The inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes.

BACKGROUND AND PURPOSE: The ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis. EXPERIMENTAL APPROACH: Experiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells. KEY RESULTS: In cultured astrocytes, DCPIB promoted the activation of a K(+) conductance mediated by two-pore-domain K(+) (K(2P) ) channels. The DCPIB effect occluded that of arachidonic acid, which activates K(2P) channels K(2P) 2.1 (TREK-1) and K(2P) 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K(2P) 2.1 and K(2P) 10.1 proteins. Moreover, DCPIB opened recombinant K(2P) 2.1 and K(2P) 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K(+) conductance in hippocampal astrocytes, but caused an increment in basal K(+) current of neurons. CONCLUSION AND IMPLICATIONS: Our results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K(+) conductance in astroglia and neuronal cells.

Water transport between CNS compartments: functional and molecular interactions between aquaporins and ion channels.

The physiological ability of the mammalian CNS to integrate peripheral stimuli and to convey information to the body is tightly regulated by its capacity to preserve the ion composition and volume of the perineuronal milieu. It is well known that astroglial syncytium plays a crucial role in such process by controlling the homeostasis of ions and water through the selective transmembrane movement of inorganic and organic molecules and the equilibration of osmotic gradients. Astrocytes, in fact, by contacting neurons and cells lining the fluid-filled compartments, are in a strategic position to fulfill this role. They are endowed with ion and water channel proteins that are localized in specific plasma membrane domains facing diverse liquid spaces. Recent data in rodents have demonstrated that the precise dynamics of the astroglia-mediated homeostatic regulation of the CNS is dependent on the interactions between water channels and ion channels, and their anchoring with proteins that allow the formation of macromolecular complexes in specific cellular domains. Interplay can occur with or without direct molecular interactions suggesting the existence of different regulatory mechanisms. The importance of molecular and functional interactions is pinpointed by the numerous observations that as consequence of pathological insults leading to the derangement of ion and volume homeostasis the cell surface expression and/or polarized localization of these proteins is perturbed. Here, we critically discuss the experimental evidence concerning: (1) molecular and functional interplay of aquaporin 4, the major aquaporin protein in astroglial cells, with potassium and gap-junctional channels that are involved in extracellular potassium buffering. (2) the interactions of aquaporin 4 with chloride and calcium channels regulating cell volume homeostasis. The relevance of the crosstalk between water channels and ion channels in the pathogenesis of astroglia-related acute and chronic diseases of the CNS is also briefly discussed.

The endocannabinoid anandamide inhibits potassium conductance in rat cortical astrocytes.

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.

Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes.

Cell-cell communication in astroglial syncytia is mediated by intracellular Ca(2+) ([Ca(2+)](i)) responses elicited by extracellular signaling molecules as well as by diverse physical and chemical stimuli. Despite the evidence that astrocytic swelling promotes [Ca(2+)](i) elevation through Ca(2+) influx, the molecular identity of the channel protein underlying this response is still elusive. Here we report that primary cultured cortical astrocytes express the transient receptor potential vanilloid-related channel 4 (TRPV 4), a Ca(2+)-permeable cation channel gated by a variety of stimuli, including cell swelling. Immunoblot and confocal microscopy analyses confirmed the presence of the channel protein and its localization in the plasma membrane. TRPV4 was functional because the selective TRPV4 agonist 4-alpha-phorbol 12,13-didecanoate (4alphaPDD) activated an outwardly rectifying cation current with biophysical and pharmacological properties that overlapped those of recombinant human TRPV4 expressed in COS cells. Moreover, 4alphaPDD and hypotonic challenge promoted [Ca(2+)](i) elevation mediated by influx of extracellular Ca(2+). This effect was abolished by low micromolar concentration of the TRPV4 inhibitor Ruthenium Red. Immunofluorescence and immunogold electron microscopy of rat brain revealed that TRPV4 was enriched in astrocytic processes of the superficial layers of the neocortex and in astrocyte end feet facing pia and blood vessels. Collectively, these data indicate that cultured cortical astroglia express functional TRPV4 channels. They also demonstrate that TRPV4 is particularly abundant in astrocytic membranes at the interface between brain and extracerebral liquid spaces. Consistent with its roles in other tissues, these results support the view that TRPV4 might participate in astroglial osmosensation and thus play a key role in brain volume homeostasis.

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance.

We show that dysregulation of the Cl- homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl- conductance were early (<1 h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl- channel blocker phloretin inhibited both the staurosporine-evoked Cl- current and AVD, and preserved cell viability. Prolonged incubation (>2 h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl- channel activity and subsequent AVD, but is independent of intracellular acidification.

Staurosporine induces apoptotic volume decrease (AVD) in ECV304 cells.

Incubation of ECV304 cells with 1 micro M staurosporine (STS) causes apoptotic cell death. In the present study, we investigate whether a significant apoptotic volume decrease (AVD) was apparent during the very early times (1 h) of the apoptotic process. Our data suggest that upregulation of Cl(-) (and possibly K(+)) channels by STS may be a very early primary event required for the subsequent onset of AVD, which results in apoptosis.

Glia-related pathomechanisms in Alzheimer's disease: a therapeutic target?

Reactive glial cell properties could contribute to pathomechanisms underlying Alzheimer's disease by favoring oxidative neuronal damage and beta-amyloid toxicity. A critical step is apparently reached when pathological glia activation is no longer restricted to microglia and includes astrocytes. By giving up their differentiated state, astrocytes may lose their physiological negative feed-back control on microglial NO production and even contribute to neurotoxic peroxynitrate formation. Another consequence is the impairment of the astrocyte-maintained extracellular ion homeostasis favoring excitotoxic damage. By the production of apolipoprotein-E, triggered by the microglial cytokine interleukine-1beta, reactive astrocytes could promote the transformation of beta-amyloid into the toxic form. A pharmacologically reinforced cAMP signaling in rat glial cell cultures depressed oxygen radical formation in microglia and their release of TNF-alpha and interleukine-1beta, feed-forward signals which mediate oxidative damage and secondary astrocyte activation. Cyclic AMP also favored differentiation and expression of a mature ion channel pattern in astrocytes improving their glutamate buffering. A deficient cholinergic signaling that increases the risk of pathological APP processing was compensated by an adenosine-mediated reinforcement of the second messenger calcium. A combination therapy with acetylcholine-esterase inhibitors together with adenosine raising pharmaca, therefore, may be used to treat cholinergic deficiency in Alzheimer's disease.

Osmosensitivity of an inwardly rectifying chloride current revealed by whole-cell and perforated-patch recordings in cultured rat cortical astrocytes.

The osmosensitivity of the inwardly rectifying Cl(-) current (I(Clh)), expressed by primary cultured rat neocortical astrocytes long-term treated with dibutyryl cyclic AMP, was investigated in the whole-cell and perforated-patch modes. In whole-cell experiments, whereas hypotonic extracellular solution (Delta=100 mOsmol) did not cause any change in I(Clh), hypertonicity produced a slowly developing, approximately 40% reversible decrease in current magnitude. By contrast, in perforated-patch experiments, exposure to a less hypertonic saline (Delta=50 mOsmol) depressed the current to approximately 50%, and hypotonicity induced a approximately 50% slow increase in I(Clh). These differences in osmosensitivity between the two experimental modes suggest that the osmoregulation of I(Clh) may be mediated by complex intracellular mechanism(s), which appear(s) to be partly compromised by the dialysis of the astrocytic cytoplasm.

Structural compatibility between the putative voltage sensor of voltage-gated K+ channels and the prokaryotic KcsA channel.

Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.

pH modulation of an inward rectifier chloride current in cultured rat cortical astrocytes.

The effects of changes in extra- and intracellular pH in the pathophysiological range (6.0-8.0) on astroglial plasma membrane ionic currents were investigated with the whole-cell patch-clamp technique. In cultured rat neocortical type-1 astrocytes differentiated by a long-term treatment with dibutyryl cyclic-AMP, exposure to an extracellular pH of 6.4 induced, as compared with the control extracellular pH at 7.3, a sustained and reversible increase in the holding current at -60mV. The rise in current was accompanied by a decrease in the apparent input resistance. Ion substitution experiments indicated that extracellular pH 6.4 upregulated the resting Cl(-) conductance, whereas an opposite effect could be observed at extracellular pH 8.0. Recordings of isolated Cl(-) currents showed that this modulation occurred on the previously identified hyperpolarization-activated, inwardly rectifying Cl(-) current, I(Clh). Extracellular acidification to pH 6.4 shifted the voltage dependence of I(Clh) activation by approximately 20mV towards more positive potentials, whereas a approximately 20mV opposite shift was observed upon exposure to extracellular pH 8.0. These effects were paralleled by an increase (extracellular pH 6.4) or decrease (extracellular pH 8.0) in the maximal conductance. Decreasing (6.0) or increasing (8.0) the intracellular pH shifted the steady-state activation of I(Clh) towards more negative or positive potentials, respectively, leaving unchanged the current sensitivity to extracellular pH modifications. The modulation of the inward rectifier Cl(-) current expressed by differentiated cultured neocortical astrocytes indicates that extra- and intracellular changes in pH occurring in a pathophysiological range may contribute to regulating Cl(-) accumulation in astroglial cells.

Single-channel analysis of a ClC-2-like chloride conductance in cultured rat cortical astrocytes.

The single-channel behavior of the hyperpolarization-activated, ClC-2-like inwardly rectifying Cl- current (IClh), induced by long-term dibutyryl-cyclic-AMP-treated cultured cortical rat astrocytes, was analyzed with the patch-clamp technique. In outside-out patches in symmetrical 144 mM Cl-solutions, openings of hyperpolarization-activated small-conductance Cl channels revealed burst activity of two equidistant conductance levels of 3 and 6 pS. The unitary openings displayed slow activation kinetics. The probabilities of the closed and conducting states were consistent with a double-barrelled structure of the channel protein. These results suggest that the astrocytic ClC-2-like Cl- current Iclh is mediated by a small-conductance Cl channel, which has the same structural motif as the Cl- channel prototype CIC-0.

Cascading glia reactions: a common pathomechanism and its differentiated control by cyclic nucleotide signaling.

A pathological glia activation, stimulated by inflammatory proteins, beta-amyloid, or brain ischemia, is discussed as a common pathogenic factor for progressive nerve cell damage in vascular and Alzheimer dementia. A critical point seems to be reached, if the cytokine-controlled microglial upregulation causes a secondary activation of astrocytes which loose the negative feedback control, are forced to give up their physiological buffering function, and may add to neuronal damage by the release of nitric oxide (NO) and by promoting toxic beta-amyloid formation. A strengthening of the cyclic adenosine-5',3'-monophosphate (cAMP) signaling exerted a differential inhibition of the stimulatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) released from cultured rat microglia, but maintained the negative feedback signal IL-6; cAMP inhibited also the release of free oxygen radicals (OR) but not of NO. Reinforcement of the NO-induced cyclic guanosine monophosphate (cGMP) increase by blockade of the phosphodiesterase (PDE) subtype-5 with propentofylline counterbalanced the toxic NO action that causes with OR neuronal damage by peroxynitrate formation. In rat cultured astrocytes, a prolonged cAMP elevation favored cell differentiation, the expression of a mature ion channel patter, and an improvement of the extracellular glutamate uptake. Cyclic AMP signaling could be strengthened by PDE blockade and by raising extracellular adenosine, which stimulates A2 receptor-mediated cAMP synthesis. Via an A1 receptor-mediated effect, elevated adenosine was found to overcome a deficient intracellular calcium mobilization resulting from an impaired muscarinic signaling at pathologically decreased acetylcholine concentrations. We suggest that pharmaca, which elevate extracellular adenosine and/or block the degradation of cyclic nucleotides, may be used to counteract glia-related neuronal damage in dementing processes.

Pathological immuno-reactions of glial cells in Alzheimer's disease and possible sites of interference.

A significant role of a pathological glial cell activation in the pathogenesis of Alzheimer's disease is supported by the growing evidence that inflammatory proteins, which are produced by reactive astrocytes, promote the transformation of diffuse beta-amyloid deposits into the filamentous, neurotoxic form. A number of vicious circles, driven by the release of TNF-a and free oxygen radicals from microglial cells, may cause an upregulated microglial activation and their production of interleukin-1 which triggers, secondarily, the crucial activation of astrocytes. Reactive functional changes of glial cells seem to be controlled by an altered balance of the second messengers Ca2+ and cAMP and can be counterregulated by the endogenous cell modulator adenosine which strengthens the cAMP-dependent signalling chain. A further reinforcement of the homeostatic adenosine effects on glial cells by pharmaca, such as propentofylline, may add to neuroprotection in Alzheimer's disease.

Protective mechanisms of adenosine in neurons and glial cells.

As illustrated in Figure 1, a disturbance of the intracellular Ca2+ homeostasis is thought to be a common pathogenic factor for the generation of secondary nerve cell damage that develops after brain trauma or stroke or during the course of neurodegenerative diseases. A neuronal Ca2+ overload which may result from an excessive glutamate-evoked membrane depolarization and consecutive Ca2+ influx as well as from an activation of metabotropic receptors and consecutive intracellular Ca2+ mobilization is known to have direct toxic effects on the cytoskeleton and the cell metabolism of neurons. In addition, a Ca(2+)-dependent activation of glial cells along with the loss of physiologically required mature astrocyte functions and with the acquisition of potentially neurotoxic microglial properties, has more recently been recognized as an additive pathogenic factor. This may provide an effective target for pharmacological interference. Specifically, the reinforcement of an endogenous homeostatic regulator, which obtained its sophisticated know-how during evolution, may provide a neuroprotective therapy which can handle the complexity of the pathological process with a minor risk of pharmacological side effects. Adenosine is such an ancient molecular signal that acts on both neurons and glial cells. In neurons, adenosine activates K+ and Cl- conductances, which limits synaptically evoked depolarization, thus counteracting the Ca2+ influx through voltage-dependent and NMDA receptor-operated ion channels. This A1 receptor-mediated effect seems to be the major action by which adenosine adds directly to the protection of neurons against Ca(2+)-dependent damage. In glial cells, the prevalent effect of adenosine is its regulatory influence on the Ca2+ and cAMP-dependent molecular signaling that determines the cellular proliferation rate, the differentiation state and related functions. When mimicking the activation of metabotropic glutamate receptors in cultures of immature rat astrocytes, which largely resemble pathologically activated astrocytes, a transient Ca2+ mobilization was initiated by adenosine. This A1 receptor-mediated Ca2+ signal caused a prolonged potentiation of the A2 receptor-mediated intracellular cAMP rise. An experimentally sustained enhancement of the cAMP signaling initiated the differentiation of cultured astrocytes and the new expression of K+ and Cl- channels which are required for the physiological astrocyte function to maintain the extracellular ion homeostasis. Evidence is accumulating that a strengthening of the cAMP signaling, which can be achieved by adenosine agonists and also by the pharmacon propentofylline (an adenosine uptake blocker and phosphodiesterase inhibitor), stimulates the mRNA production of neurotrophic factors in astrocytes. In cultured microglial cells, several days' treatment with adenosine agonists or propentofylline markedly inhibited their proliferation rate, the in vitro spontaneously occurring transformation into macrophages and their particularly high formation of free oxygen radicals. Adenosine agonists also depressed the release of the potentially toxic cytokine TNF alpha and induced programmed cell death in immunologically activated microglial cells. We conclude that a pharmacological reinforcement of the endogenous cell modulator adenosine may provide neuroprotection by counteracting neuronal Ca2+ overload, by depressing potentially neurotoxic microglial functions and by regaining physiologically required properties of differentiated astrocytes. Further information about the influence of adenosine on the molecular signaling and on ischemic brain damage is given in Refs. 37 and 38, and about the implicated possible relevance for the treatment of stroke in Ref. 39.

Support of homeostatic glial cell signaling: a novel therapeutic approach by propentofylline.

A pathological glial cell activation, which forces microglia to transform into immunocompetent cells with cytotoxic properties and astrocytes to "de-differentiate," presumably adds to neurodegenerative diseases. We examined the modulatory effect of adenosine on the Ca2+ and cAMP-dependent regulation of such reactive glial cell properties in culture and tested possibilities of pharmacologic reinforcement. A strengthening of the cAMP-signaling, as could be achieved by adenosine agonists via a Ca(2+)-dependent action, favored the differentiation of proliferating astrocytes and associated neuroprotective properties (ion homeostasis, formation of trophic factors). But potentially neurotoxic properties of microglial cells were inhibited. Adenosine depressed their proliferation rate and transformation into macrophages, their particularly high formation of reactive oxygen intermediates and the release of the cytokine TNF-alpha. Similar effects were obtained with propentofylline, which acts as selective cAMP/cGMP phosphodiesterase inhibitor and also increases the effective concentration of adenosine by blocking its cellular reuptake. The recently observed induction of microglial apoptosis by elevated extracellular adenosine levels may further contribute to limit secondary nerve cell damage related to a pathological glial cell activation.

Characterization of an inwardly rectifying chloride conductance expressed by cultured rat cortical astrocytes.

The biophysical and pharmacological properties of the inwardly rectifying Cl- conductance (IClh), expressed in rat type-1 neocortical cultured astrocytes upon a long-term treatment (1-3 weeks) with dibutyryl-cyclic-AMP (dBcAMP), were investigated with the whole-cell patch-clamp technique. Using intra- and extra-cellular solutions with symmetrical high Cl- content and with the monovalent cations replaced with N-methyl-D-glucamine, time- and voltage-dependent Cl- currents were elicited in response to hyperpolarizing voltage steps from a holding potential of 0 mV. The inward currents activated slowly and did not display any time-dependent inactivation. The rising phase of the current traces was best fitted with two exponential components whose time constants decreased with larger hyperpolarization. The steady-state activation of IClh was well described by a single Boltzmann function with a half-maximal activation potential at - 62 mV and a slope of 19 mV that yields to an apparent gating charge of 1.3. The anion selectivity sequence was Cl- = Br- = I- > F- > cyclamate > or = gluconate. External application of the putative Cl- channel blockers 4,4 diisothiocyanatostilbene-2,2 disulphonic acid or 4-acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid did not affect IClh. By contrast, anthracene-9-carboxylic acid, as well as Cd2+ and Zn2+, inhibited, albeit with different potencies, the Cl- current. Taken together, these results indicate that dBcAMP-treated cultured rat cortical astrocytes express a Cl- inward rectifier, which exhibits similar but not identical features compared with those of the cloned and heterologously expressed hyperpolarization-activated Cl- channel ClC-2.

Modulation of glial cell signaling by adenosine and pharmacological reinforcement. A neuroprotective strategy?

In view of the increasing evidence that a pathological glial activation plays a significant role in the development of neurodegenerative diseases, we investigated the underlying molecular signaling as a possible target for the pharmacological therapy. Here, we are particularly focusing on the endogenous modulation of the CA2+ and cyclic nucleotide-dependent signaling by the nucleoside adenosine and its reinforcement by the xanthine derivative propentofylline (PPF). As an experimental model, we used cultured rat microglial cells and astrocytes that are immature, show a high proliferation rate, and resemble in several aspects pathologically activated glial cells. A prolonged increase of the cellular cAMP level favored the differentiation of cultured astrocytes and associated properties required for the physiological nerve cell function. On the other hand a strengthening of the cyclic nucleotide-dependent signaling inhibited potentially neurotoxic properties of cultured microglial cells. Similar effects were obtained by treatment with propentofylline, which mimicked modulatory adenosine effects and increased the intracellular level of cAMP and cGMP. Such a pharmacological glial cell conditioning, obtained by modifying the strength and the timing of these second messengers, may provide a therapy of neurodegenerative diseases in which a pathological activation of microglial cells and astrocytes is discussed to playa pathogenic role.

Two distinct inwardly rectifying conductances are expressed in long term dibutyryl-cyclic-AMP treated rat cultured cortical astrocytes.

Long term incubation (1-3 weeks) with 250 microM dibutyryl-cyclic-AMP (dBcAMP) of pure cultured cortical astrocytes from newborn rats leads to the expression of voltage-dependent, inward-rectifying potassium (K+) and chloride (Cl-) currents which are lacking in shortly treated (4-24 h) and in control cultured astrocytes. Both conductances are already activated at the holding potential of -60 mV and are distinguishable for their gating kinetics and pharmacological sensitivity. K+ currents have a fast activation kinetic and show a time- and voltage-dependent inactivation at potentials negative to -120 mV. The conductive property of the K+ currents increases upon elevation of the extracellular K+ concentration ([K+]o) and they are reversibly blocked by extracellular 0.1 mM barium ions (Ba2+). Cl- currents are activated only at negative membrane potentials; they display a slow activation kinetic, no time-dependent inactivation and are not affected by 0.1 mM Ba2+. In individual astrocyte the K+ and Cl- conductances can be expressed singularly or in combination. The results indicate that the expression of these two conductances is controlled by a cAMP-dependent molecular signalling, presumably by regulating a late gene activation. Thus, the strengthening of this signalling would contribute to promote the maturation of less differentiated astrocytes in culture, implicating the expression of K+ and Cl- membrane conductances which may operate together in the regulation of [K+]o homeostasis via the mechanism of the local accumulation.

GTP- and GDP-analogues modulate an inwardly rectifying chloride channel in cultured hippocampal neurons.

Three different GABA-insensitive Cl- channels could be resolved in cultured hippocampal neurons using the inside-out patch clamp configuration. The most commonly observed channel revealed an inward rectification with a chord conductance of 40 pS in symmetrical Cl- solutions at a membrane potential of -50 mV and had voltage sensitive gating kinetics. Channel openings were not observed in cell-attached patch, and after excision, several minutes of perfusion of the cytoplasmic side were required before detecting the first openings. The open state probability was increased by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S 10(-4) M) and reduced by guanosine 5'-O-(2-thiophosphate) (GDP-beta-S 10(-4) M) suggesting its regulation by G proteins. This new identified chloride channel may account for the previously described voltage-sensitive, inward-rectifying whole cell Cl- current which was enhanced by adenosine in a pertussis toxin-sensitive manner.