Volume-sensitive release of organic osmolytes in the human lung epithelial cell line A549: role of the 5-lipoxygenase

2013 ◽  
Vol 305 (1) ◽  
pp. C48-C60 ◽  
Author(s):  
Jacob Bak Holm ◽  
Ryszard Grygorczyk ◽  
Ian Henry Lambert
Keyword(s):  
Cell Volume ◽  
Tyrosine Kinases ◽  
Janus Kinase ◽  
Butylated Hydroxytoluene ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Taurine Release ◽  
Diphenylene Iodonium ◽  
Lung Epithelial

Pathophysiological conditions challenge cell volume homeostasis and perturb cell volume regulatory mechanisms leading to alterations of cell metabolism, active transepithelial transport, cell migration, and death. We report that inhibition of the 5-lipoxygenase (5-LO) with AA861 or ETH 615-139, the cysteinyl leukotriene 1 receptor (CysLT1) with the antiasthmatic drug Zafirlukast, or the volume-sensitive organic anion channel (VSOAC) with DIDS blocks the release of organic osmolytes (taurine, meAIB) and the concomitant cell volume restoration following hypoosmotic swelling of human type II-like lung epithelial cells (A549). Reactive oxygen species (ROS) are produced in A549 cells upon hypotonic cell swelling by a diphenylene iodonium-sensitive NADPH oxidase. The swelling-induced taurine release is suppressed by ROS scavenging (butylated hydroxytoluene, N-acetyl cysteine) and potentiated by H2O2. Ca2+mobilization with ionomycin or ATP stimulates the swelling-induced taurine release whereas calmodulin inhibition (W7) inhibits the release. Chelation of the extracellular Ca2+(EGTA) had no effect on swelling-induced taurine release but prevented ATP-induced stimulation. H2O2, ATP, and ionomycin were unable to stimulate the taurine release in the presence of AA861 or Zafirlukast, placing 5-LO and CysLT1as essential elements in the swelling-induced activation of VSOAC with ROS and Ca2+as potent modulators. Inhibition of tyrosine kinases (genistein, cucurbitacin) reduces volume-sensitive taurine release, adding tyrosine kinases (Janus kinase) as regulators of VSOAC activity. Caspase-3 activity during hypoxia is unaffected by inhibition of 5-LO/CysLT1but reduced when swelling-induced taurine loss via VSOAC is prevented by DIDS excess extracellular taurine, indicating a beneficial role of taurine under hypoxia.

Osmotic regulation of hepatic betaine metabolism

2013 ◽  
Vol 304 (9) ◽  
pp. G835-G846 ◽  
Author(s):  
Lars Hoffmann ◽  
Gernot Brauers ◽  
Thor Gehrmann ◽  
Dieter Häussinger ◽  
Ertan Mayatepek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Volume ◽  
Tyrosine Kinases ◽  
Methylenetetrahydrofolate Reductase ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Expression Studies ◽  
Dependent Protein ◽  
Gene Expression Studies ◽  
Betaine Homocysteine Methyltransferase

Betaine critically contributes to the control of hepatocellular hydration and provides protection of the liver from different kinds of stress. To investigate how the hepatocellular hydration state affects gene expression of enzymes involved in the metabolism of betaine and related organic osmolytes, we used quantitative RT-PCR gene expression studies in rat hepatoma cells as well as metabolic and gene expression profiling in primary hepatocytes of both wild-type and 5,10-methylenetetrahydrofolate reductase (MTHFR)-deficient mice. Anisotonic incubation caused coordinated adaptive changes in the expression of various genes involved in betaine metabolism, in particular of betaine homocysteine methyltransferase, dimethylglycine dehydrogenase, and sarcosine dehydrogenase. The expression of betaine-degrading enzymes was downregulated by cell shrinking and strongly induced by an increase in cell volume under hypotonic conditions. Metabolite concentrations in the culture system changed accordingly. Expression changes were mediated through tyrosine kinases, cyclic nucleotide-dependent protein kinases, and JNK-dependent signaling. Assessment of hepatic gene expression using a customized microarray chip showed that hepatic betaine depletion in MTHFR −/− mice was associated with alterations that were comparable to those induced by cell swelling in hepatocytes. In conclusion, the adaptation of hepatocytes to changes in cell volume involves the coordinated regulation of betaine synthesis and degradation and concomitant changes in intracellular osmolyte concentrations. The existence of such a well-orchestrated response underlines the importance of cell volume homeostasis for liver function and of methylamine osmolytes such as betaine as hepatic osmolytes.

Osmotic shrinkage elicits FAK- and Src phosphorylation and Src-dependent NKCC1 activation in NIH3T3 cells

2015 ◽  
Vol 308 (2) ◽  
pp. C101-C110 ◽  
Author(s):  
Line Jee Hartmann Rasmussen ◽  
Helene Steenkær Holm Müller ◽  
Bente Jørgensen ◽  
Stine Falsig Pedersen ◽  
Else Kay Hoffmann
Keyword(s):  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Volume Regulation ◽  
Mammalian Cells ◽  
Myosin Light Chain ◽  
Janus Kinase ◽  
Nih3t3 Cells ◽  
Src Inhibitor ◽  
Nonreceptor Tyrosine Kinases

The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10–30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2–10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ∼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ∼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process.

Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

2010 ◽  
Vol 299 (4) ◽  
pp. C844-C853 ◽  
Author(s):  
Signe Skyum Kirkegaard ◽  
Ian Henry Lambert ◽  
Steen Gammeltoft ◽  
Else Kay Hoffmann
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Regulatory Volume Decrease ◽  
Janus Kinase ◽  
Cell Swelling ◽  
Hek 293 ◽  
Set Point ◽  
Phosphatase Inhibitor

The swelling-activated K+ currents ( IK,vol) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K2p), TWIK-related acid-sensitive K+ channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by IK,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 NO3−. 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.

scholarly journals Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]iand calmodulin

1999 ◽  
Vol 277 (4) ◽  
pp. C823-C832 ◽  
Author(s):  
Alexander A. Mongin ◽  
Zhaohui Cai ◽  
Harold K. Kimelberg
Keyword(s):  
Cell Swelling ◽  
Transport Systems ◽  
Channel Blockers ◽  
Organic Osmolytes ◽  
Taurine Release ◽  
Cultured Astrocytes ◽  
High K ◽  
Intracellular Ca ◽  
Free Media ◽  
Taurine Efflux

Cell swelling results in regulatory activation of multiple conductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement of extracellular and intracellular Ca2+ in volume-dependent [3H]taurine efflux from primary cultured astrocytes and compare the Ca2+ sensitivity of this efflux in slow (high K+ medium induced) and fast (hyposmotic medium induced) cell swelling. Neither Ca2+-free medium nor Ca2+-channel blockers prevented the volume-dependent [3H]taurine release. In contrast, loading cells with the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-AM suppressed [3H]taurine efflux by 65–70% and 25–30% under high-K+ and hyposmotic conditions, respectively. Fura 2 measurements confirmed that BAPTA-AM, but not Ca2+-free media, significantly reduced resting intracellular Ca2+concentration ([Ca2+]i). The calmodulin antagonists trifluoperazine and fluphenazine reversibly and irreversibly, respectively, inhibited the high-K+-induced [3H]taurine release, consistent with their known actions on calmodulin. In hyposmotic conditions, the effects were less pronounced. These data suggest that volume-dependent taurine release requires minimal basal [Ca2+]iand involves calmodulin-dependent step(s). Quantitative differences in Ca2+/calmodulin sensitivity of high-K+-induced and hyposmotic medium-induced taurine efflux are due to both the effects of the inhibitors on high-K+-induced cell swelling and their effects on transport systems and/or signaling mechanisms determining taurine efflux.

Effects of apical membrane Cl(-)-formate exchange on cell volume in rabbit proximal tubule

1990 ◽  
Vol 258 (3) ◽  
pp. F530-F536 ◽  
Author(s):  
L. Schild ◽  
P. S. Aronson ◽  
G. Giebisch
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Volume Changes ◽  
Proximal Tubule Cells ◽  
Volume Increase ◽  
Tubule Lumen ◽  
Stimulation Of

We used real-time recordings of cell volume changes to test for the role of the Cl(-)-formate exchanger in mediating NaCl entry across the apical membrane of rabbit proximal tubule cells. In the absence of extracellular Cl-, 0.5 and 5 mM formate in the tubule lumen induced an increase in cell volume of 1 and 9%, respectively. Formate-induced cell swelling was reduced by alkalinizing the tubule lumen or by addition of luminal amiloride (2 mM), indicating that the increase in cell volume results from the intracellular accumulation of Na-formate via nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. The cell volume increase induced by 0.5 mM formate was potentiated (from 1 to 4%) by Cl-, as expected for a formate-mediated stimulation of NaCl uptake via parallel Cl(-)-formate exchange and Na(+)-H+ exchange across the apical membrane. By contrast, the cell volume increase induced by 5 mM formate was attenuated (from 9 to 4%) by Cl-. The attenuating effect of Cl- on formate-induced cell swelling required the operation of the apical membrane Cl(-)-formate exchanger. The effect of 1:1 Cl(-)-formate exchange to attenuate formate-induced cell swelling can be explained if the cell possesses a volume-activated anion exit pathway, most likely at the basolateral cell membrane, that is capable of mediating the efflux of Cl- but not formate from the cell.

Cellular volume homeostasis

2004 ◽  
Vol 28 (4) ◽  
pp. 155-159 ◽  
Author(s):  
Kevin Strange
Keyword(s):  
Cell Volume ◽  
Metabolic Pathways ◽  
Cell Volume Regulation ◽  
Inorganic Ions ◽  
Organic Osmolytes ◽  
Solute Content ◽  
Signaling Mechanisms ◽  
Cellular Volume ◽  
Osmotic Water

All cells face constant challenges to their volume either through changes in intracellular solute content or extracellular osmolality. Cells respond to volume perturbations by activating membrane transport and/or metabolic processes that result in net solute loss or gain and return of cell volume to its normal resting state. This paper provides a brief overview of fundamental concepts of osmotic water flow across cell membranes, mechanisms of cell volume perturbation, the role of inorganic ions and organic osmolytes in cell volume regulation and the signaling mechanisms that regulate the activity of cell volume-sensitive transport and metabolic pathways.

Role of taurine in neural cell volume regulation

1992 ◽  
Vol 70 (S1) ◽  
pp. S356-S361 ◽  
Author(s):  
Arne Schousboe ◽  
Herminia Pasantes-Morales
Keyword(s):  
Amino Acids ◽  
Cell Volume ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Granule Neurons ◽  
Release Process ◽  
Cultured Astrocytes ◽  
Rats And Mice ◽  
The Brain

Release of taurine and other amino acids was monitored from cultured astrocytes and neurons under isomotic and hyposmotic conditions as well as during exposure of the cells to 56 mM KCl. The release was correlated with swelling, as determined by the 3-O-methylglucose method. It was shown that release of taurine from astrocytes cultured from cerebral cortex and cerebellum of rats and mice regardless of the stimulating agent is a consequence of cell swelling. The release is unrelated to depolarization. This conclusion is also valid regarding release of taurine from cerebellar granule neurons. Comparison of release of different amino acids showed that not only taurine but also to some extent glutamate, aspartate, and glycine are released during cell swelling. On the other hand, glutamine is not released under these conditions. Studies of uptake of taurine under isosmotic and hyposmotic conditions as well as the dependency of the release on sodium and temperature strongly suggest that the release process is mediated by diffusional forces and not by a reversal of the high-affinity carrier. It is proposed that taurine may play an important role as an osmotically active substance in the brain involved in cell volume regulation.Key words: swelling, taurine release, neurons, astrocytes, amino acids.

Cell volume regulation in rabbit proximal straight tubule perfused in vitro

1987 ◽  
Vol 252 (5) ◽  
pp. F922-F932 ◽  
Author(s):  
K. L. Kirk ◽  
J. A. Schafer ◽  
D. R. DiBona
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Time Course ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Physiological Role ◽  
Cell Swelling ◽  
Computer Based

Volume regulation in the perfused proximal nephron of the rabbit was examined quantitatively with a computer-based method for estimating cell volume from differential interference-contrast microscopic images of isolated nephron segments. Following a hyperosmotic challenge (290-390 mosmol), the cells shrank as simple osmometers without a subsequent regulatory volume increase. Conversely, cell swelling induced by a hyposmotic challenge (290-190 mosmol) was completely reversed with a triphasic time course in which a rapid (less than 2 min) initial volume decline was followed by secondary swelling and shrinking phases. A similar regulatory volume decrease was observed following isosmotic cell swelling that was induced by exposure to 290 mosmol, urea-containing solutions. In addition, the cells partially reversed isosmotic swelling that was induced by the luminal replacement of a relatively impermeant cation (i.e., choline) with Na+ and a concomitant increase in luminal solute entry. Our results support two conclusions. First, there exist quantitative differences between the volume regulatory behaviors of perfused and nonperfused proximal tubules, the latter of which exhibit an incomplete and monotonic reversal of hyposmotic cell swelling (M. Dellasega and J. Grantham, Am. J. Physiol. 224: 1288-1294, 1973). Second, the primary physiological role of cell volume regulation in the proximal nephron may be to minimize isosmotic cell swelling associated with acute imbalances in the rates of cell solute entry and exit.

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