RESUMO
Membrane currents of src-transformed NIH3T3 mouse fibroblasts were analyzed in comparison with their non-transformed counterparts using the patch-clamp technique. Normal NIH3T3 cells exhibit two types of Ca2+ currents and a membrane current of ohmic behaviour (current amplitude 135 pA at +30 mV) that can partially be blocked by Cd2+. Src-transformed NIH3T3 cells show an additional membrane current that becomes activated after the establishment of the whole-cell configuration with a maximum amplitude of 1040 pA at +30 mV within 30-60 s. This current then inactivates irreversibly within 5-10 min. The additional current is highly K(+)-selective and Ca(2+)-dependent but voltage-independent. It can be blocked by charybdotoxin (IC50 = 20 nM) and by internal tetraethylammonium (TEA; IC50 = 2.9 mM), but it is not sensitive to external TEA (up to 30 mM). Single-channel analysis revealed only one K+ channel type with a conductance of 37 pS at negative potentials and 18 pS at positive potentials (in symmetrical 145 mM K+ solutions), a voltage-independent open-state probability of 0.6 and the same pharmacological properties as the macroscopic KCa current. The properties of the KCa current and the underlying channels of src-transformed NIH3T3 cells are identical to those observed in ras-transformed NIH3T3 cells. In contrast, src- or ras-transformation affects differently the voltage-dependent, transient (T-type) Ca2+ current. While ras-transformation of NIH3T3 cells suppresses their T-type Ca2+ current, this current remains unchanged in src-transformed NIH3T3 cells.
Assuntos
Cálcio/química , Genes src , Potássio/química , Células 3T3 , Animais , Linhagem Celular Transformada , Membrana Celular/química , Eletroquímica , Genes ras , Canais Iônicos/química , Camundongos , Técnicas de Patch-ClampRESUMO
Gram-positive Streptococcus pneumoniae is the major pathogen causing lethal meningitis in adults. We used pneumococcal cell walls (PCW) to investigate microglial consequences of a bacterial challenge and to determine the role of serum in the activation process. PCW caused the characteristic induction of an outwardly rectifying K+ channel (IK+(OR)), together with a concomitant suppression of the constitutively expressed inward rectifier K+ current, and evoked the release of tumor necrosis factor-alpha (TNF alpha), interleukin-6 (IL-6), IL-12, KC, macrophage inflammatory protein (MIP) 1alpha and MIP-2. Serum presence strongly facilitated the PCW effects, similarly as observed for lipopolysaccharide (LPS) from gram-negative Escherichia coli. The inflammatory cytokine, interferon-gamma (IFNgamma) induced the same electrophysiological changes, but independent of serum. Recombinant LPS binding protein (LBP) could partially replace serum activity in LPS stimulations. In contrast, neither LBP nor an antibody-mediated blockade of the LPS receptor, CD14 had significant influences on PCW-inducible changes. Cell surface interactions and cofactor involvement in microglial activation by gram-positive bacteria are thus distinct from the mechanisms employed by LPS. Moreover, tyrphostin AG126, a protein kinase inhibitor that prevents activation of the mitogen-activated protein kinase, p42MAPK (ERK2), potently blocked the PCW-stimulated cytokine release while having only a limited effect on LPS-inducible cytokines. In contrast, AG126 did not influence IK+(OR) inductions. This indicates that PCW recruits more than 1 intracellular signaling pathway to trigger the various responses and that different bacterial agents signal through both common and individual routes during microglial activation.
Assuntos
Proteínas de Fase Aguda , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Glicoproteínas de Membrana , Microglia/microbiologia , Microglia/fisiologia , Animais , Animais Recém-Nascidos/metabolismo , Fenômenos Fisiológicos Sanguíneos , Proteínas de Transporte/farmacologia , Parede Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Sinergismo Farmacológico , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Interferon gama/farmacologia , Canais Iônicos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Proteínas Quinases/fisiologia , Proteínas Recombinantes , Streptococcus pneumoniae/fisiologiaRESUMO
Lipopolysaccharides derived from cell walls of Gram-negative bacteria have proven a useful tool to simulate bacterial infection of the central nervous system. Rapid activation of microglia within the brain parenchyma as well as in vitro has thereby been shown to be an early event upon bacterial or lipopolysaccharide challenges. Less is known about microglial responses to a contact with Gram-positive bacteria, such as Streptococcus pneumoniae, a lethal pathogen causing meningitis with a 30% mortality rate. In the present study, we compared lipopolysaccharide-induced microglial activation in vitro with that induced by preparations of pneumococcal cell walls. As a readout of microglial activation, we studied by patch-clamp recording the expression of outward rectifying potassium currents (IK+OR), which are known to be induced by lipopolysaccharide. We found that pneumococcal cell walls and lipopolysaccharide induced a similar type of IK+OR. Stimulation of IK+OR by pneumococcal cell walls and lipopolysaccharide involved protein synthesis since it was not induced in the presence of cycloheximide. Pharmacological characterization of the pneumococcal cell wall- and lipopolysaccharide-induced currents with specific ion channel blockers indicated for both cases expression of the charybdotoxin/margatoxin-sensitive Kv1.3 subtype of the Shaker family of voltage-dependent potassium channels. Activation of the outward currents by pneumococcal cell walls depended on the developmental stage: while lipopolysaccharide triggered IK+OR in both embryonal and postnatal microglial cells, pneumococcal cell walls had only a marginal effect on embryonal cells. This, however, does not imply that embryonic microglial cells are unresponsive to pneumococcal cell walls. In both embryonic and postnatal cells, (i) the amplitude of the constitutively expressed inward rectifying potassium current was significantly reduced, (ii) tumor necrosis factor-a was released and (iii) the cells changed their morphology, similarly as it was induced by lipopolysaccharide treatment. Thus, embryonic microglial cells are sensitive to pneumococcal cell wall challenges, but respond with a distinctly different pattern of physiological reactions. The expression of IK+OR could thus be a suitable tool to study signalling cascades selectively involved in the activation of microglia by Gram-negative and -positive cell wall components and to functionally distinguish between populations of microglial cells.
Assuntos
Encéfalo/fisiologia , Parede Celular/imunologia , Microglia/fisiologia , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/biossíntese , Streptococcus pneumoniae/imunologia , Animais , Animais Recém-Nascidos , Apamina/farmacologia , Encéfalo/citologia , Encéfalo/imunologia , Células Cultivadas , Charibdotoxina/farmacologia , Venenos Elapídicos/farmacologia , Embrião de Mamíferos , Canal de Potássio Kv1.3 , Lipopolissacarídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos , Microglia/citologia , Microglia/imunologia , Neurotoxinas/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio/fisiologia , Ratos , Venenos de Escorpião , Superfamília Shaker de Canais de PotássioRESUMO
BACKGROUND AND PURPOSE: Controlling vascular tone involves K(+) efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K(Ca)2.3 and K(Ca)3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling. EXPERIMENTAL APPROACH: Transgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K(Ca)2.3 and K(Ca)3.1 expression by immunohistochemistry and RT-PCR. K(Ca) currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K(Ca)3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry. KEY RESULTS: K(Ca)3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K(Ca)2.3 and K(Ca)3.1 mRNA expression. With 200 nM [Ca(2+)](i) , the K(Ca)2.1-2.3/K(Ca)3.1 opener NS309 increased whole-cell currents. CyPPA, a K(Ca)2.2/K(Ca)2.3 opener, was without effect. With 1 µM [Ca(2+)](i) , the K(Ca)3.1 inhibitor TRAM-34 reduced currents whereas apamin (K(Ca)2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K(Ca)3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K(Ca)3.1 and the large-conductance K(Ca) channel, K(Ca)1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%. CONCLUSION AND IMPLICATIONS: Astrocytes express functional K(Ca)3.1 channels, and these contribute to neurovascular coupling.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Astrócitos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica/métodos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
Infectious bursal disease virus (IBDV) is the causative agent of an economically significant poultry disease. IBDV infection leads to apoptosis in chicken embryos and cell cultures. Since changes in cellular ion fluxes during apoptosis have been reported, we investigated the membrane ion currents of chicken embryo fibroblasts (CEFs) inoculated with the Cu-1 strain of IBDV using the patch-clamp recording technique. Incubation of CEFs with IBDV led to marked changes in their K+ outward current properties, with respect to both the kinetics of activation and inactivation and the Ca2+ dependence of the activation. The changes occurred in a time-dependent manner and were complete after 8 h. UV-treated noninfectious virions induced the same K+ current changes as live IBDV. When CEFs were inoculated with IBDV after pretreatment with a neutralizing antibody, about 30% of the cells showed a normal K+ current, whereas the rest exhibited K+ current properties identical to or closely resembling those of IBDV-infected cells. Incubation of CEFs with culture supernatant from IBDV-infected cells from which the virus particles were removed had no influence on the K+ current. Our data strongly suggest that the K+ current changes induced by IBDV are not due to virus replication, but are the result of attachment and/or membrane penetration. Possibly, the altered K+ current may delay the apoptotic process in CEFs after IBDV infection.