Swelling-Activated Anion Channels Are Essential for Volume Regulation of Mouse Thymocytes

Channel-mediated trans-membrane chloride movement is a key process in the active cell volume regulation under osmotic stress in most cells. However, thymocytes were hypothesized to regulate their volume by activating a coupled K-Cl cotransport mechanism. Under the patch-clamp, we found that osmotic swelling activates two types of macroscopic anion conductance with different voltage-dependence and pharmacology. At the single-channel level, we identified two types of events: one corresponded to the maxi-anion channel, and the other one had characteristics of the volume-sensitive outwardly rectifying (VSOR) chloride channel of intermediate conductance. A VSOR inhibitor, phloretin, significantly suppressed both macroscopic VSOR-type conductance and single-channel activity of intermediate amplitude. The maxi-anion channel activity was largely suppressed by Gd3+ ions but not by phloretin. Surprisingly, [(dihydroindenyl)oxy] alkanoic acid (DIOA), a known antagonist of K-Cl cotransporter, was found to significantly suppress the activity of the VSOR-type single-channel events with no effect on the maxi-anion channels at 10 μM. The regulatory volume decrease (RVD) phase of cellular response to hypotonicity was mildly suppressed by Gd3+ ions and was completely abolished by phloretin suggesting a major impact of the VSOR chloride channel and modulatory role of the maxi-anion channel. The inhibitory effect of DIOA was also strong, and, most likely, it occurred via blocking the VSOR Cl− channels

Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes

ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 μM. The OGD-induced ATP release was inhibited by Gd3+ and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl− channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X7 receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd3+ and arachidonic acid. The channel was found to be permeable to ATP4− with a permeability ratio of PATP/PCl = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions

Cell death induction and protection by activation of ubiquitously expressed anion/cation channels. Part 3: the roles and properties of TRPM2 and TRPM7

Cell volume regulation (CVR) is a prerequisite for animal cells to survive and fulfill their functions. CVR dysfunction is essentially involved in the induction of cell death. In fact, sustained normotonic cell swelling and shrinkage are associated with necrosis and apoptosis, and thus called the necrotic volume increase (NVI) and the apoptotic volume decrease (AVD), respectively. Since a number of ubiquitously expressed ion channels are involved in the CVR processes, these volume-regulatory ion channels are also implicated in the NVI and AVD events. In Part 1 and Part 2 of this series of review articles, we described the roles of swelling-activated anion channels called VSOR or VRAC and acid-activated anion channels called ASOR or PAC in CVR and cell death processes. Here, Part 3 focuses on therein roles of Ca2+-permeable non-selective TRPM2 and TRPM7 cation channels activated by stress. First, we summarize their phenotypic properties and molecular structure. Second, we describe their roles in CVR. Since cell death induction is tightly coupled to dysfunction of CVR, third, we focus on their participation in the induction of or protection against cell death under oxidative, acidotoxic, excitotoxic, and ischemic conditions. In this regard, we pay attention to the sensitivity of TRPM2 and TRPM7 to a variety of stress as well as to their capability to physicall and functionally interact with other volume-related channels and membrane enzymes. Also, we summarize a large number of reports hitherto published in which TRPM2 and TRPM7 channels are shown to be involved in cell death associated with a variety of diseases or disorders, in some cases as double-edged swords. Lastly, we attempt to describe how TRPM2 and TRPM7 are organized in the ionic mechanisms leading to cell death induction and protection

Positive Inotropic Effects of ATP Released via the Maxi-Anion Channel in Langendorff-Perfused Mouse Hearts Subjected to Ischemia-Reperfusion.

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger-for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg2+ and/or H+ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP4- in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed

Lack of effect on maxi-anion channel activity of octanol, atractyloside and bongkrekic acid added to the intracellular solution in inside-out patches excised from C127 cells

<p><b>Copyright information:</b></p><p>Taken from "ATP release via anion channels"</p><p></p><p>Purinergic Signalling 2005;1(4):311-328.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096548.</p><p></p> Dashed lines indicate the zero current level. The test pulse protocol applied is shown on the top. Traces are representative of 4, 3 and 3 experiments with octanol-1 (1 mM), atractyloside (10 µM) and bongkrekic acid (10 µM), respectively. Experimental conditions were same as in Sabirov et al. []. Maxi-anion channel activity was also not affected by the addition of these drugs to the extracellular (pipette) solution (data not shown, = 2–4)

Representative current-voltage (I–V) curves composed of outward Cl currents and inward currents carried by ATP (A), ADP (B) and UTP (C)

<p><b>Copyright information:</b></p><p>Taken from "ATP release via anion channels"</p><p></p><p>Purinergic Signalling 2005;1(4):311-328.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096548.</p><p></p> The currents were recorded by ramp-clamp in macro-patches excised from C127 cells. The extracellular (pipette) solution was Cl-rich Ringer solution, and the intracellular (bath) solution was Cl-free solution containing 100 mM Na4ATP (), Na3ADP () or Na4UTP (). The reversal potentials are −16.2 ± 1.8 mV ( = 5: ), −20.3 ± 1.7 mV ( = 10: ) and −17.1 ± 0.8 mV ( = 10: ). The calculated values of P/P, P/P and P/P are 0.10 ± 0.014, 0.12 ± 0.010 and 0.09 ± 0.005, respectively

Volume-sensitive anion channels mediate osmosensitive glutathione release from rat thymocytes.

Glutathione (GSH) is a negatively charged tripeptide, which is a major determinant of the cellular redox state and defense against oxidative stress. It is assembled inside and degraded outside the cells and is released under various physiological and pathophysiological conditions. The GSH release mechanism is poorly understood at present. In our experiments, freshly isolated rat thymocytes were found to release GSH under normal isotonic conditions at a low rate of 0.82±0.07 attomol/cell/min and that was greatly enhanced under hypoosomotic stimulation to reach a level of 6.1±0.4 attomol/cell/min. The swelling-induced GSH release was proportional to the cell density in the suspension and was temperature-dependent with relatively low activation energy of 5.4±0.6 kcal/mol indicating a predominant diffusion mechanism of GSH translocation. The osmosensitive release of GSH was significantly inhibited by blockers of volume-sensitive outwardly rectifying (VSOR) anion channel, DCPIB and phloretin. In patch-clamp experiments, osmotic swelling activated large anionic conductance with the VSOR channel phenotype. Anion replacement studies suggested that the thymic VSOR anion channel is permeable to GSH(-) with the permeability ratio P(GSH)/P(Cl) of 0.32 for influx and 0.10 for efflux of GSH. The osmosensitive GSH release was trans-stimulated by SLCO/OATP substrates, probenecid, taurocholic acid and estrone sulfate, and inhibited by an SLC22A/OAT blocker, p-aminohippuric acid (PAH). The inhibition by PAH was additive to the effect of DCPIB or phloretin implying that PAH and DCPIB/phloretin affected separate pathways. We suggest that the VSOR anion channel constitutes a major part of the γ-glutamyl cycle in thymocytes and, in cooperation with OATP-like and OAT-like transporters, provides a pathway for the GSH efflux from osmotically swollen cells