Emi Maeno

Receptor‐mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD)

A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re‐adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl− channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate‐conductance Ca2+‐dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C‐mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein‐coupled Ca2+‐sensing receptor (CaR) enhances the activity of volume‐sensitive outwardly rectifying Cl− channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling‐induced and P2Y2 receptor‐mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume‐regulatory Cl− conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNFα receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma × rat glioma hybrid (NG108‐15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be abolished by prior treatment with a blocker of volume‐regulatory K+ or Cl− channels, suggesting that AVD is caused by normotonic activation of ion channels that are normally involved in RVD under hypotonic conditions. Therefore, it is likely that G protein‐coupled receptors involved in RVD regulation and death receptors triggering AVD may share common downstream signals which should give us key clues to the detailed mechanisms of volume regulation and survival of animal cells. In this Topical Review, we look at the physiological ionic mechanisms of cell volume regulation and cell death‐associated volume changes from the facet of receptor‐mediated cellular processes.

Volume-sensitive Chloride Channels Involved in Apoptotic Volume Decrease and Cell Death

Apoptosis is an essential process in organ development, tissue homeostasis, somatic cell turnover, and the pathogenesis of degenerative diseases. Apoptotic cell death occurs in response to a variety of stimuli in physiological and pathological circumstances. Efflux of K+ and Cl− leads to apoptotic volume decrease (AVD) of the cell. Both mitochondrion-mediated intrinsic, and death receptor-mediated extrinsic, apoptotic stimuli have been reported to rapidly activate Cl− conductances in a large variety of cell types. In epithelial cells and cardiomyocytes, the AVD-inducing anion channel was recently determined to be the volume-sensitive outwardly rectifying (VSOR) Cl− channel which is usually activated by swelling under non-apoptotic conditions. Blocking the VSOR Cl− channel prevented cell death in not only epithelial and cardiac cells, but also other cell types, by inhibiting the induction of AVD and subsequent apoptotic events. Ischemia-reperfusion-induced apoptotic death in cardiomyocytes and brain neurons was also prevented by Cl− channel blockers. Furthermore, cancer cell apoptosis induced by the anti-cancer drug cisplatin was recently found to be associated with augmented activity of the VSOR Cl− channel and to be inhibited by a Cl− channel blocker. The apoptosis-inducing VSOR Cl− channel is distinct from ClC-3 and its molecular identity remains to be determined.

Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase

Apoptosis is a distinct form of cell death, which requires energy. Here, we made real-time continuous measurements of the cytosolic ATP level throughout the apoptotic process in intact HeLa, PC12 and U937 cells transfected with the firefly luciferase gene. Apoptotic stimuli (staurosporine (STS), tumor necrosis factor α (TNFα), etoposide) induced significant elevation of the cytosolic ATP level. The cytosolic ATP level remained at a higher level than in the control for up to 6 h during which activation of caspase-3 and internucleosomal DNA fragmentation took place. When the STS-induced ATP response was abolished by glucose deprivation-induced inhibition of glycolysis, both caspase activation and DNA laddering were completely inhibited. Annexin V-binding induced by STS or TNFα was largely suppressed by glycolysis inhibition. Thus, it is suggested that the cells die with increased cytosolic ATP, and elevation of cytosolic ATP level is a requisite to the apoptotic cell death process.

Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency

AbstractApoptotic and necrotic blebs elicited by H2O2 were compared in terms of dynamics, structure and underlying biochemistry in HeLa cells and Clone 9 cells. Apoptotic blebs appeared in a few minutes and required micromolar peroxide concentrations. Necrotic blebs appeared much later, prior to cell permeabilization, and required millimolar peroxide concentrations. Strikingly, necrotic blebs grew at a constant rate, which was unaffected throughout successive cycles of budding and detachment. At 1 μm diameter, the necks of necrotic and apoptotic blebs were almost identical. ATP depletion was discarded as a major factor for both types of bleb. Inhibition of ROCK-I, MLCK and p38MAPK strongly decreased apoptotic blebbing but had no effect on necrotic blebbing. Taken together, these data suggest the existence of a novel structure of fixed dimensions at the neck of both types of plasma membrane blebs in epithelial cells. However, necrotic blebs can be distinguished from apoptotic blebs in their susceptibility to actomyosin kinase inhibition.

Dual roles of plasmalemmal chloride channels in induction of cell death

Even under anisotonic conditions, most cells can regulate their volume by mechanisms called regulatory volume decrease (RVD) and increase (RVI) after osmotic swelling or shrinkage, respectively. In contrast, the initial processes of necrosis and apoptosis are associated with persistent swelling and shrinkage. Necrotic volume increase (NVI) is initiated by uptake of osmolytes, such as Na+, Cl− and lactate, under conditions of injury, hypoxia, ischaemia, acidosis or lactacidosis. Persistence of NVI is caused by dysfunction of RVD due to impairment of volume-sensitive Cl− channels under conditions of ATP deficiency or lactacidosis. Both lactacidosis-induced RVD dysfunction and necrotic cell death are prevented by pretreatment of cells with the vacuolating cytotoxin-A (VacA) toxin protein purified from Helicobacter pylori, which forms a lactacidosis-resistant anion channel. Apoptotic volume decrease (AVD) is triggered by activation of K+ and Cl− conductances following stimulation with a mitochondrion-mediated or death receptor-mediated apoptosis inducer. Apoptotic cell death can be prevented by blocking the Cl− channels but not the K+-Cl− cotransporters. Thus, the volume regulatory anion channel plays, unless impaired, a cell-rescuing role in the necrotic process by ensuring RVD after swelling induced by necrotic insults, whereas normotonic activation of the anion channel plays a cell-killing role in the apoptotic process by triggering AVD following stimulation with apoptosis inducers.

Swelling-activated, cystic fibrosis transmembrane conductance regulator-augmented ATP release and Cl− conductances in murine C127 cells

A hypotonic challenge, but not cAMP stimulation, was found to induce release of ATP measured by the luciferin‐luciferase assay from both the murine mammary carcinoma cell line C127i and C127 cells stably transfected with the cDNA for human cystic fibrosis transmembrane conductance regulator (CFTR) protein (C127/CFTR). CFTR expression augmented swelling‐induced ATP release by 10–20 times under hypotonic conditions (<= 80 % osmolality). Glibenclamide failed to suppress swelling‐induced ATP release from C127/CFTR cells. In contrast, whole‐cell patch‐clamp recordings showed that both the cAMP‐activated ohmic Cl− currents and volume‐sensitive outwardly rectifying (VSOR) Cl− currents were prominently suppressed by glibenclamide. Gd3+ markedly blocked swelling‐induced ATP release but failed to suppress both cAMP‐ and swelling‐activated Cl− currents in the CFTR‐expressing cells. Even after pretreatment and during treatment with Gd3+, VSOR Cl− currents were activated normally. The continuous presence of an ATP‐hydrolysing enzyme, apyrase, in the bathing solution did not prevent activation of VSOR Cl− currents in C127/CFTR cells. The rate of regulatory volume decrease (RVD) in C127/CFTR cells was much faster than that in C127i cells. When apyrase was added to the bathing solution, the RVD rate was retarded in C127/CFTR cells. On balance, the following conclusions can be deduced. First, swelling‐induced ATP release is augmented by expression of CFTR but is not mediated by the CFTR Cl− channel. Second, swelling‐induced ATP release is not mediated by the VSOR Cl− channel. Third, the released ATP facilitated the RVD process but is not involved in the activation of VSOR Cl− channels in C127/CFTR cells.

Dysfunction of regulatory volume increase is a key component of apoptosis.

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non‐apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF‐α or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase‐3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1‐deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity‐induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.

Normotonic cell shrinkage induces apoptosis under extracellular low Cl− conditions in human lymphoid and epithelial cells

Aim:  Sustained cell shrinkage is associated with apoptosis. Apoptotic volume decrease, which is known to be induced by release of osmolytes including Cl− ions, may be an essential event for apoptosis induction. Provided any anion channels and/or anion transporters are basally functioning, there is a possibility that imposition of a driving force for Cl− efflux per se results in sustained cell shrinkage and thereby induces apoptotic death. Here, this possibility was tested by reducing the extracellular Cl− concentration.

Apoptotic Volume Decrease (AVD) Is Independent of Mitochondrial Dysfunction and Initiator Caspase Activation.

Persistent cell shrinkage is a major hallmark of apoptotic cell death. The early-phase shrinkage, which starts within 30−120 min after apoptotic stimulation and is called apoptotic volume decrease (AVD), is known to be accomplished by activation of K+ channels and volume-sensitive outwardly rectifying (VSOR) Cl− channels in a manner independent of caspase-3 activation. However, it is controversial whether AVD depends on apoptotic dysfunction of mitochondria and activation of initiator caspases. Here, we observed that AVD is induced not only by a mitochondrial apoptosis inducer, staurosporine (STS), in mouse B lymphoma WEHI-231 cells, but also by ligation of the death receptor Fas in human B lymphoblastoid SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria. Overexpression of Bcl-2 failed to inhibit the STS-induced AVD in WEHI-231 cells. These results indicate that AVD does not require the mitochondrial pathway of apoptosis. In human epithelial HeLa cells stimulated with anti-Fas antibody or STS, the AVD induction was found to precede activation of caspase-8 and caspase-9 and to be resistant to pan-caspase blockers. Thus, it is concluded that the AVD induction is an early event independent of the mitochondrial apoptotic signaling pathway and initiator caspase activation.