-1 microM) and DEANO (1 microM) potentiated the stimulation of I(K,ACh) elicited by low concentrations of ACh (1-2 nM) in rat atrial myocytes. The threshold stimulation of I(K,ACh) elicited by internal 5'-guanylylimidodiphosphate (10 microM) was also potentiated by NO donors. SNAP (1 microM) did not modify I(K,ACh) reconstituted in human embryonic kidney 293 cells, in the absence or in the presence of ACh (1 or 10 nM). Taken together, these data suggest that NO is a cGMP-independent modulator of G-protein-coupled muscarinic and beta-adrenergic receptor actions on cardiac ion channels. Although this action of NO seemed to occur at the level of G proteins, it appeared to require a component distinct from receptors, G proteins or their effectors.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Coração/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Canais de Potássio/efeitos dos fármacos , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/fisiologia , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Linhagem Celular , Separação Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Dietilaminas/farmacologia , Sinergismo Farmacológico , Eletrofisiologia , Proteínas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Miocárdio/metabolismo , Óxidos de Nitrogênio , Técnicas de Patch-Clamp , Ratos , S-Nitroso-N-Acetilpenicilamina/farmacologia , Transfecção
16.
Mol Cell Biochem ; 237(1-2): 39-46, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12236585

RESUMO

Cytoskeletal reorganization has been shown to participate in cellular remodeling and in the alterations of mechanical function of isolated cardiomyocytes during pressure overload hypertrophy. Post-translational modifications of tubulin towards stabilization of microtubules have also been described in animal models of compensatory hypertrophy, but the status of the microtubules network in end stage heart failure is not clearly established. Using a rat model of congestive heart failure (CHF) induced by aortic banding, we studied the expression of alpha- and beta-tubulin, as well as their post-translational modification and distribution in the soluble and polymerized fraction by immunoblotting. We found an accumulation of alpha- and beta-tubulin protein content specifically in the soluble fraction with no change in the polymerized fraction. Amongst the several variants of alpha-tubulin examined, only detyrosinated Glu-tubulin and deglutamylated delta2-tubulin levels were selectively increased during heart failure. Glu-tubulin accumulated in the polymerized fraction while delta2-tubulin levels were increased in the soluble fraction in CHF hearts. These results show that a profound remodeling of the microtubule network occurs in heart failure. This remodeling suggests an increase in the stability of the microtubule network which is discussed in terms of possible functional consequences.


Assuntos
Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/química , Animais , Aorta/patologia , Western Blotting , Hipertrofia , Immunoblotting , Masculino , Microtúbulos/metabolismo , Miocárdio/citologia , Isoformas de Proteínas , Ratos , Ratos Wistar , Tórax/patologia
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