Modulation of intracellular Ca2+ by glucose in MDCK cells: role of endoplasmic reticulum Ca(2+)-ATPase

1995 ◽  
Vol 268 (4) ◽  
pp. F671-F679 ◽  
Author(s):  
Y. H. Lien ◽  
X. Wang ◽  
R. J. Gillies ◽  
R. Martinez-Zaguilan
Keyword(s):  
Endoplasmic Reticulum ◽  
Mdck Cells ◽  
Cyclopiazonic Acid ◽  
Tubuloglomerular Feedback ◽  
Free Calcium ◽  
Functional Roles ◽  
Atpase Inhibitors ◽  
Dose Dependent Fashion ◽  
Cl Channels

Intracellular free calcium ([Ca2+]i) has multiple functional roles in renal epithelia, including mediating ligand- and volume-activated K+ and Cl- channels, modulating the permeability of apical membrane to Na+, and regulating tubuloglomerular feedback. We investigated glucose effects on intracellular pH (pHi) and [Ca2+]i in Madin-Darby canine kidney (MDCK) cells using fluorescent probes, SNARF-1 and fura 2, respectively. The addition of glucose decreased both pHi and [Ca2+]i in a dose-dependent fashion. Thapsigargin (TG) and cyclopiazonic acid (CPA), well-known endoplasmic reticulum (ER) Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) inhibitors, abolished the glucose-induced [Ca2+]i decrease. Without glucose, 1 microM TG induced a sustained elevation in [Ca2+]i, which increased further with glucose addition, whereas 15 microM CPA induced a transient increase in [Ca2+]i that was not affected by further addition of glucose. The sustained elevation in [Ca2+]i induced by TG was dependent on extracellular Ca2+. TG-induced [Ca2+]i increase was modulated by glucose, i.e., at higher glucose concentrations, TG induced a larger and more rapid rise in [Ca2+]i. We conclude that glucose has dual effects on [Ca2+]i regulation. Glucose alone reduces [Ca2+]i by activating ER-type Ca(2+)-ATPase, since this phenomenon is TG and CPA sensitive. In the presence of TG, glucose increases [Ca2+]i probably by increasing Ca2+ entry. Our data suggest a model in which TG activates capacitative Ca2+ entry by depletion of the ER Ca2+ pool. Glucose increases TG-induced [Ca2+]i elevation by further enhancing capacitative Ca2+ entry.

scholarly journals Oscillatory membrane potential changes in cells of mesenchymal origin: the role of an intracellular calcium regulating system

1979 ◽  
Vol 81 (1) ◽  
pp. 49-61
Author(s):  
P. G. Nelson ◽  
M. P. Henkart
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Potential ◽  
Intracellular Calcium ◽  
Calcium Ion ◽  
Surface Membrane ◽  
Ion Concentration ◽  
Free Calcium ◽  
Potassium Ions ◽  
Calcium Ion Concentration

A number of mesenchymal cells (fibroblasts, macrophages and megakaryocytes) respond to a variety of stimuli with large hyperpolarizations lasting several seconds (the H.A. response). The H.A. responses can occur as repetitive trains or oscillations. These hyperpolarizations are due to an increase of the surface membrane permeability to potassium ions which is probably mediated by an increase in the cytoplasmic free calcium ion concentration. Evidence is discussed which suggests that the source of this increased calcium, is least in part, an intracellular sequestering system, probably the endoplasmic reticulum. A model capable of producing oscillatory changes in membrane potential is proposed based on such an intracellular calcium sequestering and releasing system.

Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells

1997 ◽  
Vol 272 (4) ◽  
pp. C1241-C1249 ◽  
Author(s):  
X. Bian ◽  
F. M. Hughes ◽  
Y. Huang ◽  
J. A. Cidlowski ◽  
J. W. Putney
Keyword(s):  
Endoplasmic Reticulum ◽  
Cyclopiazonic Acid ◽  
Free Medium ◽  
Atpase Inhibitors ◽  
Induced Apoptosis

The Ca2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid, depleted intracellular Ca2+ stores, induced large increases in intracellular Ca2+ concentration, and caused apoptosis in S49 cells. Removal of extracellular Ca2+ augmented apoptosis due to thapsigargin, indicating that depletion of Ca2+ from intracellular stores is responsible for apoptosis with this agent. Overexpression of the apoptosis suppressor, Bcl-2, inhibited apoptosis due to thapsigargin but did not affect thapsigargin-induced Ca2+ signaling. Dexamethasone induced apoptosis, diminished the size of the endoplasmic reticulum Ca2+ pool, and caused a small elevation of intracellular Ca2+. However, this elevation was not due to Ca2+ influx because the increase was similar in the presence or absence of Ca2+ in the medium. Furthermore, in contrast to the results with thapsigargin, apoptosis due to dexamethasone was unchanged in a Ca2+-free medium. These results indicate that depletion of Ca2+ stores initiates a pathway leading to apoptosis. Elevations in cytoplasmic Ca2+ appears to play a lesser role than previously thought in the actions of Bcl-2 and glucocorticoids.

Ca(2+)-dependent Cl- current in canine tracheal smooth muscle cells

1995 ◽  
Vol 269 (1) ◽  
pp. C163-C169 ◽  
Author(s):  
L. J. Janssen ◽  
S. M. Sims
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cyclopiazonic Acid ◽  
Muscle Cells ◽  
Feedback System ◽  
Tracheal Smooth Muscle ◽  
Equilibrium Potential ◽  
Cl Channels ◽  
Patch Configuration

Our goal was to investigate the role of Ca2+ entry in regulating Cl- current (ICl) in smooth muscle cells from canine trachealis. When studies were done using the perforated patch configuration, depolarization elicited a dihydropyridine-sensitive Ca2+ current (ICa), followed in many cells by a sustained current. This sustained current reversed direction close to the Cl- equilibrium potential, consistent with its representing ICl. The ICl was also apparent as slowly deactivating tail currents seen upon repolarization to negative potentials. The Cl- channel blocker niflumic acid abolished both the sustained and tail currents, without affecting ICa. Several observations indicated that the ICl was dependent on Ca2+ entry. ICl was increased in magnitude when Ca2+ influx was augmented [by prolonging the depolarization or using BAY K 8644 or acetylcholine (ACh)] and decreased in magnitude when Ca2+ influx was reduced (using nifedipine). Based on these findings, we conclude that depolarization causes Ca2+ entry, with resultant elevation of cytosolic free Ca2+ concentration leading to activation of ICl (ICl(Ca)). We investigated whether Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum was involved in activation of ICl(Ca), by depleting intracellular stores of Ca2+ using cyclopiazonic acid to block the sarcoplasmic Ca(2+)-adenosinetriphosphatase and repeated stimulation with ACh. In such Ca(2+)-depleted cells, depolarization-mediated Ca2+ entry continued to activate ICl(Ca), suggesting that Ca(2+)-induced Ca2+ release was not required for its activation. We conclude that Ca2+ entry can activate Cl- channels in tracheal smooth muscle. This represents a positive-feedback system, which would promote excitation and contraction of airway muscle.

scholarly journals Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

2007 ◽  
Vol 293 (3) ◽  
pp. C938-C950 ◽  
Author(s):  
Jason I. E. Bruce ◽  
Austin C. Elliott
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Injury ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Dependent Manner ◽  
Atpase Inhibitors

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca2+]i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca2+]i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca2+]i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca2+]i, which was sensitive to antioxidants and removal of external Ca2+, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca2+-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca2+ uptake. This suggests that hydrogen peroxide promotes Ca2+ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca2+ influx. Lower concentrations of hydrogen peroxide (10–100 μM) increased [Ca2+]i and altered cholecystokinin-evoked [Ca2+]i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca2+]i also upregulated the activity of the plasma membrane Ca2+-ATPase in a Ca2+-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca2+-ATPase. This may be important in facilitating “Ca2+ overload,” resulting in cell injury associated with pancreatitis.

Endoplasmic reticulum Ca2+-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells

2000 ◽  
Vol 278 (2) ◽  
pp. C363-C371 ◽  
Author(s):  
Anna S. Gukovskaya ◽  
Sofiya Gukovsky ◽  
Stephen J. Pandol
Keyword(s):  
Endoplasmic Reticulum ◽  
Guanylate Cyclase ◽  
Cyclopiazonic Acid ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Membrane Guanylate Cyclase ◽  
Intact Cells ◽  
Pancreatic Acini ◽  
Atpase Inhibitors ◽  
Atpase Inhibition

In this study, we show that particulate guanylate cyclase (GC) is present in rat pancreatic acinar cells and is located both on plasma membrane and membranes of endoplasmic reticulum (ER). Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. The specific inhibitors of ER Ca2+-ATPase thapsigargin, 2,5-di-( t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid all activated particulate GC in pancreatic acini, both in membrane fractions and intact cells. These inhibitors also induced dephosphorylation of GC. Dose dependencies of Ca2+-ATPase inhibition and GC activation by BHQ are very similar, and those for thapsigargin partially overlap. ER Ca2+-ATPase and GC are coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca2+-ATPase, suggesting a physical association between the two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca2+-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.

scholarly journals Mobilization of Ca2+ by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone in permeabilized insulin-secreting RINm5F cells: evidence for separate uptake and release compartments in inositol 1,4,5-trisphosphate-sensitive Ca2+ pool

1993 ◽  
Vol 293 (2) ◽  
pp. 423-429 ◽  
Author(s):  
M S Islam ◽  
P O Berggren
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Concentration ◽  
Prolonged Treatment ◽  
Free Calcium ◽  
Bafilomycin A1 ◽  
Rinm5f Cells ◽  
Atpase Inhibitors ◽  
Inositol 1,4,5 Trisphosphate ◽  
Free Calcium Concentration ◽  
Uptake And Release

We characterized and directly compared the Ca(2+)-releasing actions of two inhibitors of endoplasmic-reticulum (ER) Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ), in electropermeabilized insulin-secreting RINm5F cells. Ambient free calcium concentration ([Ca2+]) was monitored by Ca(2+)-selective mini-electrodes. After ATP-dependent Ca2+ uptake, thapsigargin and tBuBHQ released Ca2+ with and EC50 of approximately 37 nM and approximately 2 microM respectively. Both agents mobilized Ca2+ predominantly from the Ins(1,4,5)P3-sensitive Ca2+ pool, and in this respect thapsigargin was more specific than tBuBHQ. The total increase in [Ca2+] obtained with thapsigargin and Ins(1,4,5)P3 was, on the average, only 7% greater than that with Ins(1,4,5)P3 alone. In contrast, the total increase in [Ca2+] obtained with tBuBHQ and Ins(1,4,5)P3 was 33% greater than that obtained with only InsP3 (P < 0.05). Although Ca2+ was rapidly mobilized by thapsigargin and tBuBHQ, complete depletion of the Ins(1,4,5)P3-sensitive Ca2+ pool was difficult to achieve. After the release by thapsigargin or tBuBHQ, Ins(1,4,5)P3 induced additional Ca2+ release. The additional Ins(1,4,5)P3-induced Ca2+ release was not altered by supramaximal concentrations of thapsigargin and tBuBHQ, or by Bafilomycin A1, an inhibitor of V-type ATPases, but was decreased by prolonged treatment with the ER Ca(2+)-ATPase inhibitors. These results suggest the existence of distinct uptake and release compartments within the Ins(1,4,5)P3-sensitive Ca2+ pool. When treated with the inhibitors, the two compartments became distinguishable on the basis of their Ca2+ permeability. Apparently, thapsigargin and tBuBHQ readily mobilized Ca2+ from the uptake compartment, whereas Ca2+ from the release compartment could be mobilized only very slowly, in the absence of Ins(1,4,5)P3.

scholarly journals Role of sarcoplasmic/endoplasmic-reticulum Ca2+-ATPases in mediating Ca2+ waves and local Ca2+-release microdomains in cultured glia

1997 ◽  
Vol 325 (1) ◽  
pp. 239-247 ◽  
Author(s):  
Peter B. SIMPSON ◽  
James T. RUSSELL
Keyword(s):  
Endoplasmic Reticulum ◽  
Release Kinetics ◽  
Spatial Relationship ◽  
Cyclopiazonic Acid ◽  
Free Medium ◽  
Multiple Domains ◽  
Slow Onset

We have characterized the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) pumps in cultured rat cortical type-1 astrocytes, type-2 astrocytes and oligodendrocytes. Perfusion with 10 μM cyclopiazonic acid (CPA) or 1 μM thapsigargin evoked a large and persistent elevation in cytosolic [Ca2+] in normal Ca2+-containing medium and a small and transient increase in nominally Ca2+-free medium. Subtraction of the response in Ca2+-free medium from that in the control revealed a slow-onset Ca2+-entry response to SERCA inhibition, which began after most of the store depletion had occurred. Thapsigargin- and CPA-induced responses propagated as Ca2+ waves, which began in several distinct cellular sites and travelled throughout the cell and through nearby cells, in confluent cultures. Propagation was supported by specialized Ca2+-release sites where the amplitude of the response was significantly higher and the rate of rise steeper. Such higher Ca2+-release kinetics were observed at these sites during Ins(1,4,5)P3-mediated Ca2+ waves in the same cells. Fluorescently tagged thapsigargin labelled SERCA pumps throughout glial cell bodies and processes. In oligodendrocyte processes, multiple domains with elevated SERCA staining were always associated with mitochondria. Our results are consistent with a model in which only a single Ca2+ store, expressing Ins(1,4,5)P3 receptors and SERCAs sensitive to both thapsigargin and CPA, is present in rat cortical glia, and indicate that inhibition of SERCA activates both Ca2+ release as a wavefront and Ca2+ entry via store-operated channels. The spatial relationship between SERCAs and mitochondria is likely to be important for regulating microdomains of elevated Ca2+-release kinetics.

Effect of m-3M3FBS on Ca2+ movement in Madin-Darby canine renal tubular cells

2009 ◽  
Vol 28 (10) ◽  
pp. 655-663 ◽  
Author(s):  
Yi-Chien Fang ◽  
Daih-Huang Kuo ◽  
Pochuen Shieh ◽  
Fu-An Chen ◽  
Chun-Chi Kuo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mdck Cells ◽  
Cyclopiazonic Acid ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Concentration Dependent Manner ◽  
Renal Tubular Cells ◽  
Renal Tubular ◽  
Darby Canine Kidney

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C (PLC) activator, on cytosolic free Ca2+ concentrations ([Ca 2+]i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended MDCK cells using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 0.1 and 20 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was decreased by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole, and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 20-μM m-3M3FBS pretreatment abolished the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin (TG) and cyclopiazonic acid (CPA). Conversely, pretreatment with TG or CPA partly reduced m-3M3FBS-induced [Ca2+]i rise. The inhibition of PLC with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in MDCK cells, m-3M3FBS induced [Ca2+]i rises by causing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels and other unidentified Ca2+ channels.

scholarly journals The Regulatory Role of GBF1 on Osteoclast Activation Through EIF2a Mediated ER Stress and Novel Marker FAM129A Induction

2021 ◽  
Vol 9 ◽  
Author(s):  
Cailing Wen ◽  
Yuheng Zhou ◽  
Yanting Xu ◽  
Huijing Tan ◽  
Caixia Pang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Early Stage ◽  
Specific Inhibitor ◽  
Brefeldin A ◽  
Vesicular Trafficking ◽  
Functional Roles ◽  
Membrane Reorganization ◽  
Cytoskeleton Remodeling ◽  
Arf Gtpases

Bone-resorbing activities of osteoclasts (OCs) are highly dependent on actin cytoskeleton remodeling, plasma membrane reorganization, and vesicle trafficking pathways, which are partially regulated by ARF-GTPases. In the present study, the functional roles of Golgi brefeldin A resistance factor 1 (GBF1) are proposed. GBF1 is responsible for the activation of the ARFs family and vesicular transport at the endoplasmic reticulum–Golgi interface in different stages of OCs differentiation. In the early stage, GBF1 deficiency impaired OCs differentiation and was accompanied with OCs swelling and reduced formation of mature OCs, indicating that GBF1 participates in osteoclastogenesis. Using siRNA and the specific inhibitor GCA for GBF1 knockdown upregulated endoplasmic reticulum stress-associated signaling molecules, including BiP, p-PERK, p-EIF2α, and FAM129A, and promoted autophagic Beclin1, Atg7, p62, and LC3 axis, leading to apoptosis of OCs. The present data suggest that, by blocking COPI-mediated vesicular trafficking, GBF1 inhibition caused intense stress to the endoplasmic reticulum and excessive autophagy, eventually resulting in the apoptosis of mature OCs and impaired bone resorption function.

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