[Show abstract][Hide abstract] ABSTRACT: The P2X7 receptor (P2X7R) induces ionotropic Ca²⁺ signalling in different cell types. It plays an important role in the immune response and in the nervous system. Here, the mechanisms underlying intracellular Ca²⁺ variations evoked by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a potent agonist of the P2X7R, in transfected HEK293 cells, are investigated both experimentally and theoretically. We propose a minimal model of P2X7R that is capable of reproducing, qualitatively and quantitatively, the experimental data. This approach was also adopted for the P2X7R variant, which lacks the entire C-terminus tail (trP2X7R). Then we introduce a biophysical model describing the Ca²⁺ dynamics in HEK293. Our model gives an account of the ionotropic Ca²⁺ influx evoked by BzATP on the basis of the kinetics model of P2X7R. To explain the complex Ca²⁺ responses evoked by BzATP, the model predicted that an impairment in Ca²⁺ extrusion flux through the plasma membrane is a key factor for Ca²⁺ homeostasis in HEK293 cells.
[Show abstract][Hide abstract] ABSTRACT: We previously observed that activation of presynaptic P2X7 receptors located on rat cerebrocortical nerve terminals induced the release of glutamate through different modes: the channel conformation allowing Ca(2+) entry triggered exocytotic release, while the receptor itself functioned as a permeation pathway for the non-exocytotic glutamate release. Considering that exocytotic and non-exocytotic glutamate release evoked by the activation of P2X7 receptors might play a role in the control of glutamatergic synapses, we investigated whether calmidazolium (which has been found to inhibit small cation currents through recombinant P2X7 receptors, but not organic molecule permeation) could distinguish between P2X7-related exocytotic and non-exocytotic modes of glutamate release. We found that calmidazolium inhibited the intrasynaptosomal Ca(2+) response to P2X7 receptor activation and the Ca(2+)-dependent exocytotic glutamate release from rat cerebrocortical nerve terminals, but was ineffective against the Ca(2+)-independent glutamate release. The P2X7 competitive antagonist A-438079 eliminated both exocytotic and non-exocytotic P2X7 receptor-evoked glutamate release. Selective inhibition of exocytotic glutamate release indicates that calmidazolium inhibits events dependent on the function of native rat P2X7 receptors as Ca(2+) channels, and suggests that it can be used as a tool to dissociate P2X7-evoked exocytotic from non-exocytotic glutamate release.
Full-text · Article · Mar 2012 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: Activation of the oxidative burst and failure of CD4(+) CD25(+) cell regulation have been implicated in idiopathic nephrotic syndrome (iNS). The intimate mechanism is, however, unknown and requires specifically focused studies. We investigated reactive oxygen species (ROS) generation [di-chlorofluorescein-diacetate (DCFDA)] fluorescence assay and the regulatory adenosine 5'-triphosphate (ATP) pathways in the blood of 41 children with iNS, utilizing several agonists and antagonists of nucleotide/nucleoside receptors, including the addition of soluble apyrase. The CD4(+) CD25(+) CD39(+) /CD73(+) expression was determined in vivo in parallel during disease activity. Overall, we found that the percentage of CD39(+) CD4(+) CD25(+) was reduced markedly in iNS by 80% (3·43±0·04% versus 13·14±0·07% of total lymphocytes, P<0·001). In these patients, reactive oxygen species (ROS) generation by polymorphonuclear neutrophils (PMN) at rest was a function of apyrase (CD39) expressed by CD4(+) CD25(+) , with higher rates in patients with very low CD39(+) CD4(+) CD25(+) levels (<7·5%). Addition of apyrase reduced ROS generation by 40% in both iNS and controls and was mainly effective in patients. The quota of ROS surviving ATP elimination was higher still in iNS. In vitro studies to limit ROS generation with adenosine analogues (2'-chloroadenosine and 5'-N-ethylcarboxamidoadenosine) produced minor effects. At variance, antagonizing ATP efflux with carbenoxolone or by antagonizing ATP effects (Brilliant Blue G, KN62 and A437089) reduced ROS generation comparable to apyrase. These results confirm a key role of ATP in the regulation of innate immunity and minimize the effect of adenosine. Decreased CD39(+) CD4(+) CD25(+) expression in iNS highlights an impairment of ATP degradation in this pathology. However, high ROS surviving ATP consumption implies a major role of other regulatory pathways.
[Show abstract][Hide abstract] ABSTRACT: The role of P2 receptors for purines/pyrimidines is not well characterized in neuroblastoma, although a variety of purinergic mRNAs/proteins are expressed in these cells. Among these, the P2Y(6) receptor is the only subtype distinguished by UDP-specific activation. In this work, after over-expressing the P2Y(6) protein in human neuroblastoma SH-SY5Y cells, we find that UDP arrests cell cycle and induces apoptosis, by counteracting the pathological functioning of neuroblastoma in vitro. UDP also causes mitochondrial damage through diffusion of cytochrome c in the cytoplasm, and stimulates caspase-3,7,8 activities, with extensive over-expression of manganese superoxide dismutase. Our data establish the direct toxic role and anti-cancer activity of UDP in a neuroblastoma cell line, and identify the P2Y(6) receptor as a novel potential target in anti-tumoural therapies. This constitutes an advancement not only in the knowledge of purinergic signalling, but also in the biological and pathological aspects of neuroblastoma in vitro.
Full-text · Article · Feb 2010 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: J. Neurochem. (2010) 113, 796–806.
The P2X7 receptor (P2X7R) is an ATP-gated cation channel whose biophysical properties remain to be unravelled unequivocally. Its activity is modulated by divalent cations and organic messengers such as arachidonic acid (AA). In this study, we analysed the differential modulation of magnesium (Mg2+) and AA on P2X7R by measuring whole-cell currents and intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) dynamics in HEK293 cells stably expressing full-length P2X7R and in cells endowed with the P2X7R variant lacking the entire C-terminus tail (trP2X7R), which is thought to control the pore activation. AA induced a robust potentiation of the P2X7R- and trP2X7R-mediated [Ca2+]i rise but did not affect the ionic currents in both conditions. Extracellular Mg2+ reduced the P2X7R- and trP2X7R-mediated [Ca2+]i rise in a dose-dependent manner through a competitive mechanism. The modulation of the magnitude of the P2X7R-mediated ionic current and [Na+]i rise were strongly dependent on Mg2+ concentration but occurred in a non-competitive manner. In contrast, in cells expressing the trP2X7R, the small ionic currents and [Na+]i signals were totally insensitive to Mg2+. Collectively, these results support the tenet of a functional structure of P2X7R possessing at least two distinct conductive pathways one for Ca2+ and another for monovalent ions, with the latter which depends on the presence of the receptor C-terminus.
Full-text · Article · Feb 2010 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC, thereby providing a possible mechanistic clue for the neuroprotective effect of CBX in vivo.
Full-text · Article · Oct 2009 · Channels (Austin, Tex.)
[Show abstract][Hide abstract] ABSTRACT: Endocannabinoids are a family of endogenous signaling molecules that modulate neuronal excitability in the central nervous system (CNS) by interacting with cannabinoid (CB) receptors. In spite of the evidence that astroglial cells also possess CB receptors, there is no information on the role of endocannabinoids in regulating CNS function through the modulation of ion channel-mediated homeostatic mechanisms in astroglial cells. We provide electrophysiological evidence that the two brain endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) markedly depress outward conductance mediated by delayed outward rectifier potassium current (IK(DR)) in primary cultured rat cortical astrocytes. Pharmacological experiments suggest that the effect of AEA does not result from the activation of known CB receptors. Moreover, neither the production of AEA metabolites nor variations in free cytosolic calcium are involved in the negative modulation of IK(DR). We show that the action of AEA is mediated by its interaction with the extracellular leaflet of the plasma membrane. Similar experiments performed in situ in cortical slices indicate that AEA downregulates IK(DR) in complex and passive astroglial cells. Moreover, IK(DR) is also inhibited by AEA in NG2 glia. Collectively, these results support the notion that endocannabinoids may exert their modulation of CNS function via the regulation of homeostatic function of the astroglial syncytium mediated by ion channel activity.
[Show abstract][Hide abstract] ABSTRACT: Glial subcellular particles (gliosomes) have been purified from rat cerebral cortex or mouse spinal cord and investigated for their ability to release glutamate. Confocal microscopy showed that gliosomes are enriched with glia-specific proteins, such as GFAP and S-100 but not neuronal proteins, such as PSD-95, MAP-2, and beta-tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin-alphaM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin, high KCl, veratrine, 4-aminopyridine, AMPA, or ATP by mechanisms involving extracellular Ca2+, Ca2+ release from intracellular stores as well as reversal of glutamate transporters. In addition, gliosomes can release glutamate also by a mechanism involving heterologous transporter activation (heterotransporters) located on glutamate-releasing and glutamate transporter-expressing (homotransporters) gliosomes. This glutamate release involves reversal of glutamate transporters and anion channel opening, but not exocytosis. Both the exocytotic and the heterotransporter-mediated glutamate release were more abundant in gliosomes prepared from the spinal cord of transgenic mice, model of amyotrophic lateral sclerosis, than in controls; suggesting the involvement of astrocytic glutamate release in the excitotoxicity proposed as a cause of motor neuron degeneration. The results support the view that gliosomes may represent a viable preparation that allows to study mechanisms of astrocytic transmitter release and its regulation in healthy animals and in animal models of brain diseases.
Full-text · Article · Feb 2009 · International Review of Neurobiology
[Show abstract][Hide abstract] ABSTRACT: The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+-dependent vesicular glutamate release and significant Ca2+-independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X(7)R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+](i) signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R-mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals.
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence indicates that astroglial syncytium plays key role in normal and pathological brain functions. Astrocytes both in vitro and in situ respond to extracellular adenine-based nucleotides via the activation of P2 receptors. Massive release of ATP from neurons and glial cells occurs as a result of pathological conditions of the brain leading to neuroinflammation and involving P2X7 receptors. In this study, we investigated whether P2X7 stimulation on cultured cortical astrocytes promoted a differential activation of mitogen-activated protein kinases (MAPKs), and whether the second messenger arachidonic acid (AA), which is also a key modulator of neuroinflammation, affected the P2X7-mediated MAPK phosphorylation. The results show that the synthetic P2X7 receptor agonist 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP), induced a concentration-dependent phosphorylation of MAPK ERK1/2, JNK and p38. Stimulation of ERK1/2, JNK and p38 phosphorylation was also obtained by pathophysiological levels of extracellularly applied AA. Interestingly, a robust potentiation of ERK1/2 phosphorylation was elicited by co-application of BzATP and AA, whereas no differences were observed in JNK or p38 phosphosignals. The kinases activation showed a differential dependence on the presence of extracellular Ca(2+). The potentiation of BzATP-mediated ERK1/2 phosphorylation was also observed in human embryonic kidney cells (HEK293) stably transfected with rat P2X7, but not in HEK cells expressing truncated P2X7 receptor lacking the full cytoplasmic carboxy-terminal or in those carrying the structurally related rat P2X2. AA and BzATP synergism in ERK1/2 activation was abolished by cyclo-oxygenase and lipoxygenase pathway inhibitors. The result that ERK1/2-mediated transduction pathway is synergistically modulated by ATP and AA signalling depicts possible novel pharmacological targets for interfering with pathological activation of astroglial cells.
Full-text · Article · Oct 2008 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X(7) receptors, it is still unclear whether neuronal functions can be attributed to P2X(7) receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X(7) receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non-neuronal cells and allows exposure of 'nude' release-regulating pre-synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X(7) receptors. Together, our findings suggest that (i) P2X(7) receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X(7) receptors are localized on glutamatergic nerve terminals; (iii) P2X(7) receptors play a significant role in ATP-evoked glutamate efflux, which involves Ca(2+)-dependent vesicular release; and (iv) the P2X(7) receptor itself constitutes a significant Ca(2+)-independent mode of exit for glutamate.
Full-text · Article · Apr 2008 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.
Full-text · Article · Dec 2007 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Starting from the experimental data on ATP evoked calcium responses in astrocytes, a biophysical model describing these phenomena was built. The simulations showed, in agreement with the experimental findings, that the intracellular calcium fluxes mediated by the P2X and P2Y purinoreceptors are responsible for the biphasic ATP evoked calcium response in astrocytes. Then, the modulation effects on the neural dynamics arising from the release of glutamate from astrocyte are also investigated. By using a minimal network model describing a neuron coupled to the astrocyte, we demonstrated that the calcium extrusion rate through the astrocyte membrane is critically involved in the generation of different firing patterns of the neuron.
[Show abstract][Hide abstract] ABSTRACT: Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long-term effects on both neurones and astroglial cells. Whether Guo also modulates the activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 microm) promotes the functional expression of an inward rectifier K+ (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up-regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo-induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo-induced up-regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that down-regulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K+ homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K+-buffering impairment of the astroglial syncytium that occurs in pathological conditions.
Full-text · Article · Aug 2006 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: In the brain, arachidonic acid (AA) plays a critical role in the modulation of a broad spectrum of biological responses, including those underlying neuroinflammation. By using microfluorometry, we investigated the action of extracellular AA in the modulation of the purinoceptor P2X7-mediated elevation of [Ca(2+)](i) in cultured neocortical type-1 astrocytes and P2X7-, P2X2-transfected human embryonic kidney (HEK) 293 cells. We report that in cultured astrocytes, AA-induced [Ca(2+)](i) elevation is coupled to depletion of intracellular Ca(2+) stores and to a sustained noncapacitative Ca(2+) entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated [Ca(2+)](i) rise evoked by the selective agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP). Pharmacological studies demonstrate that the selective P2X7 antagonists oxidized ATP and Brilliant Blue G abrogated the AA-mediated potentiation of BzATP-evoked [Ca(2+)](i) elevation. Fluorescent dye uptake experiments showed that the AA-induced increase in [Ca(2+)](i) was not due to a switch of the P2X7 receptor from channel to the pore mode of gating. The synergistic effect of AA and BzATP was also observed in HEK293 cells stably expressing rat and human P2X7 but not in rat P2X2. Control HEK293 cells responded to AA exposure only with a transient [Ca(2+)](i) elevation, whereas in those expressing the P2X7 receptor, AA elicited a potentiation of the BzATP-induced [Ca(2+)](i) rise. Together, these findings indicate that AA mediates a complex regulation of [Ca(2+)](i) dynamics also through P2X7-mediated Ca(2+) entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of [Ca(2+)](i) signaling in astroglial cells.
[Show abstract][Hide abstract] ABSTRACT: Despite the accumulating evidence that under various pathological conditions the extracellular elevation of adenine-based nucleotides and nucleosides plays a key role in the control of astroglial reactivity, how these signalling molecules interact in the regulation of astrocyte function is still largely elusive.
The action of the nucleoside adenosine in the modulation of the intracellular calcium signalling ([Ca2+]i) elicited by adenosine 5′-triphosphate (ATP)-induced activation of P2 purinoceptors was investigated on neocortical type-1 astrocytes in primary culture by using single-cell microfluorimetry.
Astrocyte challenge with ATP (1–10 μM) elicited biphasic [Ca2+]i responses consisting of an initial peak followed by a sustained elevation. The stable adenosine analogue 2-chloroadenosine (2-ClA) potentiated the transient [Ca2+]i rise induced by activation of metabotropic P2Y receptors. Among the various P1 receptor agonists tested, the nonselective agonist 5′-N-ethylcarboxamidoadenosine (NECA) mimicked the 2-ClA action, whereas the selective A1 R(−) N6-(2-phenylisopropyl)-adenosine (R-PIA), the A2A 2-[4-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680) and A3 1-deoxy-1-(6-[([3-lodophenyl]methyl)-amino]-9H-purin-9-yl)-N-methyl-β-D-ribofuranuronamide (IB-MECA) agonists were ineffective.
Application of R-PIA>NECA2-ClA depressed the [Ca2+]i plateau reversibly. Moreover, in the presence of R-PIA or 2-ClA, the prolonged [Ca2+]i signal was maintained by application of the A1 antagonist 1,3-diethyl-8-phenylxanthine (DPX). Finally, preincubation of the astrocytes with pertussis toxin abrogated the 2-ClA inhibition of the ATP-elicited sustained [Ca2+]i rise without affecting the transient [Ca2+]i potentiation.
Taken together, these findings indicate that stimulation of A1 and A2 adenosine receptors mediates a differential modulation of [Ca2+]i signalling elicited by P2 purinoceptors. Since variations in [Ca2+]i dynamics also affect cell proliferation and differentiation, our data suggest that tuning of the extracellular levels of adenosine may be relevant for the control of astrogliosis mediated by adenine nucleotides.
British Journal of Pharmacology (2004) 141, 935–942. doi:10.1038/sj.bjp.0705707
Full-text · Article · Apr 2004 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: A pathophysiological increase in free arachidonic acid (AA) is thought to regulate the channel-mediated astrocytic swelling occurring in several brain injuries. We report that in cultured rat type-1 cortical astrocytes, exposure to 10 microM AA activates an open rectifier K(+) channel, which exhibits many similarities with TREK/TRAAK members of the two-pore-domain K(+) channel family KCNK. Patch-clamp experiments showed that the current developed with a long latency and was preceded by a depression of the previously described outward rectifier K(+) conductance. Pharmacologic studies indicate that the K(+) open rectifier was differentially sensitive to classic K(+)-channel blockers (quinine, quinidine, tetraethylammonium, and barium) and was inhibited potently by gadolinium ions. The activation of this K(+) current occurred independently of the AA metabolism as pharmacologic inhibition of the lipoxygenase, cyclooxygenase, and cytochrome P450 epoxygenase signaling cascades did not alter the AA effect. Moreover, neither the neutralization of the NADPH-oxidase pathway nor scavenging intracellular free radicals modified the AA response. Finally, the AA-induced K(+) current was unaffected by protein kinase C inhibitors. The activation mechanism of the K(+) open rectifier was through an extracellular interaction of AA with the plasma membrane. RT-PCR analysis revealed that the AA-induced K(+) conductance was mediated likely by TREK-2 channels. Collectively, the results demonstrate that in cultured cortical astrocytes, pathological levels of AA directly activate an open rectifier K(+) channel, which may play a role in the control of K(+) homeostasis under pathophysiological conditions.
Full-text · Article · May 2003 · Journal of Neuroscience Research