Stage-Specific Changes in the Water,Na+, Cl- and K+ Contents of Organelles during Apoptosis,Demonstrated by a Targeted Cryo Correlative Analytical Approach

Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na⁺ and Cl^- contents of the mitochondria and a strong decrease in mitochondrial K⁺ content. During the first step in apoptotic volume decrease (AVD), Na⁺ and Cl^- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na⁺ and Cl^- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K⁺ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K⁺ contents similar to those of cells entering apoptosis but lower Na⁺ and Cl^- contents than both cells entering apoptosis and control cells.     

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl⁻ transport inhibitor DIDS and the K⁺ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl⁻ flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl⁻ fluxes rather than blocking cell shrinkage or K⁺ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.     

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K⁺ and Na⁺ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.     

持续性细胞皱缩在人上皮细胞凋亡过程中的必要性

持续性细胞皱缩是凋亡发生的一个主要标志。近期研究发现细胞皱缩在细胞凋亡过程中并不是一个被动的次要事件。在各种细胞中,包括人上皮细胞,凋亡因子（apoptogen）刺激后马上发生全细胞皱缩,又称为凋亡性容积减小（apoptotic　volumede　crease,AVD）,继而发生caspase激活、DNA片段化、细胞破裂死亡。K^＋和Cl^-通道的激活导致KCl外流,诱导AVD发生。抑制AVD发生可以抑制细胞凋亡。AVD与调节性容积增加（regulatory　volume　increase,RVI）异常相伴发生时,人上皮性HeLa细胞发生持续性细胞皱缩。RVI功能受损时,高渗本身就能诱导HeLa细胞持续性细胞皱缩,继而凋亡。即使在正常渗透压、无凋亡因子刺激的情况下,将HeLa细胞置于缺乏Na^＋或Cl。的溶液也会导致细胞持续性皱缩,继而凋亡。因此,AVD诱导和RVI异常所导致的持续性细胞皱缩是人上皮细胞发生凋亡的首要条件。     

Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease

Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C₆-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K⁺ from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K⁺ concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K⁺ concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane. water movement; major intrinsic protein; channel; enzyme     

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-alpha 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.     

Ouabain-induced perturbations in intracellular ionic homeostasis regulate death receptor-mediated apoptosis

Apoptosis is defined by specific morphological and biochemical characteristics including cell shrinkage (termed apoptotic volume decrease), a process that results from the regulation of ion channels and plasma membrane transporter activity. The Na⁺–K⁺-ATPase is the predominant pump that controls cell volume and plasma membrane potential in cells and alterations in its function have been suggested to be associated with apoptosis. We report here that the Na⁺–K⁺-ATPase inhibitor ouabain, potentiates apoptosis in the human lymphoma Jurkat cells exposed to Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not other apoptotic agents such as H₂O₂, thapsigargin or UV-C implicating a role for the Na⁺–K⁺-ATPase in death receptor-induced apoptosis. Interestingly, ouabain also potentiated perturbations in cell Ca²⁺ homeostasis only in conjunction with the apoptotic inducer FasL but not TRAIL. Ouabain did not affect alterations in the intracellular Ca²⁺ levels in response to H₂O₂, thapsigargin or UV-C. FasL-induced alterations in Ca²⁺ were not abolished in Ca²⁺-free medium but incubation of cells with BAPTA-AM inhibited both Ca²⁺ perturbations and the ouabain-induced potentiation of FasL-induced apoptosis. Our data suggest that the impairment of the Na⁺–K⁺-ATPase activity during apoptosis is linked to perturbations in cell Ca²⁺ homeostasis that modulate apoptosis induced by the activation of Fas by FasL.     

Induction of apoptosis by nitric oxide in macrophages is independent of apoptotic volume decrease

Apoptosis occurs through a sequence of specific biochemical and morphological alterations that define the progress of cell death. The changes of the mitochondrial inner membrane potential (DeltaPsi(m)), the release of cytochrome c to the cytosol, the apoptotic volume decrease (AVD) and the activation of caspases have been measured in RAW 264.7, HeLa and Jurkat T cells incubated with molecules that induce apoptosis through the mitochondrial pathway. Our data show that NO, staurosporine, etoposide and camptothecin increased DeltaPsi(m) in macrophages but not in HeLa and Jurkat cells, that exhibited a DeltaPsi(m) decrease. Moreover, the apoptosis induced by NO in macrophages, but not that promoted by staurosporine, might occur in the absence of AVD. Analysis of the sequence of apoptotic manifestations shows that DeltaPsi(m) precedes AVD and caspase activation in RAW 264.7 cells. Inhibition of AVD abrogates apoptosis in HeLa and Jurkat T cells regardless of the stimuli used. These data suggest that the changes of DeltaPsi(m) are cell-type dependent and that AVD is dispensable for apoptosis in macrophages.     

Apoptosis in C3H-10T1/2 cells: Roles of intracellular pH,protein kinase C,and the Na+/H+ antiporter

Changes in intracellular ion concentrations have been correlated with the activation of an endogenous endonuclease and thus internucleosomal DNA cleavage during apoptosis in many cell types. We investigated whether intracellular pH could play a significant role in apoptotic initiation and progression in C3H-10T1/2 cells, a cell strain that does not exhibit double-stranded DNA cleavage during apoptosis. Protein kinase C and the Na⁺/H⁺ antiporter, known regulators of intracellular pH, also were assessed for their involvement in apoptosis of C3H-10T1/2 cells. When a H⁺ ionophore was used to clamp intracellular pH to 6.0 or below, a significant level of apoptosis was induced in these cells within 6 h, whereas clamping at pH 6.75 did not induce significant amounts of apoptosis until 36 h after acidification. The acidified cells exhibited classic apoptotic morphology and chromatin condensation, similar to serum withdrawn cells, but failed to show internucleosomal DNA cleavage with electrophoresis of genomic DNA. Our results also suggest that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated inhibition of apoptosis in serum withdrawn C3H-10T1/2 cells functions through a sequential activation of protein kinase C and the Na⁺/H⁺ antiporter; thus, an alkalinization or an inhibition of acidification is involved in this apoptotic block. Serum withdrawal itself does not appear to act through a negative effect on either protein kinase C or the Na⁺/H⁺ antiporter. TPA was also capable of inhibiting the apoptosis induced by specific inhibitors of protein kinase C and the Na⁺/H⁺ antiporter, but the inhibition was successful only if the TPA was administered at least 20 min prior to the addition of the enzyme inhibitor. These results indicate that apoptosis in C3H-10T1/2 cells follows a pathway that involves intracellular acidification, but is independent of detectable endonuclease activity. J. Cell. Biochem. 67:231–240, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells placed into hyperosmotic medium

Time course of changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium with addition of 200 mM sucrose was studied. Ouabain-sensitive and ouabain-resistant Rb+ (K+) influxes were measured during regulatory cell volume increase (RVI) and apoptotic volume decrease (AVD). Microscopy of cells stained by Acrydine orange, Ethydium bromide, APOPercenrage Dye and polycaspase marker FLICA was performed. We found that initial osmotic cell shrinkage induced both RVI and AVD responses. RVI dominated at the early stage whereas AVD prevailed at the later stage. In view of the data obtained in U937 cells the current opinion that RVI "dysfunction" is a prerequisite for apoptosis and AVD (Subramanyam et al., 2010) should be revised. U937 cells are capable to trigger of apoptosis and AVD in spite of the unimpaired RVI response. It is concluded that AVD plays a significant role in preventing osmotic lysis of apoptotic cells rather than in the initiation of apoptosis.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

Staurosporine-induced apoptotic water loss is cell- and attachment-specific

Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking. In this study, we quantified intracellular water in staurosporine-treated cells using a previously described optical microscopic technique that combines volume measurements with quantitative phase analysis. Water loss was observed in detached HeLa and in adherent MDCK but not in adherent HeLa cells. At the same time, adherent HeLa and adherent MDCK cells exhibited visually similar apoptotic morphology, including fragmentation and activation of caspase-3. Morphological changes and caspase activation were prevented by chloride channel blockers DIDS and NPPB in both adherent and suspended HeLa cells, while potassium channel blocker TEA was ineffective. We conclude that staurosporine-induced dehydration is not a universal cell response but depends on the cell type and substrate attachment and can only be judged by direct water measurements. The effects of potassium or chloride channel blockers do not always correlate with the AVD.     

Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation

Cell shrinkage is an incipienthallmark of apoptosis and is accompanied by potassium releasethat decreases the concentration of intracellular potassium andregulates apoptotic progression. The plasma membrane K⁺channel recruited during apoptosis has not been characterized despite its importance as a potential therapeutic target. Here weprovide evidence that two-pore domain K⁺ (K_2P)channels underlie K⁺ efflux during apoptotic volumedecreases (AVD) in mouse embryos. These K_2P channels areinhibited by quinine but are not blocked by an array of pharmacologicalagents that antagonize other K⁺ channels. TheK_2P channels are uniquely suited to participate in theearly phases of apoptosis because they are not modulated bycommon intracellular messengers such as calcium, ATP, and arachidonic acid, transmembrane voltage, or the cytoskeleton. A K⁺channel with similar biophysical properties coordinates regulatory volume decreases (RVD) triggered by changing osmotic conditions. Wepropose that K_2P channels are the pathway by whichK⁺ effluxes during AVD and RVD and that apoptosisco-opts mechanisms more routinely employed for homeostatic cell volume regulation.

     

Swelling rather than shrinkage precedes apoptosis in serum-deprived vascular smooth muscle cells

Contrasting cell volume behaviours (swelling vs. shrinkage) are considered as criteria to distinguish necrosis from apoptosis. In this study, we employed a time-lapse, dual-image surface reconstruction technique to assess the volume of single vascular smooth muscle cells transfected with E1A-adenoviral protein (E1A-VSMC) and undergoing rapid apoptosis in the absence of growth factors or in the presence of staurosporine. After 30- to 60-min lag-phase, serum-deprived E1A-VSMC volume was increased by ~40%, which preceded maximal increments of caspase-3 activity and chromatin cleavage. Swollen cells underwent rapid apoptotic collapse, documented by plasma membrane budding, and terminated in 10–15 min by the formation of numerous apoptotic bodies. Suppression of apoptosis by inhibition of Na⁺,K⁺-ATPase and activation of cAMP signalling with ouabain and forskolin, respectively, completely abolished the swelling of serum-deprived E1A-VSMC. In contrast to serum deprivation, apoptotic collapse of staurosporine-treated E1A-VSMC preceded attenuation of their volume by ~30%. Neither transient hyposmotic swelling nor isosmtotic shrinkage triggered apoptosis. Our results show that cell shrinkage can not be considered as ubiquitous hallmark of apoptosis. The involvement of stimulus-specific cell volume perturbations in initiation and progression of apoptosis in vascular smooth muscle cells should be examined further.     

Elongation of Outer Transmembrane Domain Alters Function of Miniature K+ Channel Kcv

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.     

IKCa1 activity is required for cell shrinkage,phosphatidylserine translocation and death in T lymphocyte apoptosis

Apoptotic cell volume decrease (AVD) and exposure of phosphatidylserine (PtdSer) at the cell surface are early events in apoptosis. However, the ion channels responsible for AVD, and their relationship to PtdSer translocation and cell death are poorly understood. Real-time analysis of calcium-induced apoptosis in lymphocytes and thymocytes showed that AVD occurs rapidly, and precedes PtdSer translocation. Blockers of the K⁺ channel IKCa1 completely inhibited AVD. Blockade of IKCa1, and hence AVD, also completely prevented PtdSer translocation and cell death. Thus, IKCa1-mediated AVD is the earliest-defined essential step in calcium-induced apoptosis, required for both PtdSer translocation and cell death.     

Antagonistic anticancer effect of paclitaxel and digoxin combination

Despite the growing interest in the antitumor effect of cardiotonic steroids, combination treatments with well-established chemotherapy drugs like paclitaxel have been rarely investigated. Moreover, paclitaxel has been suggested as a Na⁺/K⁺-ATPase inhibitor. Here we investigated the effect of paclitaxel and digoxin alone or in combination on the viability of human lung (A549) and cervical cancer (HeLa) cell lines and the inhibitory effect of paclitaxel on several mammalian Na⁺/K⁺-ATPases. Although the viability of both tumor cell lines was concentration-dependently affected by digoxin treatment after 48 hours (A549 IC₅₀ = 31 nM and HeLa IC₅₀ = 151 nM), a partial effect was observed for paclitaxel, with a maximal inhibitory effect of 45% at 1000 nM with A549 and around 70% with HeLa cells (IC₅₀ = 1 nM). Although the two drugs were cytotoxic, their combined effect in HeLa cells was revealed to be antagonistic, as estimated by the combination index. No direct inhibitory effect of paclitaxel was detected in human, pig, rat, and mouse Na⁺/K⁺-ATPase enzymes, but high concentrations of paclitaxel decreased the Na⁺/K⁺-ATPase activity in HeLa cells after 48 hours without affecting protein expression. Our findings demonstrate that, under our conditions, paclitaxel and digoxin cotreatment produce antagonistic cytotoxic effects in HeLa cells, and the mechanism of action of paclitaxel does not involve a direct inhibition of Na⁺/K⁺-ATPase. More studies shall be designed to evaluate the consequences of the interaction of cardiotonic steroids and chemotherapy drugs.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis

Plasma membrane potassium (K⁺) channels are required for tumor cell proliferation and apoptosis. However, the signal transduction mechanisms underlying K⁺ channel-dependent tumor cell proliferation or apoptosis remains elusive. Using HeLa and A2780 cells as study models, we tested the hypothesis that apoptotic proteins are linked with K⁺ channel-dependent tumor cell cycle and apoptosis. The patch-clamping study using the whole-cell mode revealed two components of voltage-gated outward K⁺ currents: one is sensitive to either tetraethylammonium (TEA) or tetrandrine (Tet), a maxi-conductance Ca²⁺-activated K⁺ (BK) channel blocker, and the other is sensitive to 4-aminopyridine (4-AP), a delayed rectifier K⁺ channel blocker. MTT and flow cytometry assays showed that TEA, Tet, or iberiotoxin (Ibtx), a selective BK channel blocker, inhibited HeLa and A2780 cell proliferation in a dose-dependent manner with G₁ phase arrest. Pretreatment with TEA or Tet also induced apoptosis in HeLa and A2780 cells. However, glibenclamide (Gli), an ATP-sensitive K⁺ channel blocker, did not influence K⁺ currents, proliferation or apoptosis. Western blot analyses showed that while pretreatment of TEA and Tet produced an increase in expressions of p53, p21, and Bax, pretreatment of these two agents led to a decrease in expressions of heat shock protein (hsp)90α, hsp90β, and hsp70. Our results indicate that the blockade of BK channels results in tumor cell apoptosis and cycle arrest at G₁ phase, and the transduction pathway underlying the anti-proliferative effects is linked to the increased expression of apoptotic protein p53 and the decreased expression of its chaperone proteins hsp.