Cell swelling-induced ATP release and  gadolinium-sensitive channels

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 because gadolinium (Gd3+), an inhibitor of stretch-activated channels, suppressed ATP efflux monitored by luciferase bioluminescence. We examined the effect of Gd3+on luciferase bioluminescence and on ATP efflux from hypotonically swollen cells. We found that luciferase was inhibited by ≤10 μM Gd3+, and this may have contributed to the previously reported inhibition of ATP release. In ATP efflux experiments, luciferase inhibition could be prevented by chelating Gd3+with EGTA before luminometric ATP determinations. Using this approach, we found that 10–100 μM Gd3+, i.e., concentrations typically used to block MS channels, actually stimulated hypotonically induced ATP release from fibroblasts. Inhibition of ATP release required at least 500, 200, or 100 μM Gd3+ for fibroblasts, A549 cells, and 16HBE14o− cells, respectively. Such biphasic and cell-specific effects of Gd3+ are most consistent with its action on membrane lipids and membrane-dependent processes such as exocytosis.

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Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.6.c1790 ◽

1996 ◽

Vol 270 (6) ◽

pp. C1790-C1798 ◽

Cited By ~ 46

Author(s):

Y. Chen ◽

S. M. Simasko ◽

J. Niggel ◽

W. J. Sigurdson ◽

F. Sachs

Keyword(s):

Volume Regulation ◽

Cell Swelling ◽

Gh3 Cells ◽

Membrane Tension ◽

Hypotonic Cell Swelling ◽

Stretch Activated Channels ◽

Ca2 Channels ◽

Hypotonic Swelling ◽

Volume Decrease

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.

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Faculty Opinions recommendation of Ca2+-dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleotides.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1093974.548921 ◽

2007 ◽

Author(s):

Jens Leipziger

Keyword(s):

Atp Release ◽

A549 Cells ◽

Autocrine Stimulation ◽

Uridine Nucleotides

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ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00438.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. C569-C578 ◽

Cited By ~ 91

Author(s):

Alexander A. Mongin ◽

Harold K. Kimelberg

Keyword(s):

Amino Acid ◽

Excitatory Amino Acid ◽

Atp Release ◽

Anion Channel ◽

P2y Receptors ◽

Cell Swelling ◽

Channel Blockers ◽

Medium Osmolarity ◽

Subsequent Activation

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

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Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model

International Journal of Molecular Sciences ◽

10.3390/ijms22136910 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6910

Author(s):

Flora Szeri ◽

Valentina Corradi ◽

Fatemeh Niaziorimi ◽

Sylvia Donnelly ◽

Gwenaëlle Conseil ◽

...

Keyword(s):

Binding Site ◽

Efflux Pump ◽

Substrate Binding ◽

Functional Characterization ◽

Atp Release ◽

Homology Model ◽

Atp Binding ◽

Atp Binding Site ◽

Binding Cavity ◽

Atp Efflux

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.

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Paternal diet programs offspring health through sperm- and seminal plasma-specific pathways in mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806333115 ◽

2018 ◽

Vol 115 (40) ◽

pp. 10064-10069 ◽

Cited By ~ 58

Author(s):

Adam J. Watkins ◽

Irundika Dias ◽

Heather Tsuro ◽

Danielle Allen ◽

Richard D. Emes ◽

...

Keyword(s):

Seminal Plasma ◽

Semen Quality ◽

Protein Diet ◽

Male Mating ◽

Adult Offspring ◽

Nonalcoholic Fatty Liver ◽

Low Protein ◽

Specific Effects ◽

Underlying Mechanisms ◽

Offspring Health

The association between poor paternal diet, perturbed embryonic development, and adult offspring ill health represents a new focus for the Developmental Origins of Health and Disease hypothesis. However, our understanding of the underlying mechanisms remains ill-defined. We have developed a mouse paternal low-protein diet (LPD) model to determine its impact on semen quality, maternal uterine physiology, and adult offspring health. We observed that sperm from LPD-fed male mice displayed global hypomethylation associated with reduced testicular expression of DNA methylation and folate-cycle regulators compared with normal protein diet (NPD) fed males. Furthermore, females mated with LPD males display blunted preimplantation uterine immunological, cell signaling, and vascular remodeling responses compared to controls. These data indicate paternal diet impacts on offspring health through both sperm genomic (epigenetic) and seminal plasma (maternal uterine environment) mechanisms. Extending our model, we defined sperm- and seminal plasma-specific effects on offspring health by combining artificial insemination with vasectomized male mating of dietary-manipulated males. All offspring derived from LPD sperm and/or seminal plasma became heavier with increased adiposity, glucose intolerance, perturbed hepatic gene expression symptomatic of nonalcoholic fatty liver disease, and altered gut bacterial profiles. These data provide insight into programming mechanisms linking poor paternal diet with semen quality and offspring health.

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Abstract 99: Hypotonic Swelling Promotes Nitric Oxide Release in Cardiac Ventricular Myocytes

Circulation Research ◽

10.1161/res.115.suppl_1.99 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Luis Gonano ◽

Malena Morell ◽

Juan I Burgos ◽

Martin Vila Petroff

Keyword(s):

Nitric Oxide ◽

Cardiac Myocytes ◽

Cardiac Myocyte ◽

Contractile Function ◽

Cell Swelling ◽

Contractile Dysfunction ◽

Ischemia And Reperfusion ◽

Hypotonic Solution ◽

No Release ◽

Hypotonic Swelling

Cardiac myocyte swelling occurs in multiple pathological situations and in particular contributes to the deleterious effects of ischemia and reperfusion by promoting contractile dysfunction. We investigated whether hypotonic swelling promotes nitric oxide (NO) release in cardiac myocytes and if so, whether it impacts on swelling induced contractile dysfunction. Perfusing rat cardiac myocytes, loaded with the NO sensor DAF-FM, with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient amplitude and significantly increased DAF-FM fluorescence. When cells were exposed to the HS supplemented with 2.5 mM of the NO synthase inhibitor L-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the NOS1 inhibitor, Nitroguanidine. In addition, Colchicine (an inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either L-NAME, Nitroguandine or the guanylate cyclase inhibitor, ODQ, suggesting that NOS1-derived NO provides contractile support via a GMP-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and the HS-induced increment in ryanodine receptor (RyR2) phosphorylation at site Ser2808 suggesting that in the context of hypotonic swelling, cGMP may contribute to preserve contractile function by enhancing SR Ca2+ release. Our findings suggest a novel mechanism for NO release in cardiac myocytes with putative pathophysiological relevance in the context of ischemia and reperfusion, where it may be cardioprotective by reducing the extent of contractile dysfunction associated with hypotonic swelling.

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Protein-Lipid Interaction of Cytolytic Toxin Cyt2Aa2 on Model Lipid Bilayers of Erythrocyte Cell Membrane

Toxins ◽

10.3390/toxins12040226 ◽

2020 ◽

Vol 12 (4) ◽

pp. 226

Author(s):

Sudarat Tharad ◽

Boonhiang Promdonkoy ◽

José L. Toca-Herrera

Keyword(s):

Lipid Bilayers ◽

Erythrocyte Membrane ◽

Hemolytic Activity ◽

Membrane Lipids ◽

Binding Capacity ◽

Lipid Binding ◽

Force Microscopy ◽

Specific Effects ◽

Atomic Force ◽

Cytolytic Action

Cytolytic toxin (Cyt) is a toxin among Bacillus thuringiensis insecticidal proteins. Cyt toxin directly interacts with membrane lipids for cytolytic action. However, low hemolytic activity is desired to avoid non-specific effects in mammals. In this work, the interaction between Cyt2Aa2 toxin and model lipid bilayers mimicking the erythrocyte membrane was investigated for Cyt2Aa2 wild type (WT) and the T144A mutant, a variant with lower hemolytic activity. Quartz crystal microbalance with dissipation (QCM-D) results revealed a smaller lipid binding capacity for the T144A mutant than for the WT. In particular, the T144A mutant was unable to bind to the phosphatidylcholine lipid (POPC) bilayer. However, the addition of cholesterol (Chol) or sphingomyelin (SM) to the POPC bilayer promoted binding of the T144 mutant. Moreover, atomic force microscopy (AFM) images unveiled small aggregates of the T144A mutant on the 1:1 sphingomyelin/POPC bilayers. In contrast, the lipid binding trend for WT and T144A mutant was comparable for the 1:0.4 POPC/cholesterol and the 1:1:1 sphingomyelin/POPC/cholesterol bilayers. Furthermore, the binding of WT and T144A mutant onto erythrocyte cells was investigated. The experiments showed that the T144A mutant and the WT bind onto different areas of the erythrocyte membrane. Overall the results suggest that the T144 residue plays an important role for lipid binding.

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Effects of Prednisolone on Serum and Tissue Fluid IGF-I Receptor Activation and Post-Receptor Signaling in Humans

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2017-00696 ◽

2017 ◽

Vol 102 (11) ◽

pp. 4031-4040 ◽

Cited By ~ 9

Author(s):

Nilani Ramshanker ◽

Maiken Aagaard ◽

Rikke Hjortebjerg ◽

Thomas Schmidt Voss ◽

Niels Møller ◽

...

Keyword(s):

Messenger Rna ◽

Protein A ◽

Receptor Activation ◽

Tissue Fluid ◽

Igf Binding Proteins ◽

Downstream Signaling ◽

Specific Effects ◽

Igf I ◽

Underlying Mechanisms ◽

Double Blinded

Abstract Context Short-term glucocorticoid exposure increases serum insulinlike growth factor I (IGF-I) concentrations but antagonizes IGF-I tissue signaling. The underlying mechanisms remain unknown. Objective To identify at which levels glucocorticoid inhibits IGF-I signaling. Design and Methods Nineteen healthy males received prednisolone (37.5 mg/d) and placebo for 5 days in a randomized, double-blinded, placebo-controlled crossover study. Serum was collected on days 1, 3, and 5, and abdominal skin suction blister fluid (SBF; ~interstitial fluid) was taken on day 5 (n = 9) together with muscle biopsy specimens (n = 19). The ability of serum and SBF to activate the IGF-I receptor (IGF-IR) (bioactive IGF) and its downstream signaling proteins was assessed using IGF-IR–transfected cells. Results Prednisolone increased IGF-I concentrations and bioactive IGF in serum (P ≤ 0.001) but not in SBF, which, compared with serum, contained less bioactive IGF (~28%) after prednisolone (P < 0.05). This observation was unexplained by SBF concentrations of IGFs and IGF-binding proteins (IGFBPs) 1 to 4. However, following prednisolone treatment, SBF contained less IGFBP-4 fragments (P < 0.05) generated by pregnancy-associated plasma protein A (PAPP-A). Concomitantly, prednisolone increased SBF levels of stanniocalcin 2 (STC2) (P = 0.02) compared with serum. STC2 blocks PAPP-A from cleaving IGFBP-4. Finally, prednisolone suppressed post–IGF-IR signaling pathways at the level of insulin receptor substrate 1 (P < 0.05) but did not change skeletal muscle IGF-IR, IGF-I, or STC2 messenger RNA. Conclusion Prednisolone increased IGF-I concentrations and IGF bioactivity in serum but not in tissue fluid. The latter may relate to a STC2-mediated inhibition of PAPP-A in tissue fluids. Furthermore, prednisolone induced post–IGF-IR resistance. Thus, glucocorticoid may exert distinct, compartment-specific effects on IGF action.

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Baicalein suppresses vasculogenic mimicry through inhibiting RhoA/ROCK expression in lung cancer A549 cell line

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa075 ◽

2020 ◽

Vol 52 (9) ◽

pp. 1007-1015

Author(s):

Zhe Zhang ◽

Li Nong ◽

Menglei Chen ◽

Xiaoli Gu ◽

Weiwei Zhao ◽

...

Keyword(s):

Lung Cancer ◽

Vasculogenic Mimicry ◽

A549 Cells ◽

A549 Cell Line ◽

Traditional Herbal Medicine ◽

Underlying Mechanisms ◽

Filament Network ◽

Lung Cancer A549 Cell

Abstract Vasculogenic mimicry (VM) refers to a new tubular network of the blood supply system with abundant extracellular matrix. VM is similar to capillaries but does not involve endothelial cells. As a traditional herbal medicine commonly used in China, baicalein possesses anti-inflammatory and lipoxygenase activities. However, the effects of baicalein on the process of VM formation in non-small cell lung cancer (NSCLC) and the underlying mechanisms have remained poorly understood. In this study, baicalein was found to inhibit the viability and motility of A549 cells and induced the breakage of the cytoskeletal actin filament network. In addition, baicalein significantly decreased the formation of VM and downregulated the expressions of VM-associated factors, such as VE-cadherin, EphA2, MMP14, MMP2, MMP9, PI3K and LAMC2, similar to the effects of ROCK inhibitors. Indeed, baicalein inhibited RhoA/ROCK expression in vitro and in vivo, suggesting the underlying mechanisms of reduced VM formation. Collectively, baicalein suppressed the formation of VM in NSCLC by targeting the RhoA/ROCK signaling pathway, indicating that baicalein might serve as an emerging drug for NSCLC.

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