scholarly journals Luminal Ca2+ regulates passive Ca2+ efflux from the intracellular stores of hepatocytes

1998 ◽  
Vol 334 (2) ◽  
pp. 431-435 ◽  
Author(s):  
Mike D. BEECROFT ◽  
Colin W. TAYLOR
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Specific Activity ◽  
Critical Threshold ◽  
Similar Amount ◽  
Half Time ◽  
Atp Level ◽  
Simultaneous Addition ◽  
Inositol 1,4,5 Trisphosphate ◽  
The Many

Ca2+ uptake into the intracellular stores of permeabilized hepatocytes was entirely dependent on ATP and substantially inhibited by either ionomycin or thapsigargin, although both were required for complete inhibition. Unidirectional efflux of 45Ca2+ after removal of ATP from cells loaded to steady state (1.60±0.12 nmol/106 cells) was monoexponential and occurred with a half-time of 103±10 s. However, the 45Ca2+ content of the stores did not return to their pre-ATP level, but reached a plateau at 0.12±0.04 nmol/106 cells. A similar amount of Ca2+ was trapped within the stores when Ca2+ uptake was prevented by thapsigargin and chelation of Ca2+; at all temperatures between 2 °C and 37 °C; and after stores had first been loaded with unlabelled Ca2+. Simultaneous addition of inositol 1,4,5-trisphosphate (InsP3) and inhibition of Ca2+ uptake reduced the amount of trapped Ca2+ to a level consistent with InsP3 rapidly and more completely emptying a fraction of the stores that could be only partially emptied by the passive leak. After dilution of the specific activity of the 45Ca2+ under conditions that maintained the steady-state activities of the pumps and leaks, the stores rapidly lost their entire 45Ca2+ content. We conclude that the channel responsible for mediating the leak of Ca2+ abruptly closes when the luminal [Ca2+] of the intracellular stores falls below a critical threshold corresponding to about 7% of their steady-state loading. Whereas InsP3 is capable of completely emptying a fraction of the stores, regulation of the passive leak by luminal [Ca2+] is likely to prevent it from completely emptying them; such regulation may ensure that the many other functions of Ca2+ within the endoplasmic reticulum are not compromised.

scholarly journals Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ from permeabilized insulin-secreting cells

1984 ◽  
Vol 223 (2) ◽  
pp. 467-473 ◽  
Author(s):  
T J Biden ◽  
M Prentki ◽  
R F Irvine ◽  
M J Berridge ◽  
C B Wollheim
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Plasma Membranes ◽  
Cell Protein ◽  
Mitochondrial Activity ◽  
Rinm5f Cells ◽  
Cation Composition ◽  
Inositol 1,4,5 Trisphosphate ◽  
Insulin Secreting Cells ◽  
Stimulation Of

A possible role in secretory processes is proposed for inositol 1,4,5-triphosphate (IP3), based upon investigations of the Ca2+ steady state maintained by ‘leaky’, insulin-secreting RINm5F cells. These cells had been treated with digitonin to permeabilize their plasma membranes and thereby ensure that only intracellular Ca2+ buffering mechanisms were active. When placed in a medium with a cation composition resembling that of the cytosol, cells rapidly took up Ca2+ as measured by a Ca2+-specific minielectrode. Two Ca2+ steady states were observed. A lower level of around 120nM required ATP-dependent Ca2+ uptake and was probably determined by the endoplasmic reticulum. The higher steady state (approx. 800 nM), seen only in the absence of ATP, was shown to be due to mitochondrial activity. IP3 specifically released Ca2+ accumulated in the ATP-dependent pool, but not from mitochondria, since Ca2+ release was demonstrated in the presence of the respiratory poison antimycin. The IP3-induced Ca2+ release was rapid, with 50% of the response being seen within 15s. The apparent Km was 0.5 microM and maximal concentrations of IP3 (2.5 microM) produced a peak Ca2+ release of 10 nmol/mg of cell protein, which was followed by re-uptake. A full Ca2+ response was seen if sequential pulses of 2.5 microM-IP3 were added at 20 min intervals, although there was a slight (less than 20%) attenuation if the intervening period was decreased to 10 min. These observations could be related to the rate of IP3 degradation which, in this system, corresponded to a 25% loss of added 32P label within 2 min, and a 75% loss within 20 min. The results suggest that IP3 might act as a link between metabolic, cationic and secretory events during the stimulation of insulin release.

scholarly journals Ca2+ entry into PC12 cells initiated by ryanodine receptors or inositol 1,4,5-trisphosphate receptors

1998 ◽  
Vol 329 (2) ◽  
pp. 349-357 ◽  
Author(s):  
L. Deborah BENNETT ◽  
D. Martin BOOTMAN ◽  
J. Michael BERRIDGE ◽  
R. Timothy CHEEK
Keyword(s):  
Endoplasmic Reticulum ◽  
Pc12 Cells ◽  
Ryanodine Receptors ◽  
Half Time ◽  
Free Medium ◽  
Excitable Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Insp3 Receptors ◽  
Universal Mechanism ◽  
Stimulation Of

Capacitative Ca2+ entry (CCE) is a universal mechanism for refilling intracellular Ca2+ stores in electrically non-excitable cells. The situation in excitable cells is less clear, however, since they may rely on other entry mechanisms for Ca2+-store refilling. In the present study we investigated CCE in intact PC12 cells, using acetylcholine to bring about activation of InsP3 receptors (InsP3Rs), caffeine to activate ryanodine receptors (RyRs) and thapsigargin to inhibit sarco/endoplasmic reticulum Ca2+-ATPase pumps. We found that depletion of the InsP3-, caffeine- or thapsigargin-sensitive stores promoted Ca2+ entry, suggesting that stimulation of either InsP3Rs or RyRs can activate CCE. The CCE pathways activated by InsP3Rs, RyRs and thapsigargin appeared to be independent at least in part, since their effects were found to be additive. However, CCE triggered by caffeine, acetylcholine or thapsigargin progressively diminished with time. The decay of CCE caused by one agent also inhibited subsequent responses to the others, suggesting that some component of the CCE pathway is common to all intracellular Ca2+ stores. The magnitude of CCE stimulated by InsP3Rs or RyRs was related to the size of the stores; the InsP3-sensitive store was smaller than the RyR-sensitive store and triggered a smaller entry component. However, both stores filled with a similar half time (about 1 min), and both could be filled more rapidly by depolarization-induced Ca2+ entry through voltage-operated channels. A significant basal Ca2+ influx was apparent in PC12 cells. The basal entry component may be under the control of the InsP3-sensitive Ca2+ store, since short incubations in Ca2+-free medium depleted this store.

scholarly journals The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

2021 ◽  
Vol 118 (10) ◽  
pp. e2010789118
Author(s):  
Yihan Shen ◽  
Nagendra Babu Thillaiappan ◽  
Colin W. Taylor
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fluorescence Intensity ◽  
Small Cluster ◽  
Store Depletion ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cytosolic Domains ◽  
Intracellular Ca ◽  
The Many ◽  
Orai Channels

Increases in cytosolic Ca2+ concentration regulate diverse cellular activities and are usually evoked by opening of Ca2+ channels in intracellular Ca2+ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP3), IP3 receptors coordinate the contributions of these two Ca2+ sources by mediating Ca2+ release from the endoplasmic reticulum (ER). Loss of Ca2+ from the ER then activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca2+ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca2+ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

SPECKLE STATISTICS IN THE PHOTON LOCALIZATION TRANSITION

2010 ◽  
Vol 24 (12n13) ◽  
pp. 1950-1988 ◽  
Author(s):  
Azriel Z. Genack ◽  
Jing Wang
Keyword(s):  
Steady State ◽  
Probability Distributions ◽  
Speckle Pattern ◽  
Frequency Variation ◽  
Localization Transition ◽  
Classical Waves ◽  
Speckle Patterns ◽  
The Many ◽  
Photon Localization ◽  
State Propagation

We review the statistics of speckle in the Anderson localization transition for classical waves. Probability distributions of local and integrated transmission and of the evolution of the structure of the speckle pattern are related to their corresponding correlation functions. Steady state and pulse transport can be described in terms of modes whose speckle patterns are obtained by decomposing the frequency variation of the transmitted field. At the same time, transmission can be purposefully manipulated by adjusting the incident field and the eigenchannels of the transmission matrix can be found by analyzing sets of speckle patterns for different inputs. The many aspects of steady state propagation are reflected in diverse, but simply related, parameters so that a single localization parameter encapsulates the character of transport on both sides of the divide separating localized from diffusive waves.

Drug Interactions with Warfarin: Studies with Dichloralphenazone, Chloral Hydrate and Phenazone (Antipyrine)

1971 ◽  
Vol 40 (4) ◽  
pp. 351-364 ◽  
Author(s):  
A. Breckenridge ◽  
M. L'E. Orme ◽  
S. Thorgeirsson ◽  
D. S. Davies ◽  
R. V. Brooks
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Urinary Excretion ◽  
Binding Sites ◽  
Half Life ◽  
Chloral Hydrate ◽  
Maximal Velocity ◽  
Protein Binding Sites ◽  
Drug Oxidation ◽  
State Plasma

1. Administration of dichloralphenazone, a complex of chloral hydrate and phenazone (antipyrine) caused a fall in steady-state plasma warfarin concentration and loss of anticoagulant control in five subjects. 2. This effect of dichloralphenazone is due to stimulation of the drug-oxidizing enzymes of the liver endoplasmic reticulum by antipyrine, the non-hypnotic part of the complex. Administration of antipyrine caused a fall in steady-state plasma warfarin concentration in five subjects, a shortening of the plasma warfarin half-life, with increased urinary excretion of the metabolites of 14C-labelled warfarin in two subjects and increased urinary excretion of 6β-hydroxycortisol which is formed in the liver endoplasmic reticulum. 3. Administration of chloral hydrate, the hypnotic part of dichloralphenazone, caused no change in anticoagulant control but a fall in steady-state plasma warfarin concentration in five subjects. This is due to the accumulation of trichloroacetic acid which displaces warfarin from plasma protein binding sites. 4. Individual differences in the extent of enzyme induction have been shown to be related to the subjects' rates of drug oxidation. 5. In the rat administration of dichloralphenazone and antipyrine, but not chloral hydrate, caused shortening of pentobarbitone sleeping time and of the plasma [14C]pentobarbitone half-life, shortening of the zoxazolamine paralysis time and increase in the maximal velocity of N-demethylation of ethylmorphine.

scholarly journals Triosephosphate isomerase deficiency: consequences of an inherited mutation at mRNA, protein and metabolic levels

2005 ◽  
Vol 392 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Judit Oláh ◽  
Ferenc Orosz ◽  
László G. Puskás ◽  
László Hackler ◽  
Margit Horányi ◽  
...  
Keyword(s):  
Steady State ◽  
Triosephosphate Isomerase ◽  
Specific Activity ◽  
Energy State ◽  
Dihydroxyacetone Phosphate ◽  
Peptidase Activity ◽  
Mrna Levels ◽  
Regulatory Enzymes ◽  
Computational Data ◽  
Fructose 1,6 Bisphosphate

Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. Two Hungarian compound heterozygote brothers inherited the same TPI mutations (F240L and E145Stop), but only the younger one suffers from neurodegeneration. In the present study, we determined the kinetic parameters of key glycolytic enzymes including the mutant TPI for rational modelling of erythrocyte glycolysis. We found that a low TPI activity in the mutant cells (lower than predicted from the protein level and specific activity of the purified recombinant enzyme) is coupled with an increase in the activities of glycolytic kinases. The modelling rendered it possible to establish the steady-state flux of the glycolysis and metabolite concentrations, which was not possible experimentally due to the inactivation of the mutant TPI and other enzymes during the pre-steady state. Our results showed that the flux was 2.5-fold higher and the concentration of DHAP (dihydroxyacetone phosphate) and fructose 1,6-bisphosphate increased 40- and 5-fold respectively in the erythrocytes of the patient compared with the control. Although the rapid equilibration of triosephosphates is not achieved, the energy state of the cells is not ‘sick’ due to the activation of key regulatory enzymes. In lymphocytes of the two brothers, the TPI activity was also lower (20%) than that of controls; however, the remaining activity was high enough to maintain the rapid equilibration of triosephosphates; consequently, no accumulation of DHAP occurs, as judged by our experimental and computational data. Interestingly, we found significant differences in the mRNA levels of the brothers for TPI and some other, apparently unrelated, proteins. One of them is the prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases. We found that the peptidase activity of the affected brother was reduced by 30% compared with that of his neurologically intact brother.

scholarly journals Inositol 1,4,5 trisphosphate releases calcium from specialized sites within Limulus photoreceptors

1987 ◽  
Vol 104 (4) ◽  
pp. 933-937 ◽  
Author(s):  
R Payne ◽  
A Fein
Keyword(s):  
Endoplasmic Reticulum ◽  
Injection Site ◽  
Intracellular Calcium ◽  
Subcellular Distribution ◽  
Smooth Endoplasmic Reticulum ◽  
Image Intensifier ◽  
Photoreceptor Cells ◽  
Calcium Stores ◽  
Inositol 1,4,5 Trisphosphate ◽  
Probable Site

We have investigated the subcellular distribution and identity of inositol trisphosphate (InsP3)-sensitive calcium stores in living Limulus ventral photoreceptor cells, where light and InsP3 are known to raise intracellular calcium. We injected ventral photoreceptor cells with the photoprotein aequorin and viewed its luminescence with an image intensifier. InsP3 only elicited detectable aequorin luminescence when injected into the light-sensitive rhabdomeral (R)-lobe where aequorin luminescence induced by light was also confined. Calcium stores released by light and InsP3 are therefore localized to the R-lobe. Within the R-lobe, InsP3-induced aequorin luminescence was further confined around the injection site, due to rapid dilution and/or degradation of injected InsP3. Prominent cisternae of smooth endoplasmic reticulum are uniquely localized within the cell beneath the microvillar surface of the R-lobe (Calman, B., and S. Chamberlain, 1982, J. Gen. Physiol., 80:839-862). These cisternae are the probable site of InsP3 action.

Endurance training enhances lactate clearance during hyperlactatemia

1989 ◽  
Vol 257 (5) ◽  
pp. E782-E789 ◽  
Author(s):  
C. M. Donovan ◽  
M. J. Pagliassotti
Keyword(s):  
Steady State ◽  
Endurance Training ◽  
Specific Activity ◽  
Lactate Production ◽  
Lactate Clearance ◽  
Lactate Removal ◽  
Lactate Levels ◽  
Infusion Period ◽  
Mixed Venous ◽  
Blood Lactate Levels

Constant infusions of cold molar lactate (178.0 +/- 1.6 mumol.kg-1.min-1), [U-14C]lactate (0.50 muCi/min), and [6-3H]glucose (0.5 muCi/min) were employed to study the effects of endurance training (running 1 h/day, at 38 m/min, 10% grade) on lactate clearance in resting, hyperlactatemic rats. Before infusion, resting blood lactate levels were not significantly different between controls, 1.10 +/- 0.04 mM, and trained animals, 1.16 +/- 0.04 mM. Lactate levels increased significantly during the infusion period, attaining steady-state mixed venous concentrations of 11.32 +/- 0.24 mM and 5.44 +/- 0.09 mM, respectively, for controls and trained animals. Lactate clearance rates, based on net lactate removal (i.e., not tracer-estimated lactate removal), were twofold greater in trained animals vs. controls, 33.0 +/- 0.7 and 15.4 +/- 0.4 ml.kg-1. min-1, respectively. Lactate specific activity values during the infusion period were not significantly different between controls, 22,243 +/- 236 dpm/mumol, and trained animals, 21,270 +/- 374 dpm/mumol, indicating similar endogenous dilution of the pyruvate-lactate pool. For both control and trained animals, essentially 100% of the 14C infused as lactate was recovered as either glucose or CO2; however, trained animals demonstrated a 25% greater rate of gluconeogenesis. At a given lactate production rate, trained animals maintain lower lactate levels through enhanced clearance via gluconeogenesis and oxidation.

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