Mario Nobile

ATP-induced, sustained calcium signalling in cultured rat cortical astrocytes: evidence for a non-capacitative, P2X7-like-mediated calcium entry

The receptor mechanisms regulating the ATP‐induced free cytosolic Ca2+ concentration ([Ca2+]i) changes in cultured rat cortical type‐1 astrocytes were analyzed using fura‐2‐based Ca2+ imaging microscopy. Upon prolonged ATP challenge (1–100 μM), astroglial cells displayed a biphasic [Ca2+]i response consisting of an initial peak followed by a sustained elevation. Suramin and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid blocked both components, albeit to a different extent. By contrast, the selective P2X7 antagonist oxidized ATP irreversibly abrogated the sustained [Ca2+]i signal without affecting the transient phase. Finally, astrocyte challenge with the selective P2X7 agonist 3′‐O‐(4‐benzoyl)benzoyl‐ATP evoked a sustained [Ca2+]i elevation, which occluded that induced by ATP. We can conclude that in cultured cortical astrocytes the ATP‐mediated sustained [Ca2+]i rise does not implicate capacitative Ca2+ entry but involves Ca2+ influx through P2X7‐like receptors.

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 ≥ 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. GLIA 21:217–227, 1997.

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization.

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 μmol/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.

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release.

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X7 receptors, it is still unclear whether neuronal functions can be attributed to P2X7 receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X7 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 P2X7 receptors. Together, our findings suggest that (i) P2X7 receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X7 receptors are localized on glutamatergic nerve terminals; (iii) P2X7 receptors play a significant role in ATP‐evoked glutamate efflux, which involves Ca2+‐dependent vesicular release; and (iv) the P2X7 receptor itself constitutes a significant Ca2+‐independent mode of exit for glutamate.

Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia.

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. They also provide a possible mechanistic clue for the neuroprotective effect of CBX in vivo.

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

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 P2X7R 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.

Differential modulation of ATP‐induced calcium signalling by A1 and A2 adenosine receptors in cultured cortical astrocytes

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.

Potentiation of Native and Recombinant P2X7-Mediated Calcium Signaling by Arachidonic Acid in Cultured Cortical Astrocytes and Human Embryonic Kidney 293 Cells

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 [Ca2+]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 [Ca2+]i elevation is coupled to depletion of intracellular Ca2+ stores and to a sustained noncapacitative Ca2+ entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated [Ca2+]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 [Ca2+]i elevation. Fluorescent dye uptake experiments showed that the AA-induced increase in [Ca2+]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 [Ca2+]i elevation, whereas in those expressing the P2X7 receptor, AA elicited a potentiation of the BzATP-induced [Ca2+]i rise. Together, these findings indicate that AA mediates a complex regulation of [Ca2+]i dynamics also through P2X7-mediated Ca2+ entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of [Ca2+]i signaling 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 (I Clh), 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 I Clh is mediated by a small‐conductance Cl− channel, which has the same structural motif as the Cl− channel prototype ClC‐0.

Arachidonic acid activates an open rectifier potassium channel in cultured rat cortical astrocytes.

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 μM 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.