Role of N‐ and L‐type calcium channels in depolarization‐induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals

Multiple types of voltage‐activated calcium (Ca²⁺) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca²⁺ influx through N‐ and L‐ type voltage‐activated Ca²⁺ channels in sympathetic neurons to the depolarization‐induced activation of tyrosine hydroxylase (TH), the rate‐limiting enzyme in norepinephrine (NE) synthesis, and the depolarization‐induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both ω‐conotoxin GVIA (1 μM), a specific blocker of N‐type channels, and nimodipine (1 μM), a specific blocker of L‐type Ca²⁺ channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K⁺, indicating that Ca²⁺ influx through both types of channels contributes to enzyme activation. In contrast, K⁺ stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by ω‐conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K⁺ stimulation of NE release from both ganglia and irises was also blocked completely when ω‐conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca²⁺ influx through N‐ and L‐type Ca²⁺ channels. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 137–148, 1999     

Characterization of ATP release from cultures enriched in cholinergic amacrine-like neurons.

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of alpha-amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca(2+) dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and omega-Agatoxin IVA, but not by omega-Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca(2+) influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca(2+) channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A(1) receptor antagonist, showing that the release is tonically inhibited by the adenosine A(1) receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation.     

Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A2A receptors in striatal projection neurons

D₁- and D₂-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A₁-type receptors are located in both neuron classes, and adenosine A_2A-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca²⁺-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D₁-type receptors increase, while D₂-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, Ca_V1 Ca²⁺-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A₁- and A_2A-receptors have not been compared observing their actions on Ca²⁺-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca²⁺-currents by A₁-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A₁- and A_2A-receptors. We demonstrate that A₁-type receptors reduced Ca²⁺-currents in all SPNs tested. However, A_2A-type receptors enhanced Ca²⁺-currents only in half tested neurons. Intriguingly, to observe the actions of A_2A-type receptors, occupation of A₁-type receptors had to occur first. However, A₁-receptors decreased Ca_V2 Ca²⁺-currents, while A_2A-type receptors enhanced current through Ca_V1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.     

Prostaglandin E2 stimulates glutamate receptor‐dependent astrocyte neuromodulation in cultured hippocampal cells

Recent Ca²⁺ imaging studies in cell culture and in situ have shown that Ca²⁺ elevations in astrocytes stimulate glutamate release and increase neuronal Ca²⁺ levels, and that this astrocyte‐neuron signaling can be stimulated by prostaglandin E₂ (PGE₂). We investigated the electrophysiological consequences of the PGE₂‐mediated astrocyte‐neuron signaling using whole‐cell recordings on cultured rat hippocampal cells. Focal application of PGE₂ to astrocytes evoked a Ca²⁺ elevation in the stimulated cell by mobilizing internal Ca²⁺ stores, which further propagated as a Ca²⁺ wave to neighboring astrocytes. Whole‐cell recordings from neurons revealed that PGE₂ evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca²⁺ wave and was mediated through both N‐methyl‐D ‐aspartate (NMDA) and non‐NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE₂‐evoked Ca²⁺ elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 221–229, 1999     

Lysophosphatidic acid‐induced Ca2+ mobilization in the neural retina of chick embryo

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca²⁺ concentration ([Ca²⁺]_i) with Fura‐2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1–100 μM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca²⁺]_i in a dose‐dependent manner, with an EC₅₀ value of 9.2 μM. The Ca²⁺ rise was also evoked in a Ca²⁺‐free medium, suggesting that release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) was induced by LPA. U‐73122, a blocker of phospholipase C (PLC), inhibited the Ca²⁺ rise to LPA. Pertussis toxin partially inhibited the Ca²⁺ rise to LPA, indicating that G_i/G_o protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca²⁺ rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca²⁺ response to LPA were the same as those of the Ca²⁺ response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca²⁺ rise in the E3 chick retina. Our results show that LPA induces Ca²⁺ mobilization in the embryonic chick retina during neurogenesis. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 495–504, 1999     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Role of Adenosine Receptors in Modulation of Synaptic Transmission

Mechanisms of the effect of adenosine on transmitter release were investigated. Exogenous adenosine reduced transmitter release via P1 receptors. This reduction was not affected by changes in the [Ca²⁺], but was abolished by a P1 blocker, theophylline, and Ba²⁺. The effects of Ba²⁺ ions may be due to the involvement of A_2areceptors. Blockers of the A-type (catechol and 4-aminopyridine) and BK-type (iberiotoxin) potassium channels enhanced transmitter release and abolished the effect of adenosine. The findings suggest a possibility of A_2a channels to be coupled to BK and A potassium channels.     

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca²⁺]_i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca²⁺]_i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca²⁺]_i that was dependent on an intracellular source of Ca²⁺, while prolonged activation of P2Y receptors induced a rise in [Ca²⁺]_i that was dependent on intra‐ and extracellular sources of Ca²⁺. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca²⁺ entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca²⁺]_i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca²⁺]_i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca²⁺]_i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.     

Electroconvulsive therapy: a novel hypothesis for the involvement of purinergic signalling

It is proposed that ATP is released from both neurons and glia during electroconvulsive therapy (ECT) and that this leads to reduction of depressive behaviour via complex stimulation of neurons and glia directly via P2X and P2Y receptors and also via P1 receptors after extracellular breakdown of ATP to adenosine. In particular, A₁ adenosine receptors inhibit release of excitatory transmitters, and A_2A and P2Y receptors may modulate the release of dopamine. Sequential ECT may lead to changes in purinoceptor expression in mesolimbic and mesocortical regions of the brain implicated in depression and other mood disorders. In particular, increased expression of P2X7 receptors on glial cells would lead to increased release of cytokines, chemokines and neurotrophins. In summary, we suggest that ATP release following ECT involves neurons, glial cells and neuron–glial interactions acting via both P2 and after breakdown to adenosine via P1 receptors. We suggest that ecto-nucleotidase inhibitors (increasing available amounts of ATP) and purinoceptor agonists may enhance the anti-depressive effect of ECT.     

Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

Glial cell regulation of neuronal activity and blood flow in the retina by release of gliotransmitters

Astrocytes in the brain release transmitters that actively modulate neuronal excitability and synaptic efficacy. Astrocytes also release vasoactive agents that contribute to neurovascular coupling. As reviewed in this article, Müller cells, the principal retinal glial cells, modulate neuronal activity and blood flow in the retina. Stimulated Müller cells release ATP which, following its conversion to adenosine by ectoenzymes, hyperpolarizes retinal ganglion cells by activation of A1 adenosine receptors. This results in the opening of G protein-coupled inwardly rectifying potassium (GIRK) channels and small conductance Ca²⁺-activated K⁺ (SK) channels. Tonic release of ATP also contributes to the generation of tone in the retinal vasculature by activation of P2X receptors on vascular smooth muscle cells. Vascular tone is lost when glial cells are poisoned with the gliotoxin fluorocitrate. The glial release of vasoactive metabolites of arachidonic acid, including prostaglandin E₂ (PGE₂) and epoxyeicosatrienoic acids (EETs), contributes to neurovascular coupling in the retina. Neurovascular coupling is reduced when neuronal stimulation of glial cells is interrupted and when the synthesis of arachidonic acid metabolites is blocked. Neurovascular coupling is compromised in diabetic retinopathy owing to the loss of glial-mediated vasodilation. This loss can be reversed by inhibiting inducible nitric oxide synthase. It is likely that future research will reveal additional important functions of the release of transmitters from glial cells.     

Caffeine has a dual influence on NMDA receptor–mediated glutamatergic transmission at the hippocampus

Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A₁R and A₂R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca²⁺ homeostasis. We found that caffeine (30–200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A₁Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A_2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A_2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca²⁺ transients in neuronal cell culture, an action mimicked by the A₁R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca²⁺ homeostasis.

     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

The Adenosine Receptor Agonist,APNEA, Increases Calcium Influx into Rat Cortical Synaptosomes through N-Type Channels Associated with A2a Receptors

N⁶-2-(4-aminophenyl)ethyladenosine (APNEA) is a nonselective adenosine receptor agonist known to have a high affinity for the adenosine A₁ and A₃ receptors. It was found to be able to dose-dependently increase the sustained (4 min) Ca²⁺ influx into rat cortical synaptosomes while 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5-N-methyluronamide (Cl-IB-MECA), a selective A₃ agonist has no effect. However, this effect of APNEA was not affected by the presence of 8-cyclopentyl-l,3-dimethylxanthine (CPT), a selective A₁ antagonist; but instead completely abolished by 8-(3-chlorostyryl)caffeine (CSC), a selective A_2a antagonist, or -conotoxin GVIA. These results show that in the rat cortex, presynaptic A_2a receptors can mediate neurotransmitter release by increasing Ca²⁺ influx through the N-type calcium channels. A₁ and A₃ receptors appear not to be involved.     

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), as measured with the Ca²⁺-dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P₁ receptors in mediating this response. The increase in [Ca²⁺]_i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca²⁺]_i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′-[γ-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) ～ adenosine 5′-[β-thio]diphosphate ? adenosine 5′-[αβ-methylene] triphosphate, adenosine 5′-[βγ-imido]triphosphate. This profile is not characteristic of either the P_2X or the conventional P_2Y receptors. The Ca²⁺ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P_2Y nucleotide receptors (P_2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley-Liss, Inc.     

Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K⁺) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca²⁺-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca²⁺ concentration, or increase of extracellular K⁺ to a depolarizing level that opens astrocytic L-channels for Ca²⁺ and thereby also increase intra-astrocytic Ca²⁺ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K⁺ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K⁺ concentrations.     

Release of arachidonic acid via Ca2+ increase stimulated by pyrophosphonucleotides and bradykinin in mammary tumour cells

The relationship between the increase of intracellular Ca²⁺ and the release of arachidonic acid by bradykinin and pyrophosphonucleotides was studied in cultured mammary tumour cells, MMT060562. Bradykinin, ATP, UTP and UDP induced an increase of intracellular Ca²⁺ and the release of arachidonic acid from phospholipids into the extracellular fluid. Release of arachidonic acid was also induced by the application of the Ca²⁺ ionophore, A23187. Liberation of arachidonic acid by bradykinin and ATP was reduced by mepacrine, a blocker of phospholipase A₂ and W-7, a calmodulin antagonist. It is suggested that the increase in cytosolic Ca²⁺-induced release of arachidonic acid occurs through activation of calmodulin-dependent phospholipase A₂.     

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion‐current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage‐clamp technique. All agonists elicited Cl^? currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl^? (F_Cl) currents, while AII activated an oscillatory response; a robust Ca²⁺ influx linked specifically to F_Cl activation elicited an inward current (I_iw,Ca) which was carried mainly by Cl^? ions, through channels with a sequence of permeability of SCN^? > I^? > Br^? > Cl^?. Like F_Cl, I_iw,Ca was not dependent on oocyte [Ca²⁺]_i; instead both were eliminated by preventing [Ca²⁺]_i increase in the follicular cells, and also by U73122 and 2‐APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F_Cl and I_iw,Ca were produced by the expected, PLC‐stimulated Ca²⁺‐release and Ca²⁺‐influx, respectively, and by the opening of I_Cl(Ca) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca²⁺‐influx appeared to be driven through store‐operated, calcium‐like channels. The AII response, which is also known to require PLC activation, did not activate I_iw,Ca and was strictly dependent on oocyte [Ca²⁺]_i increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap‐junction channels. J. Cell. Physiol. 227: 3457–3470, 2012. © 2011 Wiley Periodicals, Inc.     

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca²⁺-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca²⁺-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca²⁺ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca²⁺ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca²⁺-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.     

GABA Release Modified by Adenosine Receptors in Mouse Hippocampal Slices under Normal and Ischemic Conditions

The excitatory glutamatergic neurons in the hippocampus are modulated by inhibitory GABA-releasing interneurons. The neuromodulator adenosine is known to inhibit the presynaptic release of neurotransmitters and to hyperpolarize postsynaptic neurons in the hippocampus, which would imply that it is an endogenous protective agent against cerebral ischemia and excitotoxic neuronal damage. Interactions of the GABAergic and adenosinergic systems in regulating neuronal excitability in the hippocampus is of crucial importance, particularly under cell-damaging conditions. We now characterized the effects of adenosine receptor agonists and antagonists on the release of preloaded [³H]GABA from hippocampal slices prepared from adult (3-month-old) mice, using a superfusion system. The effects were tested both under normal conditions and in ischemia induced by omitting glucose and oxygen from the superfusion medium. Basal and K⁺-evoked GABA release in the hippocampus were depressed by adenosinergic compounds. Under normal conditions activation of both adenosine A₁ and A_2A receptors by the agonists R(-)N⁶-(2-phenylisopropyl)adenosine and CGS 21680 inhibited the K⁺-evoked release, which effects were blocked by their specific antagonists, 8-cyclopentyl-1,3-dipropyl-xanthine and 3,7-dimethyl-1-propargylxanthine, respectively. Under ischemic conditions the release of both GABA and adenosine is markedly enhanced. The above receptor agonists then depressed both the basal and K⁺-evoked GABA release, only the action of A_2A receptors being however receptor-mediated. The demonstrated depression of GABA release by adenosine in the hippocampus could be deleterious to neurons and contribute to excitotoxicity.