Kristian Arild Poulsen

Deregulation of apoptotic volume decrease and ionic movements in multidrug-resistant tumor cells: role of chloride channels

Changes in cell volume and ion gradients across the plasma membrane play a pivotal role in the initiation of apoptosis. Here we explore the kinetics of apoptotic volume decrease (AVD) and ion content dynamics in wild-type (WT) and multidrug-resistant (MDR) Ehrlich ascites tumor cells (EATC). In WT EATC, induction of apoptosis with cisplatin (5 muM) leads to three distinctive AVD stages: an early AVD(1) (4-12 h), associated with a 30% cell water loss; a transition stage AVD(T) ( approximately 12 to 32 h), where cell volume is partly recovered; and a secondary AVD(2) (past 32 h), where cell volume was further reduced. AVD(1) and AVD(2) were coupled to net loss of Cl(-), K(+), Na(+), and amino acids (ninhydrin-positive substances), whereas during AVD(T), Na(+) and Cl(-) were accumulated. MDR EATC was resistant to cisplatin, showing increased viability and less caspase 3 activation. Compared with WT EATC, MDR EATC underwent a less pronounced AVD(1,) an augmented AVD(T), and a delay in induction of AVD(2). Changes in AVD were associated with inhibition of Cl(-) loss during AVD(1), augmented NaCl uptake during AVD(T), and a delay of Cl(-) loss during AVD(2). Application of the anion channel inhibitor NS3728 inhibited AVD and completely abolished the differences in AVD, ionic movements, and caspase 3 activation between WT and MDR EATC. Finally, the maximal capacity of volume-regulated anion channel was found to be strongly repressed in MDR EATC. Together, these data suggest that impairment of AVD, primarily via modulation of NaCl movements, contribute to protection against apoptosis in MDR EATC.

TMEM16F (Anoctamin 6), an anion channel of delayed Ca2+ activation

Members of the TMEM16 (Anoctamin) family of membrane proteins have been shown to be essential constituents of the Ca2+-activated Cl− channel (CaCC) in many cell types. In this study, we have investigated the electrophysiological properties of mouse TMEM16F. Heterologous expression of TMEM16F in HEK293 cells resulted in plasma membrane localization and an outwardly rectifying ICl,Ca that was activated with a delay of several minutes. Furthermore, a significant Na+ current was activated, and the two permeabilities were correlated according to PNa = 0.3 PCl. The current showed an EC50 of 100 µM intracellular free Ca2+ concentration and an Eisenman type 1 anion selectivity sequence of PSCN > PI > PBr > PCl > PAsp. The mTMEM16F-associated ICl,Ca was abolished in one mutant of the putative pore region (R592E) but retained in two other mutants (K616E and R636E). The mutant K616E had a lower relative permeability to iodide, and the mutant R636E had an altered anion selectivity sequence (PSCN = PI = PBr = PCl > PAsp). Our data provide evidence that TMEM16F constitutes a Ca2+-activated anion channel or a pore-forming subunit of an anion channel with properties distinct from TMEM16A.

Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca2

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6’s physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich–Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca2+ is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium.

Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na+/H+ exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716–19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na+/H+ antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg425, which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.

Pinpointing differences in cisplatin-induced apoptosis in adherent and non-adherent cancer cells.

Platinum compounds are used in the treatment of cancer. We demonstrate that cisplatin-induced (10 µM) apoptosis (caspase-3 activity) is pronounced within 18 hours in non-adherent Ehrlich ascites tumour cells (EATC), whereas there is no increase in caspase-3 activity in the adherent Ehrlich LettrÉ ascites tumour cells (ELA). Loss of KCl and cell shrinkage are hallmarks in apoptosis and has been shown in EATC. However, we find no reduction in cell volume and only a minor loss of K+ which is accompanied by net uptake of Na+ following 18 hours cisplatin exposure in ELA. Glutathione and taurine have previously been demonstrated to protect cells from apoptosis. We find, however, that increase or decrease in the cellular content of glutathione and taurine has no effect on cisplatin-induced cell death in EATC and ELA. Nevertheless, knock-down of the taurine transporter TauT leads to a significant increase in apoptosis in ELA following cisplatin exposure. We find that cytosolic accumulation of cisplatin is similar in EATC and ELA. However, the nuclear accumulation and DNA-binding of cisplatin is significant lower in ELA compared to EATC. We suggest three putative reasons for the observed cisplatin insensitivity in the adherent tumor cells (ELA) compared to the non-adherent tumor cells (EATC): less nuclear cisplatin accumulation, increased TauT activity, and decreased anion and water loss.

HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange

The Ca(2+)-independent phospholipase A(2) VI (iPLA(2)-VI) and the Na(+)/H(+) exchanger isoform 1 (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH (pH(o)) in ischemia-reperfusion injury and death and in regulation and function of iPLA(2)-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% O(2), 8 mM K(+), and 20 mM lactate) at pH(o) 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA(2)-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content. iPLA(2)-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pH(o)-dependent manner during SI/R. This also affected the subcellular localization of iPLA(2)-VIA. Thus, the mode of cell death and the roles and regulation of iPLA(2)-VI and NHE1 are at least in part determined by the pH(o) during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of iPLA(2)-VIA and NHE1 during cardiac I/R.

Limited Energy Supply in Müller Cells Alters Glutamate Uptake

The viability of retinal ganglion cells (RGC) is essential for the maintenance of visual function. RGC homeostasis is maintained by the surrounding retinal glial cells, the Müller cells, which buffer the extracellular concentration of neurotransmitters and provide the RGCs with energy. This study evaluates if glucose-deprivation of Müller cells interferes with their ability to remove glutamate from the extracellular space. The human Müller glial cell line, Moorfields/Institute of Ophthalmology-Müller 1, was used to study changes in glutamate uptake. Excitatory amino acid transporter (EAAT) proteins were up-regulated in glucose-deprived Müller cells and glutamate uptake was significantly increased in the absence of glucose. The present findings revealed an up-regulation of EAAT1 and EAAT2 in glucose-deprived Müller cells as well as an increased ability to take up glutamate. Hence, glucose deprivation may result in an increased ability to protect RGCs from glutamate-induced excitotoxicity, whereas malfunction of glutamate uptake in Müller cells may contribute to retinal neurodegeneration.

Interaction between VEGF and Calcium-Independent Phospholipase A2 in Proliferation and Migration of Retinal Pigment Epithelium

Purpose: Inhibition of VEGF in the eye is an important treatment modality for reducing proliferation and migration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Additionally, previous studies suggest calcium-independent phospholipase A2 group VIA (iPLA2-VIA) to be a potential regulator of cell proliferation and migration, and evidence show abundant expression of iPLA2-VIA in RPE cells. The aim of the present study was to evaluate the potential role of iPLA2-VIA in VEGF-induced proliferation and migration of RPE cells. Materials and methods: The human RPE cell line, ARPE-19, was used in all assays. To explore the role of iPLA2-VIA in VEGF-induced RPE proliferation and migration, iPLA2-VIA inhibition by the iPLA2-VIA specific inhibitor, bromoenol lactone, was done. RPE cell proliferation and migration were evaluated by measurements of incorporated radioactive thymidine in DNA and by a Boyden chamber technique, respectively. A luciferase assay monitored the VEGF-induced iPLA2-VIA transcriptional activity. Western blot analysis and an activity assay were used to detect the protein levels and activity of iPLA2-VIA respectively after treatment with VEGF. Results: RPE cells treated with VEGF showed significant increased proliferation and migration. Furthermore, inhibition of iPLA2-VIA significantly reduced the spontaneous proliferation and migration as well as the VEGF-induced proliferation and migration. Finally, inhibition of iPLA2-VIA reduced the VEGF-induced iPLA2-VIA-activity, -protein level, and -promoter activity. Conclusions: A significant interaction between VEGF and iPLA2-VIA in the regulation of RPE cells appears to be relevant in elucidating the exact mechanisms of action in the proliferative and migratory phenotype of RPE cells in AMD.

Cyclooxygenase-2 immunoreactivity in collagenous colitis.

Collagenous colitis (CC) is an inflammatory bowel disease of unknown aetiology and pathogenesis. In ulcerative colitis and Crohns disease, prostaglandins may be involved in the pathogenesis of inflammation, and increased expression of cyclo‐oxygenase‐2 (COX‐2) has been detected. The purpose of this study was to examine the presence and cellular localization of COX‐2 in colonic mucosa of patients with CC. Using immunohistochemistry, immunoflouresence and Western blot analysis, COX‐2 expression was evaluated in colonic mucosal biopsies from 10 patients with active untreated CC, and compared with samples from eight normal controls, and samples from eight patients with ulcerative colitis or Crohns disease. Specimens from patients with CC expressed COX‐2 protein in increased amounts compared with controls, but similar to patients with ulcerative colitis and Crohns disease. COX‐2 expression was localized to the mononuclear cells of the lamina propria. COX‐2 expression was most evident in macrophages. Co‐localization of COX‐2 and macrophages was increased in number in comparison with controls. In conclusion COX‐2 is expressed in increased amounts primarily in the macrophage subpopulation of the inflammatory infiltrate of lamina propria in CC. Increased recruitment of macrophages, increased expression of COX‐2 and increased prostaglandin synthesis may be involved in the pathogenesis of CC.