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1.
Neurochem Res ; 42(9): 2537-2550, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28434165

RESUMO

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a 'docked' counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.


Assuntos
Astrócitos/metabolismo , Conexinas/fisiologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Conexinas/agonistas , Conexinas/antagonistas & inibidores , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia
2.
J Biol Chem ; 290(13): 8039-47, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25666621

RESUMO

The mechanisms of HCO3(-)-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H(+) pump by Cl(-) removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3-6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3(-)-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.


Assuntos
Equilíbrio Ácido-Base , Astrócitos/metabolismo , Bicarbonatos/metabolismo , ATPases Vacuolares Próton-Translocadoras/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Nervo Óptico/citologia , Ratos Wistar
3.
J Biol Chem ; 289(38): 26058-26073, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25086040

RESUMO

Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.


Assuntos
Astrócitos/metabolismo , Conexina 43/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Benzenossulfonatos/farmacologia , Carbenoxolona/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Etídio/metabolismo , Corantes Fluorescentes/metabolismo , Gadolínio/farmacologia , Junções Comunicantes/metabolismo , Potenciais da Membrana , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/metabolismo , Xenopus laevis
4.
J Neurophysiol ; 114(5): 3014-22, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26400258

RESUMO

Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.


Assuntos
Conexina 43/metabolismo , Conexinas/metabolismo , Proteína Quinase C/metabolismo , Animais , Benzofenantridinas/farmacologia , Conexina 30 , Indóis/farmacologia , Maleimidas/farmacologia , Potenciais da Membrana , Camundongos , Fosforilação , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Ratos , Xenopus laevis
5.
J Membr Biol ; 245(2): 77-87, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22383044

RESUMO

The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant. TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions. In contrast, TonEBP activity and TauT transcription are significantly increased following hyperosmotic exposure. Swelling-induced ROS production in NIH3T3 fibroblasts is generated by NOX4 and by increasing total ROS, by either exogenous application of H(2)O(2) or overexpressing NOX4, we demonstrate that TonEBP activity and taurine influx are regulated negatively by ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low-sodium/hypo-osmotic conditions by direct modulation of TauT.


Assuntos
Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Estresse Fisiológico/fisiologia , Animais , Células Cultivadas , Regulação para Baixo , Peróxido de Hidrogênio/metabolismo , Soluções Hipertônicas , Soluções Hipotônicas , Cinética , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Células NIH 3T3 , Osmose , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Sódio/metabolismo , Taurina/genética , Taurina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Equilíbrio Hidroeletrolítico
6.
Cell Physiol Biochem ; 28(6): 1099-110, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22178999

RESUMO

Maintaining cell volume is critical for cellular function yet shift in cell volume is a prerequisite for mitosis and apoptosis. The ubiquitously and evolutionary conserved serine/threonine kinase CK2 promotes cell survival and suppresses apoptosis. The present review describes how mammalian cells regulate the cellular content of the major cellular organic osmolyte, taurine with emphasis on CK2 mediated regulation of active taurine uptake and volume-sensitive taurine release. Furthermore, we discuss how CK2-mediated regulation of taurine homeostasis is potentially involved in cellular functions such as proliferation and survival.


Assuntos
Caseína Quinase II/metabolismo , Taurina/metabolismo , Animais , Tamanho Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Amino Acids ; 40(4): 1091-106, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20827495

RESUMO

Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume is a critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na(+) dependent transporter TauT and taurine release are increased and reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA(2) activity, exposure to exogenous reactive oxygen species as well as inhibition of protein tyrosine phosphotases (PTP) potentiate the swelling-induced taurine loss. Inhibition of PI3K and PTEN reduces and potentiates swelling-induced taurine release, respectively. Inhibition of CK2 has no effect on PLA(2) activity and ROS production by NADPH oxidase, whereas it lifts the effect of PTEN and PTP inhibition. It is suggested that CK2 regulates the taurine release downstream to known swelling-induced signal transducers including PLA(2), NADPH oxidase and PI3K.


Assuntos
Caseína Quinase II/metabolismo , Fibroblastos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Taurina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Carcinoma de Ehrlich/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Tamanho Celular/efeitos dos fármacos , Expressão Gênica , Homeostase , Cinética , Camundongos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Células NIH 3T3 , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Equilíbrio Hidroeletrolítico/efeitos dos fármacos
8.
Nat Commun ; 9(1): 1032, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29531223

RESUMO

The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.


Assuntos
Axônios/metabolismo , Ácido Glutâmico/metabolismo , Isquemia/metabolismo , Bainha de Mielina/metabolismo , Animais , Vesículas Citoplasmáticas , Feminino , Humanos , Isquemia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo
9.
FEBS Lett ; 588(8): 1446-57, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24503060

RESUMO

Connexin 43 (Cx43) hemichannels may form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules and presumably a range of smaller physiologically relevant molecules. However, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of biological molecules smaller than ethidium, such as glutamate, lactate, and glucose, did not permeate the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was observed in oocytes expressing Cx30. Exposure to divalent cation free solutions induced a robust membrane conductance in Cx30-expressing oocytes but none in Cx43-expressing oocytes. C-terminally truncated Cx43 (M257) displayed increased dye uptake and, unlike wild type Cx43 channels, conducted current. Neither Cx30 nor Cx43 acted as water channels in their hemichannel configuration. Our results demonstrate that connexin hemichannels have isoform-specific permeability profiles and that dye uptake cannot be equaled to permeability of smaller physiologically relevant molecules in given settings.


Assuntos
Potenciais de Ação , Permeabilidade da Membrana Celular , Conexina 43/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Conexina 30 , Junções Comunicantes/fisiologia , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Ácido Láctico/metabolismo , Potenciais da Membrana , Camundongos , Ratos , Água/metabolismo , Xenopus
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