Mechanisms of glutamate release from astrocytes: gap junction “hemichannels”, purinergic receptors and exocytotic release

Neurochemistry International 45 (2004) 259–264

Mechanisms of glutamate release from astrocytes: gap junction “hemichannels”, purinergic receptors and exocytotic release Vladimir Parpura a,∗ , Eliana Scemes b , David C. Spray b a

Department of Cell Biology and Neuroscience, Center for Nanoscale Science and Engineering, University of California, Riverside, CA 92521, USA b Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA Received 14 October 2003; received in revised form 16 December 2003; accepted 16 December 2003

Abstract Neuronal exocytotic release of glutamate at synapses involves a highly specialized vesicular apparatus, consisting of a variety of proteins connected to the vesicles or required for vesicular fusion to the presynaptic membrane. Astrocytes also release glutamate, and recent evidence indicates that this release can modify neuronal function. Several mechanisms have been proposed for astrocytic release of glutamate under pathological conditions, such as reversal of glutamate transporters and opening of volume sensitive ion channels. In this review we limit our discussion to findings supporting the exocytotic release of glutamate, as well as two new pathways implicated in this release, the ionotropic (P2X) purinergic receptors and gap junction hemichannels. © 2004 Elsevier Ltd. All rights reserved. Keywords: Astrocytes; Glutamate release; P2X receptors; Connexin “hemichannels”; Ca2+ ; Vesicular glutamate transporters; SNAREs; Exocytosis

Astrocytes release many neuroligands, including the neurotransmitter glutamate. Mechanisms that have been implicated in release of glutamate from astrocytes are, in chronological order of their discovery: reversal of uptake by glutamate transporters (Szatkowski et al., 1990), anion channels opened by cell swelling (Kimelberg et al., 1990), Ca2+ -dependent exocytosis (Parpura et al., 1994), diffusional release through ionotropic purinergic receptors (P2XRs) (Duan et al., 2003) and functional “hemichannels” or unpaired half gap junction channels (or connexons) on the cell surface (Ye et al., 2003). In this overview, we concentrate on studies providing evidence for glutamate release through P2X receptors, gap junction hemichannels and through exocytosis or vesicular release (pathways schematized in Fig. 1).

1. Non-vesicular release of glutamate: P2X receptors and/or connexin “hemichannels” Two very recent studies using similar experimental paradigms have proposed that quite different processes mediate the non-vesicular release of glutamate from astrocytes: the P2X7 receptor (P2X7 R) (Duan et al., 2003) and ∗

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0197-0186/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2003.12.011

“hemichannels” or connexons formed by the gap junction protein connexin43 (Cx43) (Ye et al., 2003). Because the evidence in support of these pathways in astrocytic glutamate release is so recent, we provide below a summary of these findings as well as an overview of the current status of research regarding P2X receptors and gap junction “hemichannels.” 1.1. Purinergic receptors, especially P2X7 R The P2X receptors are ionotropic, where binding of agonist opens ionic channels, as distinguished from the metabotropic P2Y receptors, in which ligand binding is coupled through G proteins to intracellular second messenger generation. There are at least nine P2X receptor subtypes: homomeric P2X1 , P2X2 P2X3 , P2X4 , P2X5 , P2X6 , P2X7 , and heteromeric P2X2/3 and P2X1/5 receptors (North, 2002). Each of these channel types (with the exception of P2X6 , has been reported to be expressed in astrocytes in vivo or in vitro either under normal conditions or following injury (Fumagalli et al., 2003; Franke et al., 2001; Kukley et al., 2001). All P2X receptors form Ca2+ permeable channels when activated; however, P2X2 , P2X2/3 , P2X4 , P2X5 , and P2X7 receptors have been reported to undergo a remarkable “dilation” during prolonged exposure to agonist, allowing the uptake (and presumed release) of large molecules. Such behavior led to initial characterization of channels with

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Fig. 1. Three mechanisms postulated for glutamate release from astrocytes that are the subject of this overview. (A) Gap junctions formed primarily of Cx43 proteins interconnect astrocytes; glutamate release has been postulated to occur through purinergic type P2X7 receptors and through Cx43 hemichannels on the unapposed astrocyte surface. (B) Astrocytes possess SNARE proteins known to mediate exocytosis, synaptobrevin II (red), syntaxin I (green) and SNAP-23 (blue). Tetanus toxin (TeTx) that cleaves specifically synaptobrevin II inhibits release of glutamate (Glu) from astrocytes. The storage of glutamate in acidic compartments (vesicles) requires the presence of V-type H+ -ATPase (brown), which can be blocked by bafilomycin A1 (BA1 ), and also of vesicular glutamate transporters (pink), both found in astrocytes.

this property as “permeabilizing pores” (or P2Z receptors; Burnstock, 1990; Hickman et al., 1996; Steinberg et al., 1987; Di Virgilio, 1995). In some cell types, the high permeability of these channels allows the diffusion of molecules with molecular weights as high as 900 Da, whereas in other cell types their permeability may be restricted to smaller molecules (Ralevic and Burnstock, 1998; North, 2002). Cloning of the P2X7 receptor in 1996 revealed that it possessed the “dilation pore” property of the P2Z receptor when expressed in mammalian cells (Surprenant et al., 1996), although this property has not yet been fully observed in cRNA-injected Xenopus oocytes (Petrou et al., 1997; Boldt et al., 2003). Biophysical properties of P2X7 receptors include little or no voltage sensitivity in channel gating, blockade by KN-62, Brilliant Blue G and prolonged exposure to oxidized ATP (oATP) (North, 2002), as well as channel closure (or inhibition of channel opening) by divalent cations and acidification (Virginio et al., 1997) and by the chloride channel blockers 4-acetamido-4 -isothiocyanato-stilbene-2,2 -disulfonate (SITS) and 4,4 -diisothiocyanato-stilbene-2,2 -disulfonate (DIDS) (Soltoff et al., 1993). Conductance and cell uptake of fluorescent molecules are enhanced under low divalent conditions; although other P2X receptors show dye uptake upon activation, the high sensitivity to moderate divalent cation concentrations is a hallmark feature of P2X7 receptors. In cultured astrocytes, activation of P2X7 R was shown to induce an increased current that was amplified by low Ca2+ and Mg2+ solution (Duan et al., 2003). Following activation, the uptake of the 457 Da fluorescent dye lucifer yellow (LY), which had been shown by Ballerini et al. (1996) to enter astrocytes upon exposure to 3 -O-(4-benzoyl)benzoyl ATP (BzATP), was also increased in low divalent cation solution (Duan et al., 2003). Both current and dye uptake were blocked by prolonged pretreatment with oxidized ATP. Un-

der voltage-clamp conditions, Duan et al. (2003) found little or no voltage sensitivity to the BzATP-induced currents and calculated the P2X7 R permeability sequence for normal substrates (relative permeabilities in parentheses) as: Na+ (1); Cl− (0.34); l-glutamate (0.15); l-aspartate (0.16). Basal release of radiolabeled glutamate was found to be increased about two- to three-fold in Ca2+ /Mg2+ free solution and this increase was amplified by addition of BzATP (100 ␮M) or ATP (300 ␮M), with a higher degree of amplification occurring in Ca2+ /Mg2+ free solution. Release of glutamate from astrocytes was found to be blocked by oATP, pyridoxal phosphate-6-azophenyl-2 -4 -disulfonic acid (PPADS; a P2 receptor antagonist) and DIDS (an anion channel blocker). Based on the similarity of these results to properties expected for P2X7 R, Duan et al. (2003) concluded that these purinoceptors may represent a major pathway of astrocytic glutamate release. 1.2. Connexin “hemichannels” Gap junction channels, formed by docking of the two connexons provided by apposed cell membranes, are voltage sensitive intercellular channels that display some degree of ionic selectivity although allowing the transfer of molecules with molecular weights up to about 1 kDa. In astrocytes, Cx43 is the main protein forming gap junctional intercellular channels, although other gap junction channel types can contribute a small portion of the intercellular channels (see Dermietzel et al., 2002). The conductance of gap junction channels shows bell-shaped voltage sensitivity with the fully open state maximally occupied at zero transjunctional voltage (Spray et al., 1981) and the maximal closure induced by long and large transjunctional voltages involving closure to a partially conducting residual conductance state (Moreno et al., 1994); steepness of voltage sensitivity and degree of total block are characteristic properties of each connexin type (see Spray et al., 2002). Gap junction channels are closed by intracellular acidification (where apparent pKa is also connexin-specific) and by certain alcohols (heptanol and octanol), halothane, glyccerhetinic acid derivatives including carbenoxolone (for review see Spray et al., 2002), and by certain chloride channel blockers (e.g., flufenamic acid), but not others (e.g., SITS, DIDS) (Srinivas and Spray, 2003). Quinine blocks gap junctions formed of some connexins (e.g., Cx36, Cx50), but channels formed of other connexins are insensitive (e.g., Cx43) (Srinivas et al., 2001). Connexons, or hemichannels, are hexameric units composed of gap junction proteins (connexins) that are hypothesized to exist and be activatable on the cell surface in the absence of cell pairing to form complete gap junction channels. Only recently have the connexons been viewed as forming functional hemichannels in astrocytes (Hofer and Dermietzel, 1998; Contreras et al., 2002; Stout et al., 2002). However, much earlier studies attributed large conductance anion channels in myoblasts to hemichannels (Blatz

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and Magleby, 1983; Schwarze and Kolb, 1984), which are now known to be due to expression of plasmalemmal voltage-dependent anion channels (plVDAC; Buettner et al., 2000). The so-called “suicide channels” induced by ATP in macrophages and macrophage cell lines were also hypothesized to be hemichannels formed of Cx43 (Beyer and Steinberg, 1991), but these are now known to be P2X, and most likely P2X7 receptors, as summarized in the section above. The formation of functional hemichannels in skate and teleost horizontal cells (possibly by Cx52.6; Zoidl et al., 2004) and in Xenopus oocytes exogenously expressing certain connexins (particularly Cx46) are well documented (Malchow et al., 1993; DeVries and Schwartz, 1992; Pfahnl and Dahl, 1999; Paul et al., 1991; Ebihara and Steiner, 1993). Excised patch recordings from Xenopus oocytes has permitted high temporal resolution of channel transitions and has provided further evidence that channel gating by acidification is direct, not requiring other molecules (Trexler et al., 1999). Moreover, such studies have indicated several biophysical properties of hemichannels, including channel opening under low divalent cation concentrations and a characteristic voltage sensitivity, where channels are closed at resting membrane potentials (below about −10 to 0 mV), only opening upon significant membrane depolarization (e.g., Trexler et al., 1996). It is important to point out that the voltage dependence demonstrated for hemichannels in oocyte expression studies (closure at resting membrane potential and opening only at or beyond 0 mV) would not be expected to allow significant flux of ions or small molecules under normal conditions. In HeLa cells transfected with Cx43 and green fluorescent protein (GFP)-tagged Cx43 constructs, hemichannel currents with this voltage sensitivity were recently observed, yet dye uptake under resting conditions was attributed to this pathway (Contreras et al., 2003); effects of divalent cation removal on the conductance was also surprisingly modest in this study. The recent wave of reports claiming hemichannel function induced in cardiac myocytes and astrocytes under ischemic conditions and by low divalent cation concentrations have provided evidence that hemichannels are permeable to LY under these conditions, but have also shown properties not expected from the Xenopus expression studies, including absence of voltage sensitivity and presence of dye uptake at normal resting potential (John et al., 1999; Kondo et al., 2000; Contreras et al., 2002; Liu et al., 1995), and lack of requirement of reduced divalent cations for hemichannel opening (Contreras et al., 2002, 2003). Moreover, blockade of “hemichannel” currents by pharmacological agents that close gap junction channels has generally been incomplete, whereas other agents without demonstrated effect on gap junctions (such as La3+ and Gd3+ ) have been found to cause more complete channel closure. Similarly to what was described above for the P2X7 R, Ye et al. (2003) reported that the uptake of LY and the release of glutamate by cultured astrocytes was potentiated by re-

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ducing extracellular divalent concentration. These authors showed that LY uptake and glutamate release were greatly reduced by carbenoxolone, heptanol, octanol, ␣-GA (compounds used to block gap junction channels). However, contrary to what was described by Duan et al. (2003), the P2X7 R antagonists oATP and PPADS and the chloride channel blocker DIDS did not alter the release of glutamate from astrocytes when lowering extracellular divalent cations (Ye et al., 2003). Unfortunately, Ye et al. (2003) did not report the effects of oATP, PPADS, and DIDS on LY uptake, although a similar disparity with the study by Duan et al. (2003) would be expected. Nevertheless, based on the similarity of these results to properties expected for gap junction hemichannels, Ye et al. (2003) concluded that this may represent a major pathway of astrocyte glutamate release under conditions of altered extracellular divalent ions (e.g., ischemia). 1.3. Further experiments to resolve the issue The lack of voltage dependence noted in certain reports of Cx43 hemichannel currents observed in cultured astrocytes when lowering extracellular divalents and the robust uptake of lucifer yellow at normal resting potentials (e.g., Stout and Charles, 2003; Stout et al., 2002) are not consistent with characteristics of gap junction hemichannels measured in Xenopus oocyte expression studies or with the recent report characterizing hemichannel currents induced in HeLa cells by transfection with Cx43 and GFP-tagged Cx43 constructs (Contreras et al., 2003). Moreover, while the demonstration that glutamate release from cultured astrocytes under low divalent conditions is blocked by a range of gap junction channel blockers may provide evidence that is compelling to some for the involvement of gap junction hemichannels in the process (Ye et al., 2003), the contrary reports that glutamate release is either blocked or not affected by DIDS, PPADS, and oATP (compare Duan et al., 2003; with Ye et al., 2003) indicate that the involvement of P2X7 receptors remains possible. The most likely way to resolve these conflicting results may involve use of astrocytes from P2X7 R null and Cx43 null mice. However, given the overlapping properties of P2X7 receptors and gap junction hemichannels, it would seem prudent to expand criteria for assigning mechanism of uptake of dyes under low divalent conditions to minimally include potent P2X7 receptor and gap junction channel blockers. Moreover, attributing dye flux to the “hemichannel” pathway without such demonstration should be discouraged, because of the known similarity of many of the biophysical properties with those of P2X receptors. 2. Ca2+ -dependent release of glutamate: exocytosis in astrocytes There is compelling evidence emerging in support of exocytosis as a mechanism that underlies physiological

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Ca2+ -dependent release of neurotransmitters from astrocytes. The first evidence for Ca2+ -dependent glutamate release from astrocytes came from purified cortical astrocytic culture (Parpura et al., 1994). Bradykinin caused intracellular Ca2+ elevations and glutamate release from astrocytes, and consequential N-methyl-d-aspartate (NMDA) receptor-mediated intracellular Ca2+ increases in adjacent neurons. This observation of Ca2+ -dependent glutamate release and glutamate-mediated astrocyte-neuron signaling is not limited to cultured astrocytes, but has been pharmacologically confirmed in acute hippocampal slices as well (Bezzi et al., 1998). Ca2+ -dependent glutamate release from astrocytes shows supportive evidence for exocytosis. Most notably, it exhibits a Ca2+ dependency and the presence of exocytotic secretory machinery. 2.1. Ca2+ dependency Intracellular Ca2+ elevations in astrocytes were sufficient and necessary to cause glutamate release (Parpura et al., 1994). Hence, addition of Ca2+ ionophore ionomycin in presence of external calcium ions, but not in their absence, caused glutamate release from astrocytes. Additionally, other stimuli that directly increased astrocytic internal Ca2+ levels, such as mechanical stimulation (Parpura et al., 1994; Araque et al., 1998a,b), UV light stimulation (Parpura et al., 1994) and photolysis of Ca2+ cages (Araque et al., 1998b; Parpura and Haydon, 2000), all caused glutamate release. Moreover, manipulations of internal Ca2+ in astrocytes using calcium chelator, BAPTA (Araque et al., 1998a; Bezzi et al., 1998), or pre-incubation of cells with thapsigargin, an inhibitor of Ca2+ -ATPase specific for internal stores (Araque et al., 1998a), led to reduction of evoked glutamate release from astrocytes. Thus, it seems that the internal stores represent the predominant source of calcium ions necessary for glutamate release form astrocytes. This is consistent with reported either absence or inactivity of voltage-gated Ca2+ channels (VGCC) in astrocytes (Carmignoto et al., 1998). However, since astrocytes express various VGCC (MacVicar, 1984; Latour et al., 2003) and transient receptor potential genes (TRP) (Pizzo et al., 2001), the source of calcium ions in this release will have to be studied in detail. Indeed, recent work reported in the form of an abstract (Hua et al., 2002) suggests a dual source for Ca2+ in exocytotic glutamate release from astrocytes, both the release of calcium ions from internal stores and their entry from extracellular space. The amount of Ca2+ in synaptic terminals is critical for shaping synaptic transmission due to Ca2+ regulation of neurotransmitter release at the terminal. Seminal work by Dodge and Rahamimoff (1967) had established the quantitative relationship between neuronal Ca2+ levels and neurotransmitter release by determining the slope of this relationship on a logarithm–logarithm plot, commonly referred as the Hill coefficient. This coefficient indicates the cooperativity of calcium ions in the release of neurotransmitter at synaptic terminals, and has been reported to be

between 2 and 4 (Augustine and Eckert, 1984; Smith et al., 1985; Stanley, 1986; Zhong and Wu, 1991; Trudeau et al., 1998). Such basic quantitative assessment of the secretory apparatus in cultured astrocytes has been recently initiated (Parpura and Haydon, 2000), showing that: (i) the threshold level of intracellular Ca2+ necessary to stimulate glutamate release from astrocytes is much lower than that in neurons (Ca2+ elevations from the resting level of about 80 nM to only 140 nM caused glutamate release) and (ii) the Hill coefficient for glutamate release is 2.1–2.7. 2.2. Exocytotic secretory machinery ␣-Latrotoxin, a component of black widow spider venom, stimulates glutamate release from astrocytes (Parpura et al., 1995b). Because ␣-latrotoxin also induces vesicle fusion and neuronal transmitter release (Ushkaryov et al., 1992; Petrenko, 1993), these data suggest that astrocytes may release transmitters using a mechanism similar to the neuronal secretory process. Indeed, astrocytes express SNARE proteins know to mediate exocytosis at presynaptic terminals (Fig. 1): synaptobrevin II, and its non-neuronal homologue cellubrevin, syntaxin I and SNAP-23 (Parpura et al., 1995a; Jeftinija et al., 1997; Hepp et al., 1999; Maienschein et al., 1999; Araque et al., 2000; Pasti et al., 2001). Clostridial toxins can cleave some of these proteins (Parpura et al., 1995a; Jeftinija et al., 1997) and also inhibit Ca2+ -dependent release of glutamate from astrocytes (Jeftinija et al., 1997; Bezzi et al., 1998; Araque et al., 2000; Pasti et al., 2001). The Ca2+ sensor for glutamate release in astrocytes is most likely synaptotagmin. This is in good agreement with the presence of neurexins, as indicated by the action of ␣-latrotoxin, and with the Hill coefficient for glutamate release. Some additional proteins of neuronal secretory machinery such as synapsin I and rab3a were also found in astrocytes (Maienschein et al., 1999). An immunoelectron microscopic study indicated that these exocytotic proteins in astrocytes are often associated with electronlucent or dense-core vesicular structures, the diameters of which are less uniform than those reported in neurons (Maienschein et al., 1999). Consistent with the possibility that astrocytes can exhibit release via dense-core vesicles, a Ca2+ -dependent release of secretogranin II, a marker for dense-core vesicles, had been demonstrated (Calegari et al., 1999). Additionally, exocytotic release of atrial natriuretic peptide had been recently reported, confirming the presence of exocytosis through dense-core vesicles in cultured astrocytes (Krzan et al., 2003). The storage of glutamate in synaptic vesicles requires the presence of vacuolar type H+ -ATPase (V-ATPase) and vesicular glutamate transporters (VGLUTs). Astrocytic Ca2+ -dependent glutamate release can be reduced with bafilomycin A1 (Araque et al., 2000; Bezzi et al., 2001; Pasti et al., 2001) which specifically interferes with V-ATPase leading to alkalinization of vesicular lumen, and collapse of the proton gradient necessary for VGLUTs to transport

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glutamate into vesicles. Recently, there have been reports indicating the presence of VGLUTs in astrocytes (Fremeau et al., 2002; Ni et al., 2003). The process of exocytosis relies on an increase of intracellular Ca2+ levels that, through action on a fusion complex, causes quantal release of neurotransmitter due to vesicular fusion. Indeed, quantal-like events were recorded from cultured astrocytes using biosensor cells expressing functional NMDA receptors, consistent with vesicular exocytosis (Pasti et al., 2001). The exocytotic cues we discussed support the existence of vesicular exocytosis in astrocytes. A conclusive demonstration will require direct observation of vesicular fusions with the plasma membrane, and visualizing spatio-temporal characteristics of exocytosis in astrocytes together with defining the location of release sites and their morphological correlates in situ.

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