Annexins sense changes in intracellular pH during hypoxia

2007 ◽  
Vol 409 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Katia Monastyrskaya ◽  
Fabian Tschumi ◽  
Eduard B. Babiychuk ◽  
Deborah Stroka ◽  
Annette Draeger
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Fluorescent Proteins ◽  
Annexin A2 ◽  
Transport Systems ◽  
Physiological Parameter ◽  
Membrane Association ◽  
Ph Dependent

The pHi (intracellular pH) is an important physiological parameter which is altered during hypoxia and ischaemia, pathological conditions accompanied by a dramatic decrease in pHi. Sensors of pHi include ion transport systems which control intracellular Ca2+ gradients and link changes in pHi to functions as diverse as proliferation and apoptosis. The annexins are a protein family characterized by Ca2+-dependent interactions with cellular membranes. Additionally, in vitro evidence points to the existence of pH-dependent, Ca2+-independent membrane association of several annexins. We show that hypoxia promotes the interaction of the recombinant annexin A2–S100A10 (p11) and annexin A6 with the plasma membrane. We have investigated in vivo the influence of the pHi on the membrane association of human annexins A1, A2, A4, A5 and A6 tagged with fluorescent proteins, and characterized this interaction for endogenous annexins present in smooth muscle and HEK (human embryonic kidney)-293 cells biochemically and by immunofluorescence microscopy. Our results show that annexin A6 and the heterotetramer A2–S100A10 (but not annexins A1, A4 and A5) interact independently of Ca2+ with the plasma membrane at pH 6.2 and 6.6. The dimerization of annexin A2 within the annexin A2–S100A10 complex is essential for the pH-dependent membrane interaction at this pH range. The pH-induced membrane binding of annexins A6 and A2–S100A10 might have consequences for their functions as membrane organizers and channel modulators.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

scholarly journals Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

2021 ◽  
Vol 22 (13) ◽  
pp. 7232
Author(s):  
Gloria Lazzeri ◽  
Carla L. Busceti ◽  
Francesca Biagioni ◽  
Cinzia Fabrizi ◽  
Gabriele Morucci ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Light Chain ◽  
Adrenergic Receptors ◽  
Cell Damage ◽  
Protective Effects ◽  
Autophagy Flux ◽  
Brain Areas ◽  
Indirect Consequence

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane β2-adrenergic receptors (ARs). Evidence indicates that β2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

scholarly journals Purinergic Modulation of Interleukin-1β Release from Microglial Cells Stimulated with Bacterial Endotoxin

1997 ◽  
Vol 185 (3) ◽  
pp. 579-582 ◽  
Author(s):  
Davide Ferrari ◽  
Paola Chiozzi ◽  
Simonetta Falzoni ◽  
Stefania Hanau ◽  
Francesco Di  Virgilio
Keyword(s):  
Plasma Membrane ◽  
P2x7 Receptor ◽  
Purinergic Receptor ◽  
Present Report ◽  
Physiological Role ◽  
Bacterial Endotoxin ◽  
Microglial Cells

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.

scholarly journals Vascular Dysfunction Induced by Mercury Exposure

2019 ◽  
Vol 20 (10) ◽  
pp. 2435 ◽  
Author(s):  
Tetsuya Takahashi ◽  
Takayoshi Shimohata
Keyword(s):  
Oxidative Stress ◽  
Vascular Dysfunction ◽  
Brain Parenchyma ◽  
Transport Systems ◽  
Mehg Exposure ◽  
In Vivo Models ◽  
The Central Nervous System ◽  
The Brain

Methylmercury (MeHg) causes severe damage to the central nervous system, and there is increasing evidence of the association between MeHg exposure and vascular dysfunction, hemorrhage, and edema in the brain, but not in other organs of patients with acute MeHg intoxication. These observations suggest that MeHg possibly causes blood–brain barrier (BBB) damage. MeHg penetrates the BBB into the brain parenchyma via active transport systems, mainly the l-type amino acid transporter 1, on endothelial cell membranes. Recently, exposure to mercury has significantly increased. Numerous reports suggest that long-term low-level MeHg exposure can impair endothelial function and increase the risks of cardiovascular disease. The most widely reported mechanism of MeHg toxicity is oxidative stress and related pathways, such as neuroinflammation. BBB dysfunction has been suggested by both in vitro and in vivo models of MeHg intoxication. Therapy targeted at both maintaining the BBB and suppressing oxidative stress may represent a promising therapeutic strategy for MeHg intoxication. This paper reviews studies on the relationship between MeHg exposure and vascular dysfunction, with a special emphasis on the BBB.

Intracellular pH-temperature interactions of hepatocytes from American eels

1983 ◽  
Vol 245 (1) ◽  
pp. R32-R37
Author(s):  
P. J. Walsh ◽  
T. W. Moon
Keyword(s):  
Intracellular Ph ◽  
Acclimation Temperature ◽  
C Cells ◽  
Medium Ph ◽  
American Eel ◽  
Temperature Changes ◽  
American Eels ◽  
Temperature Interactions

The effects of acclimation temperature and acute temperature changes on the intracellular pH (pHi) of hepatocytes isolated from the American eel, Anguilla rostrata, were studied by the measurement of the distribution ratio of dimethyloxizolidinedione (DMO). Varying the concentration of DMO (10(-7) to 10(-4) M) did not affect estimates of pHi, indicating that DMO acts as an ideal pHi probe in eel hepatocytes. In vitro studies yielded values of liver cell pHi identical to those previously measured in vivo (in vitro pHi = 7.556 +/- 0.010; in vivo pHi = 7.570 +/- 0.049 at 20 degrees C); hepatocyte pHi varied inversely with acclimation temperature (5-20 degrees C) in a manner consistent with alphastat regulation (delta pH/delta T = -0.0182 +/- 0.021). During acute temperature increases (5-20 degrees C) and decreases (20-5 degrees C) hepatocytes regulated pHi to the appropriate (acclimated) value within 30-45 min posttransfer under conditions of constant medium pH (pHe). The effects of medium pH were also studied, and although patterns of pHi regulation differed between 5 and 20 degrees C cells, a pHi difference consistent with alphastat regulation was maintained between 5 and 20 degrees C cells over the pHe range 7.8-8.3.

Doxorubicin-Loaded Micelles Based on Folic Acid Conjugated pH-Dependent Thermo-Sensitive Copolymer: In Vitro and In Vivo Evaluation

2015 ◽  
Vol 15 (8) ◽  
pp. 5553-5558 ◽  
Author(s):  
Hongli Zhao ◽  
Liang Tao ◽  
Ronghua Yu ◽  
Huihui Yuan ◽  
Minbo Lan
Keyword(s):  
Folic Acid ◽  
Ph Dependent

scholarly journals Studies of insulin resistance in patients with clinical and subclinical hypothyroidism

2009 ◽  
Vol 160 (5) ◽  
pp. 785-790 ◽  
Author(s):  
Eirini Maratou ◽  
Dimitrios J Hadjidakis ◽  
Anastasios Kollias ◽  
Katerina Tsegka ◽  
Melpomeni Peppa ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
Subclinical Hypothyroidism ◽  
Tolerance Test ◽  
Oral Glucose ◽  
Model Assessment ◽  
Increased Risk

ObjectiveAlthough clinical hypothyroidism (HO) is associated with insulin resistance, there is no information on insulin action in subclinical hypothyroidism (SHO).Design and methodsTo investigate this, we assessed the sensitivity of glucose metabolism to insulin both in vivo (by an oral glucose tolerance test) and in vitro (by measuring insulin-stimulated rates of glucose transport in isolated monocytes with flow cytometry) in 21 euthyroid subjects (EU), 12 patients with HO, and 13 patients with SHO.ResultsAll three groups had comparable plasma glucose levels, with the HO and SHO having higher plasma insulin than the EU (P<0.05). Homeostasis model assessment index was increased in HO (1.97±0.22) and SHO (1.99±0.13) versus EU (1.27±0.16, P<0.05), while Matsuda index was decreased in HO (3.89±0.36) and SHO (4.26±0.48) versus EU (7.76±0.87, P<0.001), suggesting insulin resistance in both fasting and post-glucose state. At 100 μU/ml insulin: i) GLUT4 levels on the monocyte plasma membrane were decreased in both HO (215±19 mean fluorescence intensity, MFI) and SHO (218±24 MFI) versus EU (270±25 MFI, P=0.03 and 0.04 respectively), and ii) glucose transport rates in monocytes from HO (481±30 MFI) and SHO (462±19 MFI) were decreased versus EU (571±15 MFI, P=0.04 and 0.004 respectively).ConclusionsIn patients with HO and SHO: i) insulin resistance was comparable; ii) insulin-stimulated rates of glucose transport in isolated monocytes were decreased due to impaired translocation of GLUT4 glucose transporters on the plasma membrane; iii) these findings could justify the increased risk for insulin resistance-associated disorders, such as cardiovascular disease, observed in patients with HO or SHO.

scholarly journals Evaluation of 3-l- and 3-d-[18F]Fluorophenylalanines as PET Tracers for Tumor Imaging

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 6030
Author(s):  
Felicia Krämer ◽  
Benedikt Gröner ◽  
Chris Hoffmann ◽  
Austin Craig ◽  
Melanie Brugger ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tumor Imaging ◽  
Human Tumor ◽  
Transport Systems ◽  
Acid Decarboxylase ◽  
Tumor Xenografts ◽  
Tumor Delineation ◽  
Mcf 7

Purpose: The preclinical evaluation of 3-l- and 3-d-[18F]FPhe in comparison to [18F]FET, an established tracer for tumor imaging. Methods: In vitro studies were conducted with MCF-7, PC-3, and U87 MG human tumor cell lines. In vivo µPET studies were conducted in healthy rats with/without the inhibition of peripheral aromatic l-amino acid decarboxylase by benserazide pretreatment (n = 3 each), in mice bearing subcutaneous MCF-7 or PC-3 tumor xenografts (n = 10), and in rats bearing orthotopic U87 MG tumor xenografts (n = 14). Tracer accumulation was quantified by SUVmax, SUVmean and tumor-to-brain ratios (TBrR). Results: The uptake of 3-l-[18F]FPhe in MCF-7 and PC-3 cells was significantly higher relative to [18F]FET. The uptake of all three tracers was significantly reduced by the suppression of amino acid transport systems L or ASC. 3-l-[18F]FPhe but not 3-d-[18F]FPhe exhibited protein incorporation. In benserazide-treated healthy rats, brain uptake after 42–120 min was significantly higher for 3-d-[18F]FPhe vs. 3-l-[18F]FPhe. [18F]FET showed significantly higher uptake into subcutaneous MCF-7 tumors (52–60 min p.i.), while early uptake into orthotopic U87 MG tumors was significantly higher for 3-l-[18F]FPhe (SUVmax: 3-l-[18F]FPhe, 107.6 ± 11.3; 3-d-[18F]FPhe, 86.0 ± 4.3; [18F]FET, 90.2 ± 7.7). Increased tumoral expression of LAT1 and ASCT2 was confirmed immunohistologically. Conclusion: Both novel tracers enable accurate tumor delineation with an imaging quality comparable to [18F]FET.

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