scholarly journals ATP Dependence of the ICl, swell Channel Varies with Rate of Cell Swelling

1999 ◽  
Vol 113 (3) ◽  
pp. 441-456 ◽  
Author(s):  
Tamara Bond ◽  
Srisaila Basavappa ◽  
Michael Christensen ◽  
Kevin Strange
Keyword(s):  
Cell Metabolism ◽  
Neuroblastoma Cells ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Hyperbolic Function ◽  
Channel Activation ◽  
Atp Production ◽  
Intracellular Atp ◽  
Atp Concentration ◽  
Current Activation

Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4–5 min with solutions containing (μM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25–35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15–55%/min in the absence of intracellular ATP, current activation was slow (0.3–0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4–6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4–6 pA/pF per min) in the absence of ATP when cells were swollen at rates ≥65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.

A volume-activated taurine channel in skate hepatocytes: membrane polarity and role of intracellular ATP

1994 ◽  
Vol 267 (2) ◽  
pp. G285-G291 ◽  
Author(s):  
N. Ballatori ◽  
T. W. Simmons ◽  
J. L. Boyer
Keyword(s):  
Basolateral Membrane ◽  
Rate Coefficients ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Metabolic Inhibitor ◽  
Antimycin A ◽  
Taurine Release ◽  
Intracellular Atp ◽  
Taurine Uptake

Osmoregulation in isolated hepatocytes and perfused livers of the little skate (Raja erinacea), an osmoconforming marine elasmobranch, is mediated in part by the uptake or release of the intracellular osmolyte taurine. To further characterize the efflux mechanism, [14C]taurine release and Na(+)-independent uptake were assessed after cell swelling in hypotonic media containing 0.1-100 mM taurine. Rate coefficients for [14C]taurine uptake (0.016 +/- 0.002 min-1) and efflux (0.015 +/- 0.003 min-1) were similar and independent of extracellular taurine concentration, indicating that a taurine-permeable channel mediates the release of this amino acid after cell swelling. Volume-activated taurine uptake and efflux were both blocked by pretreatment with the metabolic inhibitors 2,4-dinitrophenol, antimycin A, and KCN plus iodoacetate, by the sulfhydryl-reactive compound N-ethylmaleimide and the transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. [14C]taurine release via this channel was immediately blocked if isotonicity was restored or a membrane-permeable metabolic inhibitor (0.5 mM 2,4-dinitrophenol) was added at different times after a hypotonic stimulus. Similar, although delayed, effects were noted with antimycin A and KCN plus iodoacetate. When isolated perfused skate livers were exposed to hypotonic stimuli, all of the taurine was released into the sinusoidal circulation, but not into bile, an effect that was also blocked by restoring isotonicity. These findings demonstrate that taurine efflux from skate hepatocytes after cell swelling is mediated by a channel. This channel is localized to the basolateral membrane and appears to require the continual presence of intracellular ATP for its function.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel

1998 ◽  
Vol 275 (2) ◽  
pp. C416-C422 ◽  
Author(s):  
Carolyn L. Cannon ◽  
Srisaila Basavappa ◽  
Kevin Strange
Keyword(s):  
Ionic Strength ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Anion Channel ◽  
Intracellular Concentration ◽  
Cell Swelling ◽  
Channel Activation ◽  
Ovary Cells ◽  
Whole Cell Patch Clamp ◽  
Current Activation

Cell swelling activates an outwardly rectifying anion channel termed VSOAC (volume-sensitive organic osmolyte/anion channel). Regulation of VSOAC by intracellular electrolytes was characterized in Chinese hamster ovary cells by whole cell patch clamp. Elevation of intracellular CsCl concentration from 40 to 180 mM resulted in a concentration-dependent decrease in channel activation. Activation of VSOAC was insensitive to the salt gradient across the plasma membrane, the intracellular concentration of specific anions or cations, and the total intracellular concentration of cations, anions, or electrolytes. Comparison of cells dialyzed with either CsCl or Na2SO4solutions demonstrated directly that VSOAC activation is modulated by intracellular ionic strength (μi). The relative cell volume at which VSOAC current activation was triggered, termed the channel volume set point, decreased with decreasing ionic strength. At μi = 0.04, VSOAC activation occurred spontaneously in shrunken cells. The rate of VSOAC activation was nearly 50-fold higher in cells with μi = 0.04 vs. those with μi = 0.18. We propose that μi modulates the volume sensor responsible for channel activation.

Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes

2006 ◽  
Vol 291 (1) ◽  
pp. H71-H80 ◽  
Author(s):  
J. M. Quayle ◽  
M. R. Turner ◽  
H. E. Burrell ◽  
T. Kamishima
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Atp Synthase ◽  
Femoral Artery ◽  
Mitochondrial Membrane ◽  
Cell Metabolism ◽  
Metabolic Inhibitors ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Atp Production

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 ∼35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 μM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-d-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 μM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.

scholarly journals Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 60
Author(s):  
Juan Vélez ◽  
Zahady Velasquez ◽  
Liliana M. R. Silva ◽  
Ulrich Gärtner ◽  
Klaus Failing ◽  
...  
Keyword(s):  
Host Cell ◽  
Cryptosporidium Parvum ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Host Cells ◽  
Metabolic Inhibitors ◽  
Low Oxygen ◽  
Metabolic Signatures

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

scholarly journals DDR1 Affects Metabolic Reprogramming in Breast Cancer Cells by Cross-Talking to the Insulin/IGF System

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 926
Author(s):  
Veronica Vella ◽  
Marika Giuliano ◽  
Maria Luisa Nicolosi ◽  
Maria Giovanna Majorana ◽  
Małgorzata Anna Marć ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Cells ◽  
Cell Metabolism ◽  
Growth Factor Receptor ◽  
Metabolic Reprogramming ◽  
Atp Production ◽  
Igf System ◽  
Putative Target ◽  
Collagen Receptor ◽  
Discoidin Domain Receptor 1

The insulin receptor isoform A (IR-A), a dual receptor for insulin and IGF2, plays a role in breast cancer (BC) progression and metabolic reprogramming. Notably, discoidin domain receptor 1 (DDR1), a collagen receptor often dysregulated in cancer, is involved in a functional crosstalk and feed forward loop with both the IR-A and the insulin like growth factor receptor 1 (IGF1R). Here, we aimed at investigating whether DDR1 might affect BC cell metabolism by modulating the IGF1R and/or the IR. To this aim, we generated MCF7 BC cells engineered to stably overexpress either IGF2 (MCF7/IGF2) or the IR-A (MCF7/IR-A). In both cell models, we observed that DDR1 silencing induced a significant decrease of total ATP production, particularly affecting the rate of mitochondrial ATP production. We also observed the downregulation of key molecules implicated in both glycolysis and oxidative phosphorylation. These metabolic changes were not modulated by DDR1 binding to collagen and occurred in part in the absence of IR/IGF1R phosphorylation. DDR1 silencing was ineffective in MCF7 knocked out for DDR1. Taken together, these results indicate that DDR1, acting in part independently of IR / IGF1R stimulation, might work as a novel regulator of BC metabolism and should be considered as putative target for therapy in BC.

scholarly journals Temporal Quantitative Proteomics Analysis of Neuroblastoma Cells Treated with Bovine Milk-Derived Extracellular Vesicles Highlights the Anti-Proliferative Properties of Milk-Derived Extracellular Vesicles

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 750
Author(s):  
Pamali Fonseka ◽  
Taeyoung Kang ◽  
Sing Chee ◽  
Sai V. Chitti ◽  
Rahul Sanwlani ◽  
...  
Keyword(s):  
High Risk ◽  
Extracellular Vesicles ◽  
Quantitative Proteomics ◽  
Cell Metabolism ◽  
Bovine Milk ◽  
Neuroblastoma Cells ◽  
Treatment Strategies ◽  
Treatment Resistance ◽  
Label Free ◽  
The Impact

Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

Characterization of ATP-sensitive potassium channels in freshly dissociated rabbit aortic endothelial cells

1997 ◽  
Vol 272 (5) ◽  
pp. H2507-H2511 ◽  
Author(s):  
C. Katnik ◽  
D. J. Adams
Keyword(s):  
Endothelial Cells ◽  
Single Channel ◽  
Muscle Tone ◽  
Cell Metabolism ◽  
Katp Channels ◽  
K Channel ◽  
Membrane Hyperpolarization ◽  
Aortic Endothelial Cells ◽  
Intracellular Atp ◽  
Aortic Endothelial

ATP-sensitive potassium (KATP) channels represent a class of K+ channel regulated by intracellular ATP and serve to transduce changes in cell metabolism into changes in membrane potential. The presence of an KATP conductance has recently been demonstrated in freshly dissociated endothelial cells from rabbit arteries. In the present study, the single-channel activity underlying the KATP conductance in rabbit aortic endothelial cells was examined. Unitary currents were evoked in response to lowering intracellular ATP concentration or application of the K(+)-channel activator levcromakalim and were inhibited by the sulfonylurea drug glibenclamide. Exposure of the cytoplasmic face of an inside-out membrane patch to a solution containing 0.1 mM ATP produced single-channel events with unitary conductances of approximately 150 and approximately 25 pS that were inhibited by either 6 mM ATP or 10 microM glibenclamide. A small conductance channel was also activated in cell-attached patches by bath-applied levcromakalim (25 microM). Activation of endothelial cell KATP channels, and subsequent membrane hyperpolarization, may contribute to endothelium-dependent regulation of vascular smooth muscle tone in response to changes in levels of intracellular metabolites.

The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding

1994 ◽  
Vol 267 (5) ◽  
pp. C1203-C1209 ◽  
Author(s):  
P. S. Jackson ◽  
R. Morrison ◽  
K. Strange
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Pipette Solution ◽  
Organic Osmolyte ◽  
Atp Levels ◽  
Patch Pipette ◽  
Taurine Efflux

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.

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