RESUMEN

Use of null mutant mice is a powerful way to evaluate the role of specific proteins in brain function. Studies performed on knockout mice have revealed some unexpected roles of the gap junction proteins (connexins). Thus, analyses of gene expression in connexin43 (Cx43) null brains indicated that deletion of a single gene (Gja1) induced expression level change of numerous other genes located on all chromosomes and involved in a wide diversity of functional pathways. The significant overlap between alterations in gene expression level, control and coordination in Cx43 knockout and knockdown astrocytes raised the possibility that Gja1 represents a transcriptomic node of gene regulatory networks. However, conditional deletion of Gja1 in astrocytes of two mouse strains resulted in remarkably different phenotypes. In order to evaluate the influence of the genetic background on the transcriptome, we performed microarray studies on brains of GFAP-Cre:Cx43(f/f) C57Bl/6 and 129/SvEv mice. The surprisingly low number of Cx43 core genes (regulated in all Cx43 nulls regardless of strain) and the high number of differently regulated genes in the two Cx43 conditional knockouts indicate high influence of mouse strain on brain transcriptome. This article is part of a Special Issue entitled Electrical Synapses.

Asunto(s)

Encéfalo/crecimiento & desarrollo , Conexina 43/genética , Conexina 43/fisiología , Transcriptoma/genética , Transcriptoma/fisiología , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Biomarcadores , Western Blotting , Células Cultivadas , Cromosomas/genética , Cromosomas/ultraestructura , Interpretación Estadística de Datos , Regulación hacia Abajo , Perfilación de la Expresión Génica , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Especificidad de la Especie

RESUMEN

Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 microM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.

Asunto(s)

Permeabilidad de la Membrana Celular , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Purinérgicos P2/metabolismo , Transducción de Señal , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Carbenoxolona/farmacología , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Conexinas/efectos de los fármacos , Conexinas/genética , Relación Dosis-Respuesta a Droga , Ácido Flufenámico/farmacología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/enzimología , Humanos , Macrófagos/metabolismo , Mefloquina/farmacología , Potenciales de la Membrana , Ratones , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Oocitos , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Transducción de Señal/efectos de los fármacos , Xenopus , Familia-src Quinasas/metabolismo

RESUMEN

Microglia, the tissue macrophages of the central nervous system (CNS), intimately interact with neurons physically and through soluble factors that can affect microglial activation state and neuronal survival and physiology. We report here a new mechanism of interaction between these cells, provided by the formation of gap junctions composed of connexin (Cx) 36. Among eight Cxs tested, expression of Cx36 mRNA and protein was found in microglial cultures prepared from human and mouse, and Cx45 mRNA was found in mouse microglial cultures. Electrophysiological measurements found coupling between one-third of human or mouse microglial pairs that averaged below 30 pico-Siemens and displayed electrical properties consistent with Cx36 gap junctions. Importantly, similar frequency of low-strength electrical coupling was also obtained between microglia and neurons in cocultures prepared from neocortical or hippocampal rodent tissue. Lucifer yellow dye coupling between neurons and microglia was observed in 4% of pairs tested, consistent with the low strength and incidence of electrical coupling. Cx36 expression level and/or the degree of coupling between microglia did not significantly change in the presence of activating agents, including lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha, except for some reduction of Cx36 protein when exposed to the latter two agents. Our findings that intercellular coupling occurs between neuronal and microglial populations through Cx36 gap junctions have potentially important implications for normal neural physiology and microglial responses in neuronopathology in the mammalian CNS.

Asunto(s)

Comunicación Celular/fisiología , Conexinas/metabolismo , Microglía/metabolismo , Neuronas/metabolismo , Telencéfalo/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Técnicas de Cocultivo , Conexinas/genética , Conexinas/ultraestructura , Encefalitis/metabolismo , Encefalitis/fisiopatología , Colorantes Fluorescentes , Gliosis/metabolismo , Gliosis/fisiopatología , Humanos , Mediadores de Inflamación/farmacología , Isoquinolinas , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Microglía/ultraestructura , Neuronas/ultraestructura , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Telencéfalo/citología , Proteína delta-6 de Union Comunicante

RESUMEN

Astrocytes express gap junction proteins and multiple types of P2Y receptors (P2YRs) that contribute to the propagation of intercellular Ca(2+) waves (ICW). To gain access to the role played by gap junctional communication in ICW propagation generated by P2YR activation, we selectively expressed P2Y(1,2,4)R subtypes and Cx43 in the human 1321N1 astrocytoma cell line, which lacks endogenous P2 receptors. Fluorescence recovery after photobleaching revealed that 1321N1 cells are poorly dye-coupled and do not propagate ICW. Forced expression of Cx43 in 1321N1 cells (which did not show functional hemichannels) increased dye coupling and allowed short-range ICW transmission that was mainly mediated by intercellular diffusion of Ca(2+) generated in the stimulated cells. Astrocytoma clones expressing each of the P2YR subtypes were also able to propagate ICWs that were likely dependent on IP(3) generation. These waves exhibited properties particular to each P2YR subtype. Co-expression of eGFP-hCx43 and P2Y(1)R modified the properties of P2Y(1)R-generated ICW to those characteristics of P2Y(2)R. Increased coupling in P2Y(4)R clones induced by expression of eGFP-hCx43 abolished the ICWs observed in uncoupled P2Y(4)R clones. No changes in the behavior of ICWs generated in P2Y(2)R clones were observed after forced expression of Cx43. These data indicate that in 1321N1 cells gap junctional communication provides intercellular integration of Ca(2+) signals generated by P2YR activation, thus coordinating the propagation of intercellular calcium waves.

Asunto(s)

Astrocitos/metabolismo , Señalización del Calcio/genética , Membrana Celular/metabolismo , Uniones Comunicantes/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Astrocitoma , Calcio/metabolismo , Comunicación Celular/genética , Línea Celular Tumoral , Células Clonales/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Difusión , Recuperación de Fluorescencia tras Fotoblanqueo , Colorantes Fluorescentes , Uniones Comunicantes/efectos de los fármacos , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Líquido Intracelular/metabolismo , Ratones , Ratas , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y2

RESUMEN

Astrocytes are coupled to one another by gap junction channels that allow the diffusion of ions and small molecules throughout the interconnected syncytium. In astrocytes, gap junctions are believed to participate in spatial buffering removing the focal excess of potassium resultant from intense neuronal activity by current loops through the syncytium and are also implicated in the propagation of astrocytic calcium waves, a form of extraneuronal signaling. Gap junctions can be modulated by several factors, including elevation of extracellular potassium concentration. Because K(+) elevation is a component of spinal cord injury, we evaluated the extent to which cultured spinal cord astrocytes is affected by K(+) levels and obtained evidence suggesting that a Ca(2+)-calmodulin (CaM) protein kinase is involved in the K(+)-induced increased coupling. Exposure of astrocytes to high K(+) solutions induced a dose-dependent increase in dye coupling; such increased coupling was greatly attenuated by reducing extracellular Ca(2+) concentration, prevented by nifedipine, and potentiated by Bay-K-8644. These results indicate that K(+)-induced increased coupling is mediated by a signaling pathway that is dependent on the influx of Ca(2+) through L-type Ca(2+) channels. Evidence supporting the participation of the CaM kinase pathway on K(+)-induced increased coupling was obtained in experiments showing that calmidazolium and KN-93 totally prevented the increase in dye and electrical coupling induced by high K(+) solutions. Because no changes in connexin43 expression levels or distribution were observed in astrocytes exposed to high K(+) solutions, we propose that the increased junctional communication is related to an increased number of active channels within gap junction plaques.

Asunto(s)

Astrocitos/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Comunicación Celular/efectos de los fármacos , Uniones Comunicantes/metabolismo , Potasio/farmacología , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Calmodulina/antagonistas & inhibidores , Células Cultivadas , Conexina 43/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/metabolismo , Colorantes Fluorescentes , Uniones Comunicantes/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Transducción de Señal/efectos de los fármacos , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo

Asunto(s)

Bronquios/efectos de los fármacos , Citocinas/farmacología , Uniones Comunicantes/efectos de los fármacos , Bronquios/citología , Bronquios/metabolismo , Comunicación Celular/efectos de los fármacos , Conexinas/genética , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Colorantes Fluorescentes/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Isoquinolinas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

We investigated the possible role of ion channels and transporters in cell volume control using Aplysia brasiliana ventricular tissues exposed to a 26% hyposmotic shock, by assessing changes in wet weight, intracellular water and ionic contents. Thirty minutes after the shock, the wet weight of isolated ventricles increase about 20% above control levels and then attain near original weight within 60 min after the shock. At the time when the wet weight returned to control values, intracellular water and KCl contents are decreased by 22 and 20%, respectively. The K(+) channel blockers, 4-AP and TEA, but not the cotransport blockers, hydrochlorothiazide and furosemide, greatly affect the magnitude of wet weight gain and the time course of weight recovery, indicating that KCl loss occur through conductive pathways. Intracellular recordings performed on ventricular myocytes during exposure to the osmotic shock showed an immediate membrane hyperpolarization and blockade of spontaneous electrical activity; diastolic membrane potential recover over time and spontaneous action potentials are completely restored 60 min after the hyposmotic shock. Because significant weight loss is observed during the exposure of ventricular tissues to 26% hypo-ionic, but isosmotic saline, it is suggested that ventricular volume restoration is accomplished by two distinct but simultaneously occurring processes: a volume-dependent and a volume-independent mechanism. Because wet weight restoration is completely prevented by exposing ventricular tissue to a Ca(2+)-free hyposmotic solution, we postulate that both processes involved in A. brasiliana ventricular weight restoration are Ca(2+)-dependent mechanisms.

Asunto(s)

Aplysia/fisiología , Presión Osmótica , Función Ventricular , 4-Aminopiridina/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Electrofisiología , Furosemida/farmacología , Ventrículos Cardíacos/anatomía & histología , Ventrículos Cardíacos/efectos de los fármacos , Hidroclorotiazida/farmacología , Técnicas In Vitro , Canales Iónicos/metabolismo , Tamaño de los Órganos , Potasio/metabolismo , Sodio/metabolismo , Tetraetilamonio/farmacología

RESUMEN

The gap junction protein connexin43 (Cx43) is the primary component of intercellular channels in cardiac tissue and in astrocytes, the most abundant type of glial cells in the brain. Mice in which the gene for Cx43 is deleted by homologous recombination die at birth, due to profound hypertrophy of the ventricular outflow tract and stenosis of the pulmonary artery. Despite this significant cardiovascular abnormality, brains of connexin43 null [Cx43 (-/-)] animals are shown to be macroscopically normal and to display a pattern of cortical lamination that is not detectably different from wildtype siblings. Presence of Cx40 and Cx45 in brains and astrocytes cultured from both Cx43 (-/-) mice and wildtype littermates was confirmed by RT-PCR, Northern blot analyses and by immunostaining; Cx46 was detected by RT-PCR and Northern blot analyses. Presence of Cx26 in astrocyte cultures was indicated by RT-PCR and by Western blot analysis, although we were unable to resolve whether it was contributed by contaminating cells; Cx30 mRNA was detected by Northern blot in long term (2 weeks) but not fresh cultures of astrocytes. These studies thus reveal that astrocyte gap junctions may be formed of multiple connexins. Presumably, the metabolic and ionic coupling provided by these diverse gap junction types may functionally compensate for the absence of the major astrocyte gap junction protein in Cx43 (-/-) mice, providing whatever intercellular signaling is necessary for brain development and cortical lamination.

Asunto(s)

Astrocitos/fisiología , Conexina 43/genética , Uniones Comunicantes/fisiología , Animales , Elementos sin Sentido (Genética) , Astrocitos/química , Astrocitos/citología , Northern Blotting , Southern Blotting , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Química Encefálica/genética , Comunicación Celular/fisiología , Células Cultivadas , Conexina 43/análisis , Conductividad Eléctrica , Femenino , Uniones Comunicantes/química , Regulación del Desarrollo de la Expresión Génica , Genotipo , Proteína Ácida Fibrilar de la Glía/análisis , Heterocigoto , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/fisiología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Voltage-dependent anion channels (VDACs) are pore-forming proteins (porins) that form the major pathway for movement of adenine nucleotides through the outer mitochondrial membrane. Electrophysiological studies indicate that VDAC-like channel activity is also prevalent in the cell membranes of many mammalian cells. However, the multitopological localization of porins outside the mitochondrion has remained an extremely controversial issue. Herein, we show that usage of two alternative first exons of the murine VDAC-1 gene leads to expression of two porins differing within their N termini. One porin (plasmalemmal VDAC-1) harboring a hydrophobic leader peptide is primarily targeted through the Golgi apparatus to the cell membrane. In contrast, the second isoform lacking the N-terminal leader (mitochondrial VDAC-1) is translocated more efficiently into the outer mitochondrial membrane. Thus, our data provide unique genetic evidence in favor of a multitopological localization of a mitochondrial porin.

Asunto(s)

Porinas/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Secuencia de Bases , Células COS , ADN Complementario , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Células PC12 , Técnicas de Placa-Clamp , Porinas/genética , Isoformas de Proteínas/genética , ARN Mensajero/genética , Ratas , Fracciones Subcelulares/metabolismo , Canal Aniónico 1 Dependiente del Voltaje , Canales Aniónicos Dependientes del Voltaje

RESUMEN

Electrophysiological properties of gap junction channels and mechanisms involved in the propagation of intercellular calcium waves were studied in cultured spinal cord astrocytes from sibling wild-type (WT) and connexin43 (Cx43) knock-out (KO) mice. Comparison of the strength of coupling between pairs of WT and Cx43 KO spinal cord astrocytes indicates that two-thirds of total coupling is attributable to channels formed by Cx43, with other connexins contributing the remaining one-third of junctional conductance. Although such a difference in junctional conductance was expected to result in the reduced diffusion of signaling molecules through the Cx43 KO spinal cord syncytium, intercellular calcium waves were found to propagate with the same velocity and amplitude and to the same number of cells as between WT astrocytes. Measurements of calcium wave propagation in the presence of purinoceptor blockers indicate that calcium waves in Cx43 KO spinal cord astrocytes are mediated primarily by extracellular diffusion of ATP; measurements of responses to purinoceptor agonists revealed that the functional P2Y receptor subtype is shifted in the Cx43 KO astrocytes, with a markedly potentiated response to ATP and UTP. Thus, the reduction in gap junctional communication in Cx43 KO astrocytes leads to an increase in autocrine communication, which is a consequence of a functional switch in the P2Y nucleotide receptor subtype. Intercellular communication via calcium waves therefore is sustained in Cx43 null mice by a finely tuned interaction between gap junction-dependent and independent mechanisms.

Asunto(s)

Astrocitos/fisiología , Calcio/fisiología , Conexina 43/fisiología , Uniones Comunicantes/fisiología , Receptores Purinérgicos P2/fisiología , Médula Espinal/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Conexina 43/deficiencia , Conexina 43/genética , Uniones Comunicantes/efectos de los fármacos , Heptanol/farmacología , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Suramina/farmacología , Uridina Trifosfato/farmacología

RESUMEN

It has become evident that astrocytes play major roles in central nervous system (CNS) function. Because they are endowed with ion channels, transport pathways, and enzymatic intermediates optimized for ionic uptake, degradation of metabolic products, and inactivation of numerous substances, they are able to sense and correct for changes in neural microenvironment. Besides this housekeeping role, astrocytes modulate neuronal activity either by direct communication through gap junctions or through the release of neurotransmitters and/or nucleotides affecting nearby receptors. One prominent mode by which astrocytes regulate their own activity and influence neuronal behavior is via Ca2+ signals, which may be restricted within one cell or be transmitted throughout the interconnected syncytium through the propagation of intercellular calcium waves. This review aims to outline the most recent advances regarding the active communication of astrocytes that is encoded by intracellular calcium variation.

Asunto(s)

Adenosina Difosfato Ribosa/análogos & derivados , Astrocitos/fisiología , Señalización del Calcio/fisiología , Adenosina Difosfato Ribosa/fisiología , Adenosina Trifosfato/fisiología , Animales , Calcineurina/fisiología , Calcio/metabolismo , Canales de Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Comunicación Celular , Conexinas/fisiología , ADP-Ribosa Cíclica , Espacio Extracelular/metabolismo , Uniones Comunicantes/fisiología , Células Gigantes/metabolismo , Ácido Glutámico/fisiología , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Interleucina-1/farmacología , Transporte Iónico , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Neurotransmisores/farmacología , Neurotransmisores/fisiología , Fosfatidilinositol Diacilglicerol-Liasa , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2Y2 , Fosfolipasas de Tipo C/fisiología

RESUMEN

In mammalian astrocytes, calcium waves are transmitted between cells via both a gap junction-mediated pathway and an extracellular, P2 receptor-mediated pathway, which link the cells into a syncytium. Calcium waves in astrocytes have also been shown to evoke calcium transients in neurons, and activity in neurons can elicit calcium waves in astrocytes. In this study, we show that in primary human fetal astrocytes, the P2 receptor-mediated and gap junction-mediated pathways are differentially regulated by the cytokine IL-1beta. Confocal microscopy of astrocytes loaded with Indo-1 demonstrated that intercellular calcium wave transmission in IL-1beta-treated cultures was potentiated compared with controls. However, transmission of calcium waves via the gap junction-mediated pathway was strikingly reduced. The major component of functional gap junctions in human fetal astrocytes was demonstrated to be connexin43 (Cx43), and there was a marked reduction of junctional conductance, loss of dye coupling, loss of Cx43 protein, and down-regulation of Cx43 mRNA expression after IL-1beta treatment of cultures. Conversely, transmission of calcium waves via the P2 receptor-mediated pathway was potentiated in IL-1beta-treated cultures compared with controls. This potentiation was associated with an increase in the number of cells responsive to UTP, and with a transient increase in expression of the P2Y(2) purinoceptor mRNA. Because in inflammatory conditions of the human central nervous system IL-1beta is produced both by resident glia and by invading cells of the immune system, our results suggest that inflammatory events may have a significant impact on coordination of astrocytic function and on information processing in the central nervous system.

Asunto(s)

Astrocitos/metabolismo , Calcio/metabolismo , Uniones Comunicantes/fisiología , Interleucina-1/farmacología , Receptores Purinérgicos P2/fisiología , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Conexina 43/genética , Feto/metabolismo , Humanos , ARN Mensajero/análisis , Receptores Purinérgicos P2/genética , Uridina Trifosfato/farmacología

Asunto(s)

Uniones Comunicantes/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Neuroglía/fisiología , Animales , Moléculas de Adhesión Celular/fisiología , Humanos , Plasticidad Neuronal

RESUMEN

Gap junctions are regarded as the primary pathway underlying propagation of Ca2+ waves between astrocytes, although signaling through extracellular space may also contribute. Results obtained from astrocytes cultured from sibling Cx43 knockout (KO) and wild-type (WT) mice in six litters showed that Ca2+ waves propagated more slowly in Cx43 KO than in WT astrocytes; however, because this difference in velocity was only seen in conditions where cell confluence was higher in WT than KO astrocytes, it is attributable to differences in plating density. By contrast, density-independent differences were observed in the amplitudes of the Ca2+ responses (15% smaller in KO astrocytes) and efficacy of spread (to 14% fewer cells in KO astrocytes). Blockade of purinergic receptors with suramin reduced the velocities of the waves by 40% in WT and KO astrocytes and reduced the amplitudes by 20% and 6%, respectively. In the presence of heptanol, Ca2+ waves spread to only 30% of the cells, with a 70% reduced velocity and 30% reduced amplitude. It is concluded that the propagation of Ca2+ waves between astrocytes from Cx43 KO mice is not so greatly affected as expected by deletion of the major gap junction protein between these cells. The residual 5% coupling contributed by the additional connexins (Cx40, Cx45, and Cx46) expressed in KO astrocytes still suffices to provide a more substantial portion of Ca2+ wave propagation than does signaling through extracellular purinergic pathways. These studies demonstrate that, even with severely reduced junctional conductance, Cx43 KO astrocytes are capable of performing long-range Ca2+ wave signaling, perhaps preserving one mechanism critical to neural function.

Asunto(s)

Astrocitos/metabolismo , Calcio/metabolismo , Conexina 43/genética , Ratones Noqueados/genética , Ratones Noqueados/metabolismo , Animales , Astrocitos/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Espacio Extracelular/fisiología , Heptanol/farmacología , Ratones , Valores de Referencia , Transducción de Señal/fisiología , Suramina/farmacología

RESUMEN

When exposed to 20% and 35%, but not to 50% hyposmotic solutions, mouse astrocytes recovered their volume within a few minutes, which coincided with the activation of nonjunctional conductances. Conductance of gap junctions between astrocyte pairs also increased after exposure to a 35% hyposmotic shock; however, this effect began at 3 min after the shock, when cells had partially recovered their initial volumes. During the first minute of exposure to 20% and 35% hyposmotic stimuli, there was a transient monophasic increase in intracellular calcium levels; exposure to 50% hyposmotic solution led to intracellular Ca2+ oscillations. The differences in time courses of nonjunctional conductance changes, Ca2+ alterations, and intercellular coupling suggest that distinct second messenger pathways are involved in each response. The velocity of mechanically evoked calcium waves propagated among the astrocytes increased at 7.5 min after 35% hyposmotic shock. This increase was not seen with 20% or 50% hyposmotic stimuli and is not ascribable to the increase in junctional conductance because it was blocked by suramin, a P2 purinergic receptor antagonist. Given that the transduction pathways activated during cell swelling (e.g., generation of phospholipases, phosphokinases, arachidonic acid) exert inhibitory effects on astrocytic gap junctions (Giaume and McCarthy, 1996), it is proposed that the increased junctional conductance during hyposmotic shock is due to increased number of channels, perhaps triggered by the initial Ca2+ signals (Dolmetsch et al., 1997). As a functional consequence of the increased coupling and enhanced extracellular propagation of Ca2+ waves, spread of signaling molecules throughout the glial network is expected to be significantly enhanced during hyposmotic stress. The increased intercellular communication between mouse astrocytes in response to hyposmotic challenge thus occurs via both gap junction-dependent and -independent mechanisms and presumably provides neuroprotective effects following nervous system injury.

Asunto(s)

Astrocitos/fisiología , Comunicación Celular/fisiología , Espacio Extracelular/fisiología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Calcio/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Conductividad Eléctrica , Ratones , Ratones Endogámicos , Concentración Osmolar , Estimulación Química , Factores de Tiempo

RESUMEN

The exposure of Aplysia brasiliana to dilute seawater (90 and 80%) caused an increase of the relative weight, which returned to the original values after a few hours. Both osmotic and chloride concentrations of the hemolymph decreased on exposure to 80 and 90% dilute seawater, and after 3-h exposure there were no differences between the hemolymph and external media osmotic and chloride concentrations. In contrast to the clear regulatory capabilities reported for A. californica, A. brasiliana cannot maintain the osmolality of its body fluid in dilute media. In A. californica, osphradial receptors and neuron R15 are apparently involved in this regulatory mechanism. Perfusion of osphradium of A. brasiliana with dilute seawater (95-80%) did not affect electrical activity of the bursting neuron R15; perfusion with 70 and 60% seawater caused a transient increase in the duration of the quiescent period. In contrast to the model established for A. californica, in A. brasiliana no relationship was found between exposure of the osphradium to dilute media and electrical activity in neuron R15, which is in accordance with the lack of an osmoregulatory mechanism in this species. Such differences may reflect inherent differences in salinity tolerance between the two species.

Asunto(s)

Aplysia/fisiología , Neuronas/fisiología , Equilibrio Hidroelectrolítico , Animales , Cloruros/metabolismo , Electrofisiología , Ganglios/citología , Hemolinfa/fisiología , Cinética , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/fisiología , Concentración Osmolar , Agua de Mar

RESUMEN

The ultrastructure of the radial neuromuscular system of the trachymedusa Liriope tetraphylla was examined to determine the morphological substrate underlying crumpling behavior--the folding of the margin into the subumbrellar cavity by radial muscle contraction. These contractions are produced by the four smooth muscle bands that run the length of the peduncle and extend over the subumbrellar surface to the margin, along the radii. Axons are present in the radial system and attain appreciable density at the base of insertion of the peduncle; contact between this radial nerve net and the inner nerve ring may occur at the margin. Gap junctions were not encountered within the ectodermal radial system. These various observations are discussed with respect to the control of crumpling in this and other species of hydromedusae.

RESUMEN

1. In order to evaluate how crumpling behavior influences the swimming system in Liriope tetraphylla, extracellular recordings of muscle action potentials related to both behaviors were performed. 2. The swimming pattern of Liriope consists of irregular bursts of muscle potentials. A correlation was obtained between number of muscle potentials and burst length and between burst and interburst periods. The swimming pattern could be predicted from the burst length since a long burst was followed by a short period of quiescence and by a burst as long as the preceding one. 3. The addition of atropine to induce radial muscle contraction first caused an increase in swimming activity and then an irreversible reduction. The enhancement of swimming activity was due to the increase of burst length and of the frequency of swimming bursts (due to the decrease of the interburst period), and the reduction of swimming activity, to the enhancement of the interburst period and to the reduction of the burst length. 4. It is proposed that, when facing a noxious stimulus, Liriope will first "escape" from it by enhancing its swimming activity and, if the stimulus persists, blockade of the pacemaker system becomes accentuated as the radial muscle contractions become more sustained.

Asunto(s)

Atropina/farmacología , Conducta Animal/efectos de los fármacos , Cnidarios/fisiología , Contracción Muscular/efectos de los fármacos , Escifozoos/fisiología , Animales , Electrofisiología , Natación

RESUMEN

1. In order to evaluate how crumpling behavior influences the swimming system in Liriope tetraphylla, extracellular recordidngs of muscle action potentials related to both behaviors were performed. 2. The swimming pattern of Liriope consists of irregular bursts of muscle potentials. A correlation was obtained between number of muscle potentials and burst lenght and between burst and interburst periods. The swimming pattern could be predicted from the burst length since a long burst was followed by a short period of quiescence and by a burst as long as the preceding one. 3. The addition of atropine to induce radial muscle contraction first caused an increase in swimming activity and then an irreversible reduction. The enhancement of swimming activity was due to the increase of burst length and of the frequency of swimming bursts (due to the decrease of the interburst period), and the reduction of swimming activity, to the enhacement of the intervurst period and to the reduction of the burst length. 4. It is proposed that, when facing a noxious stimulus, Liriope will first "escape" from it by enhancing its swimming activity and, if the stimulus persists, blockade of the pacemaker system becomes accentuated as the radial muscle contractions becomes more sustained

Asunto(s)

Animales , Atropina/farmacología , Conducta Animal/efectos de los fármacos , Cnidarios/fisiología , Contracción Muscular/efectos de los fármacos , Escifozoos/fisiología , Electrofisiología , Natación

RESUMEN

Crude whole body homogenates of Liriope tetraphylla exhibit a cholinesterase particularly active on acetylthiocholine but not on butyrylthiocholine. The acetylthiocholine hydrolysis is completely blocked by neostigmine. The Michaelis-Menten constant for acetylthiocholine is 0.14 mM. The pharmacological analysis of the responses to the choline esters nicotine and atropine suggests the involvement in Liriope tetraphylla of a cholinergic mechanism in the pointing reflex. Butyrylcholine, nicotine and atropine (but not muscarinic agonists) caused the contraction of the subumbrellar radial muscles. The effects of atropine were dose-dependent and were depressed in competition with muscarinic agonists. MgCl2 interfered with the action of atropine. The results were explained by suggesting the existence, at least at the neuromuscular junction, of excitatory (nicotinic) and inhibitory (muscarinic) pre-synaptic receptors modulating the release of the (unknown) transmitter acting post-synaptically.

Asunto(s)

Hydra/fisiología , Receptores Colinérgicos/fisiología , Animales , Atropina/farmacología , Colinesterasas/metabolismo , Hydra/enzimología , Transmisión Sináptica , Factores de Tiempo