Mercury inhibits Na-K-ATPase primarily at the cytoplasmic side

1992 ◽  
Vol 262 (5) ◽  
pp. F843-F848 ◽  
Author(s):  
B. M. Anner ◽  
M. Moosmayer
Keyword(s):  
Active Transport ◽  
Metal Binding ◽  
Binding Interface ◽  
Intracellular Proteins ◽  
Active Ion Transport ◽  
A Cell ◽  
Intracellular Domains ◽  
Inside Out ◽  
K Transport ◽  
Cytoplasmic Side

The investigation of active Na-K transport inhibition by mercury is difficult to perform in a cell because of the presence of numerous other membrane and intracellular proteins modifiable by mercury. Thus purified Na-K-adenosinetriphosphatase (ATPase) molecules performing active transport in an artificial membrane are required to demonstrate unequivocally the inhibition of active transport by mercury. We made use of a single population of Na-K-ATPase liposomes filled with ATP and Na to show mercury inhibition of active 86Rb transport mediated by both the inside-out and right-side-out pumps in the same liposome. The effect of HgCl2 on the Na-K-ATPase in cell-like and reversed orientation was measured in comparison with convallatoxin. A dilution series showed that 10 microM externally added HgCl2 inhibited the active 86Rb transport at the cytoplasmic side first; at 50 microM both pump populations were blocked, indicating either membrane permeation by HgCl2 and inhibition at the internal intracellular domains or onset of extracellular action at higher HgCl2 concentration. The results show that the metal-binding interface of Na-K-ATPase molecule is profoundly implicated in active ion transport and that the intracellular part of the Na-K-ATPase molecule presents the primary target for mercury action.

scholarly journals Nanonet force microscopy for measuring forces in single smooth muscle cells of the human aorta

2017 ◽  
Vol 28 (14) ◽  
pp. 1894-1900 ◽  
Author(s):  
Alexander Hall ◽  
Patrick Chan ◽  
Kevin Sheets ◽  
Matthew Apperson ◽  
Christopher Delaughter ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
External Perturbation ◽  
Human Aorta ◽  
Cycle Times ◽  
Force Microscopy ◽  
Cell Matrix ◽  
Innovative Methods ◽  
A Cell ◽  
Inside Out

A number of innovative methods exist to measure cell–matrix adhesive forces, but they have yet to accurately describe and quantify the intricate interplay of a cell and its fibrous extracellular matrix (ECM). In cardiovascular pathologies, such as aortic aneurysm, new knowledge on the involvement of cell–matrix forces could lead to elucidation of disease mechanisms. To better understand this dynamics, we measured primary human aortic single smooth muscle cell (SMC) forces using nanonet force microscopy in both inside-out (I-O intrinsic contractility) and outside-in (O-I external perturbation) modes. For SMC populations, we measured the I-O and O-I forces to be 12.9 ± 1.0 and 57.9 ± 2.5 nN, respectively. Exposure of cells to oxidative stress conditions caused a force decrease of 57 and 48% in I-O and O-I modes, respectively, and an increase in migration rate by 2.5-fold. Finally, in O-I mode, we cyclically perturbed cells at constant strain of varying duration to simulate in vivo conditions of the cardiac cycle and found that I-O forces decrease with increasing duration and O-I forces decreased by half at shorter cycle times. Thus our findings highlight the need to study forces exerted and felt by cells simultaneously to comprehensively understand force modulation in cardiovascular disease.

Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization

2000 ◽  
Vol 113 (2) ◽  
pp. 315-324 ◽  
Author(s):  
P.C. Baciu ◽  
S. Saoncella ◽  
S.H. Lee ◽  
F. Denhez ◽  
D. Leuthardt ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Cellular Protein ◽  
Actin Stress Fiber ◽  
Independent Manner ◽  
Intracellular Proteins ◽  
Central Regions ◽  
A Cell

Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.

Role of ATP in respiratory control and active transport in tobacco hornworm midgut

1980 ◽  
Vol 238 (1) ◽  
pp. C10-C14 ◽  
Author(s):  
L. J. Mandel ◽  
T. G. Riddle ◽  
J. M. Storey
Keyword(s):  
Active Transport ◽  
Respiratory Control ◽  
Intermediate Form ◽  
Decay Kinetics ◽  
Kinetic Rates ◽  
Fresh Frozen ◽  
The Difference ◽  
Redox Level ◽  
K Transport

The intracellular ATP, ADP, AMP, and orthophosphate (Pi) levels were measured in the midgut of Manduca sexta. The nucleotide levels were identical in tissues either “fresh” frozen or equilibrate in regular (32 mM) K or low (8 mM) K solutions. The calculated [ATP]/[ADP][Pi]ratio was approximately 300 M-1, which is low compared to other tissues. Given the ability of this ratio to control the respiratory rate, it is speculated that this low value may cause the maximal uncontrolled respiration normally observed in the midgut. The kinetics to anoxia of active transport (Isc) and the redox level of the mitochondrial cytochromes were measured simultaneously in the midgut. The cytochromes became reduced with a time constant of 0.75 +/- 0.15 min, whereas that for Isc inhibition was 2.1 +/- 0.15 min after a delay of 0.25 min. The difference between these two kinetic rates indicates that an intermediate form of energy exists in this tissue to energize active K transport. Measurements of ATP levels during the transition to anoxia indicate that its decay kinetics are sufficiently slow for ATP to be the immediate energy source for active transport in this tissue.

Coupling between oxidative metabolism and active transport in the midgut of tobacco hornworm

1980 ◽  
Vol 238 (1) ◽  
pp. C1-C9 ◽  
Author(s):  
L. J. Mandel ◽  
D. F. Moffett ◽  
T. G. Riddle ◽  
M. M. Grafton
Keyword(s):  
Active Transport ◽  
Oxidative Metabolism ◽  
Consumption Rate ◽  
Respiratory Control ◽  
Tobacco Hornworm ◽  
Respiratory Enzymes ◽  
Endogenous Substrates ◽  
Redox Level ◽  
Mitochondrial Uncoupler ◽  
K Transport

Active K transport (Isc) in the midgut of tobacco hornworm Manduca sexta has been shown to be highly dependent on oxidative metabolism. However, the oxygen consumption rate (rO2) was not altered by conditions that drastically affect Isc. Respiration was normally maximal, inasmuch as uncouplers did not increase rO2. This rate could be maintained without any added substrate probably by oxidation of endogenous substrates. Additional succinate increased rO2 by 17%. Simultaneous monitoring of Isc and the redox level of the respiratory chain components demonstrated that 1) succinate (5 mM) reduced all the respiratory enzymes while increasing Isc by 17%; 2) sesamol (5 mM), a mitochondrial uncoupler, reoxidized all respiratory enzymes and inhibited Isc by about 50%; 3) cyanide (1 mM) fully reduced the cytochromes and completely inhibited Isc. These redox responses indicate that the mitochondria in this tissue are normally coupled, even if respiration is maximal and is not modulated by active transport. Mitochondria isolated from the midgut show coupling and respiratory control by ADP, appearing to behave like mitochondria from other tissues. Therefore, a cytoplasmic constraint must exist in this tissue that continually elicits an unmodulated maximal respiratory rate.

Mercury blocks Na-K-ATPase by a ligand-dependent and reversible mechanism

1992 ◽  
Vol 262 (5) ◽  
pp. F830-F836 ◽  
Author(s):  
B. M. Anner ◽  
M. Moosmayer ◽  
E. Imesch
Keyword(s):  
Metal Binding ◽  
Inhibitory Concentration ◽  
Potent Inhibitor ◽  
Inhibitory Receptor ◽  
Protein Inhibitor ◽  
Binding Interface ◽  
Cellular Expression ◽  
Metal Binding Domain ◽  
Cardioactive Steroids ◽  
Atpase Inhibition

An inhibitory receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the Na-K-adenosinetriphosphatase (ATPase) molecule. Besides cardioactive steroids, mercury is a potent inhibitor of the Na-K-ATPase activity. The half-maximal inhibitory concentration (IC50), determined within 30 min at 37 degrees C at 1 microgram protein/ml, was 200 nM, despite the presence of 1 mM EDTA; the IC50 decreased with increasing protein/inhibitor ratio, and it reached 2.7 microM at 0.1 mg protein/ml and 20 microM at 1 mg protein/ml. The IC50 for Na-K-ATPase inhibition by the diuretic compound mersalyl was 4 and 5 microM for the nondiuretic p-chloromercuribenzenesulfonic acid at 0.1 mg protein/ml. The IC50 for HgCl2 inhibition was modulated by the presence of EDTA as well as by the pump ligands Mg, Na, K, and ATP. The E2 conformation of the Na-K-ATPase molecule was more sensitive to HgCl2 than the E1 conformation. The mercury antidote 2,3-dimercapto-1-propanesulfonic acid was able to reactivate approximately 70% of the blocked enzyme. In conclusion, a metal-binding domain of the Na-K-ATPase molecule with particular high affinity for Hg(II) was described functionally in the present work. Therefore Na-K-ATPase belongs to the metal-binding proteins. Metals may modulate the cellular expression and activity of the system by interacting with its metal-binding interface.

scholarly journals C-terminal topology of gastric H+,K+-ATPase

1994 ◽  
Vol 299 (1) ◽  
pp. 59-64 ◽  
Author(s):  
S Asano ◽  
S Arakawa ◽  
M Hirasawa ◽  
H Sakai ◽  
M Ohta ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Intact Cell ◽  
Alpha Subunit ◽  
Dibutyryl Cyclic Amp ◽  
C Terminus ◽  
Streptolysin O ◽  
Cytosolic Side ◽  
Inside Out ◽  
Cytoplasmic Side ◽  
Stimulation Of

An antibody was prepared against a peptide corresponding to residues 1024-1034 (the putative C-terminus) of the alpha-subunit of hog gastric H+,K(+)-ATPase. The antibody bound to a 95 kDa band of H+,K(+)-ATPase that was solubilized in SDS, but not to that of Na+,K(+)-ATPase. It also bound to products of tryptic digestion that included C-terminal fragments of the H+,K(+)-ATPase alpha-subunit. The same amount of the antibody bound to both intact (tight) and lyophilized (leaky) inside-out gastric vesicles, indicating that its epitope is present on the cytosolic side of the vesicles. This finding was further confirmed by using fluorescence-immunolocalization techniques and streptolysin-O to permeabilize newt oxyntic cells. Stimulation of isolated newt oxyntic cells with dibutyryl cyclic AMP induces fusion of tubulovesicles with the apical membrane, so that the luminal domains of the H+,K(+)-ATPase alpha-subunit directly face the cell-suspension medium. The antibody did not bind to the stimulated intact cell, but bound to cells permeabilized with streptolysin-O, indicating that it binds from the cytoplasmic side to the C-terminus of the H+,K(+)-ATPase alpha-subunit in apical and tubulovesicular membrane, and also that the H+,K(+)-ATPase alpha-subunit has an even number of transmembrane domains.

scholarly journals Variations in the cytoskeletal interaction and posttranslational modification of the CD44 homing receptor in macrophages.

1991 ◽  
Vol 115 (5) ◽  
pp. 1283-1292 ◽  
Author(s):  
R L Camp ◽  
T A Kraus ◽  
E Puré
Keyword(s):  
Structural Changes ◽  
Inflammatory Responses ◽  
Surface Glycoprotein ◽  
Structural Variations ◽  
Cell Surface Glycoprotein ◽  
A Cell ◽  
Indirect Association ◽  
Macrophage Populations ◽  
Intracellular Domains ◽  
Two Populations

Murine CD44 is a cell surface glycoprotein that is thought to play a role in leukocyte migration. We studied the structure and expression of CD44 on two populations of macrophages: those that reside in the peritoneum of unprimed mice, and those that have been elicited to migrate into the peritoneum by the intraperitoneal injection of agents that cause localized inflammatory responses. Our studies reveal structural variations in both the extracellular and intracellular domains of CD44 expressed by these two macrophage populations. The form of CD44 in elicited macrophages has an apparent molecular mass that is approximately 5 kD greater and more heterogenous than that in resident macrophages. This structural changes is posttranslational, extracellular, and apparently reflects increases in N-linked glycosylation. It is also specific for CD44 and does not occur with several other glycoproteins examined. This novel regulation of glycosylation may play an important role in the ability of CD44 to bind to different substrates, particularly lectin-like ligands. In addition, we demonstrate that CD44 in resident macrophages is divided into two pools, one containing nonphosphorylated, cytoskeletally associated CD44, and one containing phosphorylated, unassociated CD44. In contrast, CD44 on the surface of elicited macrophages does not associate with the cytoskeleton. The attachment of CD44 to the cytoskeleton involves either direct or indirect association with actin. The regulated association of CD44 with the cytoskeleton suggests that it may influence or be influenced by macrophage mobility.

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