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Else K Hoffmann - One of the best experts on this subject based on the ideXlab platform.
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activation of the task 2 channel after Cell Swelling is dependent on tyrosine phosphorylation
American Journal of Physiology-cell Physiology, 2010Co-Authors: Signe Skyum Kirkegaard, Ian Henry Lambert, Steen Gammeltoft, Else K HoffmannAbstract:The Swelling-activated K(+) currents (I(K,vol)) in Ehrlich ascites tumor Cells (EATC) has been reported to be through the two-pore domain (K(2p)), TWIK-related acid-sensitive K(+) channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by I(K,vol) indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower Cell volume. Swelling-activated K(+) efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, Cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K(+) channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 Cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a Cell Swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the Swelling-induced phosphorylation and activation of TASK-2 in EATC.
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Swelling activated ion channels functional regulation in Cell Swelling proliferation and apoptosis
Acta Physiologica, 2006Co-Authors: A Stutzin, Else K HoffmannAbstract:Cell volume regulation is one of the most fundamental homeostatic mechanisms and essential for normal Cellular function. At the same time, however, many physiological mechanisms are associated with regulatory changes in Cell size meaning that the set point for Cell volume regulation is under physiological control. Thus, Cell volume is under a tight and dynamic control and abnormal Cell volume regulation will ultimately lead to severe Cellular dysfunction, including alterations in Cell proliferation and Cell death. This review describes the different Swelling-activated ion channels that participate as key players in the maintenance of normal steady-state Cell volume, with particular emphasis on the intraCellular signalling pathways responsible for their regulation during hypotonic stress, Cell proliferation and apoptosis.
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hypotonic Cell Swelling induces translocation of the α isoform of cytosolic phospholipase a2 but not the γ isoform in ehrlich ascites tumor Cells
FEBS Journal, 2000Co-Authors: Susanne Juhl Pedersen, Ian Henry Lambert, S M Thoroed, Else K HoffmannAbstract:We demonstrate that two isoforms of the cytosolic phospholipase A2, cPLA2alpha and cPLA2gamma, are present in Ehrlich ascites tumor Cells. Both enzymes are almost uniformly distributed throughout the Cells under control conditions, as visualized by laser-scanning confocal microscopy. Stimulation by either hypotonic Cell Swelling or addition of the Ca2+ ionophore A23187 results in translocation of cPLA2alpha, but not cPLA2gamma, to the nucleus, where it forms hot-spot-like clusters. Our group previously showed that release of radioactively labeled arachidonic acid, incorporated into the phospholipids of Ehrlich Cells, was immediately and transiently increased on hypotonic Cell Swelling [Thoroed, S.M., Lauritzen, L., Lambert, I.H., Hansen, H.S. & Hoffmann, E.K. (1997) J. Membr. Biol. 160, 47-58]. We now demonstrate that arachidonic acid is released from the nuclear fraction following hypotonic exposure. Stimulation of Ehrlich Cells with A23187 also leads to an increase in arachidonic acid release from the nucleus. However, as hypotonic Cell Swelling is not accompanied by any detectable increase in intraCellular concentration of free cytosolic Ca2+ ([Ca2+]i), stimulus-induced translocation of cPLA2alpha can also occur without elevation of [Ca2+]i. The stimulus-induced translocation of cPLA2alpha appears not to be prevented by inhibition of mitogen-activated protein (MAP) kinase activation, p38 MAP kinase, tyrosine kinases and protein kinase C, hence, phosphorylation is not crucial for the stimulus-induced translocation of cPLA2alpha. Disruption of F-actin did not affect the translocation process, thus, an intact F-actin cytoskeleton does not seem to be required for translocation of cPLA2alpha.
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characterisation of a Cell Swelling activated k selective conductance of ehrlich mouse ascites tumour Cells
The Journal of Physiology, 2000Co-Authors: Else K Hoffmann, Maria Isabel Niemeyer, Charlotte Hougaard, Finn Jorgensen, Andres Stutzin, Francisco V SepulvedaAbstract:1 The K+ and Cl− currents activated by hypotonic Cell Swelling were studied in Ehrlich ascites tumour Cells using the whole-Cell recording mode of the patch-clamp technique. 2 Currents were measured in the absence of added intraCellular Ca2+ and with strong buffering of Ca2+. K+ current activated by Cell Swelling was measured as outward current at the Cl− equilibrium potential (ECl) under quasi-physiological gradients. It could be abolished by replacing extraCellular Na+ with K+, thereby canCelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4≈ Na+≈ Li+; Cs+ appeared to be inhibitory. 3 The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and +20 mV with a permeability coefficient of around 10−6 cm s−1 with both physiological and high-K+ extraCellular solutions. 4 The class III antiarrhythmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner with an IC50 of 32 μM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich Cells. 5 Cell Swelling-activated K+ currents of Ehrlich Cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so-called background K+ channels. 6 Noise analysis of whole-Cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by Cell Swelling, measured at 0 mV under a quasi-physiological K+ gradient.
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Cell Swelling activates phospholipase a2 in ehrlich ascites tumor Cells
The Journal of Membrane Biology, 1997Co-Authors: S M Thoroed, Ian Henry Lambert, Lotte Lauritzen, Harald S Hansen, Else K HoffmannAbstract:Ehrlich ascites tumor Cells, loaded with 3H-labeled arachidonic acid and 14C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the Cells. During the first two minutes of hypo-osmotic exposure the rate of 3H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell Swelling also causes an increase in the production of 14C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A2 is activated by Cell Swelling in the Ehrlich Cells. Within the same time frame there is no Swelling-induced increase in 14C-labeled stearic acid release nor in the synthesis of phosphatidyl 14C-butanol in the presence of 14C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the Swelling induced release of 14C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A1, C and D in the Swelling-induced liberation of arachidonic acid. The Swelling-induced release of 3H-labeled arachidonic acid from Ehrlich Cells as well as the volume regulatory response are inhibited after preincubation with GDPβS or with AACOCF3, an inhibitor of the 85 kDa, cytosolic phospholipase A2. Based on these results we propose that Cell Swelling activates a phospholipase A2—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response.
Michael D Norenberg - One of the best experts on this subject based on the ideXlab platform.
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Microglia contribute to ammonia-induced astrocyte Swelling in culture
Metabolic Brain Disease, 2013Co-Authors: Kakulavarapu V. Rama Rao, Monica Brahmbhatt, Michael D NorenbergAbstract:Brain edema, a lethal complication of acute liver failure (ALF), is believed to be largely cytotoxic due to the Swelling of astrocytes. Ammonia, a principal neurotoxin in ALF, has been strongly implicated in the development of the brain edema. It was previously shown that treatment of cultured astrocytes with ammonia (5 mM NH_4Cl) results in Cell Swelling. While ammonia continues to exert a direct effect on astrocytes, it is possible that ammonia can affect other neural Cells, particularly microglia. Microglia are capable of evoking an inflammatory response, a process known to contribute to the brain edema. We therefore examined the potential role of microglia in the mechanism of ammonia-induced astrocyte Swelling. Conditioned media (CM) derived from ammonia-treated cultured microglia when added to cultured astrocytes resulted in significant Cell Swelling. Such Swelling was synergistically increased when astrocytes were additionally treated with 5 mM ammonia. CM from ammonia-treated microglia also showed significant release of oxy-radicals and nitric oxide into the CM. CM from ammonia-treated microglia containing Tempol (a superoxide scavenger) or uric acid (a peroxynitrite scavenger) when added to astrocytes resulted in marked reduction in the Cell Swelling. Together, these studies indicate that microglia contribute to the ammonia-induced astrocyte Swelling by a mechanism involving oxidative/nitrosative stress.
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na k cl cotransporter 1 in the mechanism of Cell Swelling in cultured astrocytes after fluid percussion injury
Journal of Neurochemistry, 2011Co-Authors: Arumugam R Jayakumar, Kiran S Panickar, Mitsuaki Moriyama, Michael D Norenberg, Kevin M Curtis, Xiao Y TongAbstract:Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury (TBI). An important early component of the edema associated with TBI is astrocyte Swelling (cytotoxic edema). Mechanisms for such Swelling, however, are poorly understood. Ion channels/transporters/exchangers play a major role in Cell volume regulation, and a disturbance in one or more of these systems may result in Cell Swelling. To examine potential mechanisms in TBI-mediated brain edema, we employed a fluid percussion model of in vitro barotrauma and examined the role of the ion transporter Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) in trauma-induced astrocyte Swelling as this transporter has been strongly implicated in the mechanism of Cell Swelling in various neurological conditions. Cultures exposed to trauma (3, 4, 5 atm pressure) caused a significant increase in NKCC1 activity (21%, 42%, 110%, respectively) at 3 h. At 5 atm pressure, trauma significantly increased NKCC1 activity at 1 h and it remained increased for up to 3 h. Trauma also increased the phosphorylation (activation) of NKCC1 at 1 and 3 h. Inhibition of MAPKs and oxidative/nitrosative stress diminished the trauma-induced NKCC1 phosphorylation as well as its activity. Bumetanide, an inhibitor of NKCC1, significantly reduced the trauma-induced astrocyte Swelling (61%). Silencing NKCC1 with siRNA led to a reduction in trauma-induced NKCC1 activity as well as in Cell Swelling. These findings demonstrate the critical involvement of NKCC1 in the astrocyte Swelling following in vitro trauma, and suggest that blocking NKCC1 activity may represent a useful therapeutic strategy for the cytotoxic brain edema associated with the early phase of TBI.
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marked potentiation of Cell Swelling by cytokines in ammonia sensitized cultured astrocytes
Journal of Neuroinflammation, 2010Co-Authors: Kakulavarapu Rama V Rao, Arumugam R Jayakumar, Michael D Norenberg, Xiaoying Tong, Veronica M AlvarezAbstract:Brain edema leading to high intracranial pressure is a lethal complication of acute liver failure (ALF), which is believed to be cytotoxic due to Swelling of astrocytes. In addition to the traditional view that elevated levels of blood and brain ammonia are involved in the mechanism of brain edema in ALF, emerging evidence suggests that inflammatory cytokines also contribute to this process. We earlier reported that treatment of astrocyte cultures with a pathophysiological concentration of ammonia (5 mM NH4Cl) resulted in the activation of nuclear factor-kappaB (NF-κB) and that inhibition of such activation diminished astrocyte Swelling, suggesting a key role of NF-κB in the mechanism of ammonia-induced astrocyte Swelling. Since cytokines are also well-known to activate NF-κB, this study examined for additive/synergistic effects of ammonia and cytokines in the activation of NF-κB and their role in astrocyte Swelling. Primary cultures of astrocytes were treated with ammonia and cytokines (TNF-α, IL-1, IL-6, IFN-γ, each at 10 ng/ml), individually or in combination, and Cell volume was determined by the [3H]-O-methylglucose equilibration method. The effect of ammonia and cytokines on the activation of NF-κB was determined by immunoblots. Cell Swelling was increased by ammonia (43%) and by cytokines (37%) at 24 h. Simultaneous co-treatment with cytokines and ammonia showed no additional Swelling. By contrast, cultures pretreated with ammonia for 24 h and then exposed to cytokines for an additional 24 h, showed a marked increase in astrocyte Swelling (129%). Treatment of cultures with ammonia or cytokines alone also activated NF-κB (80-130%), while co-treatment had no additive effect. However, in cultures pre-treated with ammonia for 24 h, cytokines induced a marked activation of NF-κB (428%). BAY 11-7082, an inhibitor of NF-κB, completely blocked the astrocyte Swelling in cultures pre-treated with ammonia and followed by the addition of a mixture of cytokines. Our results indicate that ammonia and a mixture of cytokines each cause astrocyte Swelling but when these agents are added simultaneously, no additive effects were found. On the other hand, when Cells were initially treated with ammonia and 24 h later given a mixture of cytokines, a marked potentiation in Cell Swelling and NF-κB activation occurred. These data suggest that the potentiation in Cell Swelling is a consequence of the initial activation of NF-κB by ammonia. These findings provide a likely mechanism for the exacerbation of brain edema in patients with ALF in the setting of sepsis/inflammation.
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ammonia induced activation of p53 in cultured astrocytes role in Cell Swelling and glutamate uptake
Neurochemistry International, 2009Co-Authors: K S Panickar, Arumugam R Jayakumar, K Rama V Rao, Michael D NorenbergAbstract:Cytotoxic brain edema, due principally to astrocyte Swelling, is a major neurological complication of the acute form of hepatic encephalopathy (HE) (acute liver failure, ALF), a condition likely caused by elevated levels of brain ammonia. Potential mediators of ammonia-induced astrocyte Swelling include oxidative/nitrosative stress (ONS), the mitochondrial permeability transition (mPT), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB), since blockade of these factors reduces the extent of astrocyte Swelling. As p53, a tumor suppressor protein and transcription factor, is a downstream target of ONS and MAPKs, we examined its potential role in the mechanism of ammonia-induced astrocyte Swelling. Astrocytes exposed to NH(4)Cl (5mM) showed increased phosphorylation (activation) of p53((Ser392)) at 1h and such phosphorylation was significantly reduced by inhibitors of MAPKs (ERK1/2, JNK and p38-MAPK), antioxidants (vitamin E, catalase, PBN, desferoxamine, MnTBAP), as well as by L-NAME, an inhibitor of nitric oxide synthase, indicating a key role of oxidative/nitrosative stress and MAPKs in the ammonia-induced activation of p53. Since p53 is known to induce the mPT and to activate NF-kappaB (factors leading to ONS and implicated in ammonia-induced astrocyte Swelling), we examined whether inhibition of p53 activation blocked mPT induction, NF-kappaB activation, as well as Cell Swelling. Pifithrin-alpha (PFT), an inhibitor of p53, blocked these processes. Impairment of astrocytic glutamate uptake is another important feature of HE and hyperammonemia. We therefore examined the potential role of p53 in the ammonia-induced inhibition of glutamate uptake and found that PFT also reversed the ammonia-induced inhibition of glutamate uptake. Our results indicate that a potentially important downstream target of ammonia neurotoxicity is p53, whose activation contributes to astrocyte Swelling and glutamate uptake inhibition, processes likely a consequence of ONS derived from the mPT and activation of NF-kappaB.
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trauma induced Cell Swelling in cultured astrocytes
Journal of Neuropathology and Experimental Neurology, 2008Co-Authors: Arumugam R Jayakumar, Kiran S Panickar, K Rama V Rao, Mitsuaki Moriyama, Pichili V B Reddy, Michael D NorenbergAbstract:Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury that account for most early deaths after traumatic brain injury. An important component of brain edema after traumatic brain injury is astrocyte Swelling (cytotoxic edema). To examine the pathophysiologic mechanisms of trauma-induced astrocyte Swelling, we used an in vitro fluid percussion trauma model. Exposure of cultured rat astrocytes to 5 atm of pressure resulted in significant Cell Swelling at 1 to 24 hours posttrauma that was maximal at 3 hours. Because oxidative/nitrosative stress, mitochondrial permeability transition (mPT), and mitogen-activated protein kinases (MAPKs) have been implicated in astrocyte Swelling in other neurologic conditions, we examined their potential roles in this model. We previously showed increased free radical generation after in vitro trauma and show here that trauma to astrocytes increased the production of nitric oxide. Trauma also induced mPT and increased phosphorylation (activation) of MAPKs (extraCellular signal-regulated kinase 1/2, c-Jun-N-terminal kinase, and p38-MAPK); these changes were diminished by antioxidants and the nitric oxide synthase inhibitor N -nitro-l-arginine methyl ester. Antioxidants, N -nitro-l-arginine methyl ester, the mPT inhibitor cyclosporin A, and inhibitors of MAPKs all significantly diminished trauma-induced astrocyte Swelling. These findings demonstrate that direct mechanical injury to cultured astrocytes brings about Cell Swelling, and that blockade of oxidative/nitrosative stress, mPT, and MAPKs significantly reduce such Swelling.
Raynald Laprade - One of the best experts on this subject based on the ideXlab platform.
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hypotonicity increases basolateral taurine permeability in rabbit proximal convoluted tubule
American Journal of Physiology-renal Physiology, 1995Co-Authors: Sylvie Breton, Mireille Marsolais, Raynald LapradeAbstract:The permeabilities of the basolateral membrane of rabbit proximal convoluted tubule (PCT) to taurine (PTau) and glucose (PGlc) were estimated under control and hypotonic conditions using the initial rate of increase in Cellular volume (CV) induced on isotonic replacement of 40 mM mannitol by one or the other of these substrates. Under control conditions, addition of taurine led to an increase in CV at an initial rate of 7.1 +/- 1.7%/min, leading to a Cell Swelling of 30.2 +/- 4.8% after 5 min (n = 6). Addition of glucose led to an increase in CV at an initial rate of 30.0 +/- 3.8%/min, leading to a Cell Swelling of 25.7 +/- 3.1% after 5 min (n = 7). After a period of recovery of 5 min in the absence of taurine or glucose, a 40 mosmol/kg hypotonic shock induced a Cell Swelling of 14.2 +/- 1.3 and 16.1 +/- 5.2%, respectively, followed by an almost complete volume regulatory decrease after 5 min. At that time, addition of taurine under continuous hypotonicity induced an increase in CV at an initial rate 2.57...
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involvement and source of calcium in volume regulatory decrease of collapsed proximal convoluted tubule
American Journal of Physiology-renal Physiology, 1992Co-Authors: Sylvie Breton, J S Beck, J Cardinal, G Giebisch, Raynald LapradeAbstract:We examined the role of Ca2+ in the volume regulatory decrease (VRD) of rabbit collapsed proximal tubules. Reduction of bath osmolality by 125 mosmol/kgH2O led to an initial Cell Swelling of 62.3 +...
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volume regulation and intraCellular calcium in the rabbit proximal convoluted tubule
American Journal of Physiology-renal Physiology, 1991Co-Authors: J S Beck, Sylvie Breton, Raynald LapradeAbstract:The hypothesis that an increase of calcium leads to activation of calcium-activated ionic conductances during Cell Swelling was examined in the isolated perfused proximal convoluted tubule of the r...
Ian Henry Lambert - One of the best experts on this subject based on the ideXlab platform.
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activation of the task 2 channel after Cell Swelling is dependent on tyrosine phosphorylation
American Journal of Physiology-cell Physiology, 2010Co-Authors: Signe Skyum Kirkegaard, Ian Henry Lambert, Steen Gammeltoft, Else K HoffmannAbstract:The Swelling-activated K(+) currents (I(K,vol)) in Ehrlich ascites tumor Cells (EATC) has been reported to be through the two-pore domain (K(2p)), TWIK-related acid-sensitive K(+) channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by I(K,vol) indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower Cell volume. Swelling-activated K(+) efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, Cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K(+) channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 Cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a Cell Swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the Swelling-induced phosphorylation and activation of TASK-2 in EATC.
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activation of pla2 isoforms by Cell Swelling and ischaemia hypoxia
Acta Physiologica, 2006Co-Authors: Ian Henry Lambert, Stine F Pedersen, Kristian Arild PoulsenAbstract:Phospholipase A2 (PLA2) activity is increased in mammalian Cells in response to numerous stimuli such as osmotic challenge, oxidative stress and exposure to allergens. The increased PLA2 activity is seen as an increased release of free, polyunsaturated fatty acids, e.g. arachidonic acid and membrane-bound lysophospholipids. Even though arachidonic acid acts as a second messenger in its own most mammalian Cells seem to rely on oxidation of the fatty acid into highly potent second messengers via, e.g. cytochrome P450, the cyclo-oxygenase, or the lipoxygenase systems for downstream signalling. Here, we review data that illustrates that stress-induced PLA2 activity involves various PLA2 subtypes and that the PLA2 in question is determined by the Cell type and the physiological stress condition.
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regulation of the Cellular content of the organic osmolyte taurine in mammalian Cells
Neurochemical Research, 2004Co-Authors: Ian Henry LambertAbstract:Change in the intraCellular concentration of osmolytes or the extraCellular tonicity results in a rapid transmembrane water flow in mammalian Cells until intraCellular and extraCellular tonicities are equilibrated. Most Cells respond to the osmotic Cell Swelling by activation of volume-sensitive flux pathways for ions and organic osmolytes to restore their original Cell volume. Taurine is an important organic osmolyte in mammalian Cells, and taurine release via a volume-sensitive taurine efflux pathway is increased and the active taurine uptake via the taurine specific taurine transporter TauT decreased following osmotic Cell Swelling. The Cellular signaling cascades, the second messengers profile, the activation of specific transporters, and the subsequent time course for the readjustment of the Cellular content of osmolytes and volume vary from Cell type to Cell type. Using Ehrlich ascites tumor Cells, NIH3T3 mouse fibroblasts and HeLa Cells as biological systems, it is revealed that phospholipase A2-mediated mobilization of arachidonic acid from phospholipids and subsequent oxidation of the fatty acid via lipoxygenase systems to potent eicosanoids are essential elements in the signaling cascade that is activated by Cell Swelling and leads to release of osmolytes. The Cellular signaling cascade and the activity of the volume-sensitive taurine efflux pathway are modulated by elements of the cytoskeleton, protein tyrosine kinases/phosphatases, GTP-binding proteins, Ca2+/calmodulin, and reactive oxygen species and nucleotides. Serine/threonine phosphorylation of the active taurine uptake system TauT or a putative regulator, as well as change in the membrane potential, are important elements in the regulation of TauT activity. A model describing the Cellular sequence, which is activated by Cell Swelling and leads to activation of the volume-sensitive efflux pathway, is presented at the end of the review.
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hypotonic Cell Swelling induces translocation of the α isoform of cytosolic phospholipase a2 but not the γ isoform in ehrlich ascites tumor Cells
FEBS Journal, 2000Co-Authors: Susanne Juhl Pedersen, Ian Henry Lambert, S M Thoroed, Else K HoffmannAbstract:We demonstrate that two isoforms of the cytosolic phospholipase A2, cPLA2alpha and cPLA2gamma, are present in Ehrlich ascites tumor Cells. Both enzymes are almost uniformly distributed throughout the Cells under control conditions, as visualized by laser-scanning confocal microscopy. Stimulation by either hypotonic Cell Swelling or addition of the Ca2+ ionophore A23187 results in translocation of cPLA2alpha, but not cPLA2gamma, to the nucleus, where it forms hot-spot-like clusters. Our group previously showed that release of radioactively labeled arachidonic acid, incorporated into the phospholipids of Ehrlich Cells, was immediately and transiently increased on hypotonic Cell Swelling [Thoroed, S.M., Lauritzen, L., Lambert, I.H., Hansen, H.S. & Hoffmann, E.K. (1997) J. Membr. Biol. 160, 47-58]. We now demonstrate that arachidonic acid is released from the nuclear fraction following hypotonic exposure. Stimulation of Ehrlich Cells with A23187 also leads to an increase in arachidonic acid release from the nucleus. However, as hypotonic Cell Swelling is not accompanied by any detectable increase in intraCellular concentration of free cytosolic Ca2+ ([Ca2+]i), stimulus-induced translocation of cPLA2alpha can also occur without elevation of [Ca2+]i. The stimulus-induced translocation of cPLA2alpha appears not to be prevented by inhibition of mitogen-activated protein (MAP) kinase activation, p38 MAP kinase, tyrosine kinases and protein kinase C, hence, phosphorylation is not crucial for the stimulus-induced translocation of cPLA2alpha. Disruption of F-actin did not affect the translocation process, thus, an intact F-actin cytoskeleton does not seem to be required for translocation of cPLA2alpha.
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Cell Swelling activates phospholipase a2 in ehrlich ascites tumor Cells
The Journal of Membrane Biology, 1997Co-Authors: S M Thoroed, Ian Henry Lambert, Lotte Lauritzen, Harald S Hansen, Else K HoffmannAbstract:Ehrlich ascites tumor Cells, loaded with 3H-labeled arachidonic acid and 14C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the Cells. During the first two minutes of hypo-osmotic exposure the rate of 3H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell Swelling also causes an increase in the production of 14C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A2 is activated by Cell Swelling in the Ehrlich Cells. Within the same time frame there is no Swelling-induced increase in 14C-labeled stearic acid release nor in the synthesis of phosphatidyl 14C-butanol in the presence of 14C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the Swelling induced release of 14C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A1, C and D in the Swelling-induced liberation of arachidonic acid. The Swelling-induced release of 3H-labeled arachidonic acid from Ehrlich Cells as well as the volume regulatory response are inhibited after preincubation with GDPβS or with AACOCF3, an inhibitor of the 85 kDa, cytosolic phospholipase A2. Based on these results we propose that Cell Swelling activates a phospholipase A2—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response.
Ryszard Grygorczyk - One of the best experts on this subject based on the ideXlab platform.
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membrane reserves and hypotonic Cell Swelling
The Journal of Membrane Biology, 2006Co-Authors: Nicolas Groulx, Francis Boudreault, S N Orlov, Ryszard GrygorczykAbstract:To accommodate expanding volume (V) during hyposmotic Swelling, animal Cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intraCellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o−, CHO and NIH 3T3 Cells were evaluated by reconstructing Cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by Swelling Cells in extreme 98% hypotonic (∼6 mOsm) solution until membrane rupture; all Cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 ± 0.2-fold and V 10.7 ± 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10°C) reduced SA and V increases of A549 Cells to 1.7 ± 0.3-fold and 4.4 ± 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate Swelling in 50% hypotonicity. Thus, mammalian Cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme Swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various Cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian Cells.
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Cell Swelling induced atp release is tightly dependent on intraCellular calcium elevations
The Journal of Physiology, 2004Co-Authors: Francis Boudreault, Ryszard GrygorczykAbstract:Mechanical stresses release ATP from a variety of Cells by a poorly defined mechanism(s). Using custom-designed flow-through chambers, we investigated the kinetics of Cell Swelling-induced ATP secretion, Cell volume and intraCellular calcium changes in epithelial A549 and 16HBE14o− Cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from Cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o− Cells, then declined to baseline within the next 15 min. Whereas the release time course had a similar pattern for the three Cell types, the peak rates differed significantly (294 ± 67, 70 ± 22 and 17 ± 2.8 pmol min−1 (106 Cells)−1, for A549, 16HBE14o− and NIH/3T3, respectively). The concomitant volume changes of substrate-attached Cells were analysed by a 3-dimensional Cell shape reconstruction method based on images acquired from two perpendicular directions. The three Cell types swelled at a similar rate, reaching maximal expansion in 1 min 45 s, but differed in the duration of the volume plateau and regulatory volume decrease (RVD). These experiments revealed that ATP release does not correlate with either Cell volume expansion and the expected activation of stretch-sensitive channels, or with the activation of volume-sensitive, 5-nitro-2-(3-phenylpropylamino) benzoic acid-inhibitable anion channels during RVD. By contrast, ATP release was tightly synchronized, in all three Cell types, with cytosolic calcium elevations. Furthermore, loading A549 Cells with the calcium chelator BAPTA significantly diminished ATP release (71% inhibition of the peak rate), while the calcium ionophore ionomycin triggered ATP release in the absence of Cell Swelling. Lowering the temperature to 10°C almost completely abolished A549 Cell Swelling-induced ATP release (95% inhibition of the peak rate). These results strongly suggest that calcium-dependent exocytosis plays a major role in mechanosensitive ATP release.
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Cell Swelling induced atp release and gadolinium sensitive channels
American Journal of Physiology-cell Physiology, 2002Co-Authors: Francis Boudreault, Ryszard GrygorczykAbstract:ATP release induced by hypotonic Swelling is an ubiquitous phenomenon in eukaryotic Cells, but its underlying mechanisms are poorly defined. A mechanosensitive (MS) ATP channel has been implicated ...
