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 Mitochondrial–Endoplasmic Reticulum Interplay: A Lifelong On–Off Relationship?

Contact ◽  
2019 ◽  
Vol 2 ◽  
pp. 251525641986122 ◽  
Author(s):  
Corina T. Madreiter-Sokolowski ◽  
Roland M. Malli ◽  
Wolfgang F. Graier
Keyword(s):  
Endoplasmic Reticulum ◽  
Adenosine Triphosphate ◽  
Therapeutic Strategies ◽  
Mitochondrial Activity ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Potential Target ◽  
Pathological Conditions ◽  
Elevated Glucose ◽  
Stimulation Of

This article comments recent publications that highlight an intriguing importance of specific settings in the interaction between the mitochondria and the endoplasmic reticulum to ensure cell-specific functions like the responsiveness to elevated glucose in pancreatic β-cells. Hence, alterations of the mitochondria–endoplasmic reticulum communications under various pathological conditions like aging or cancer often come with enhanced Ca2+ transfer that, in turn, yields stimulation of basal mitochondrial activity to meet the increasing adenosine triphosphate demand of the very cell. Such observations identify mitochondria-associated membranes as potential target for new therapeutic strategies against aging or cancer.

scholarly journals Histamine and a guanine nucleotide increase calcium permeability in pig aortic microsomal fractions

1990 ◽  
Vol 267 (1) ◽  
pp. 105-109 ◽  
Author(s):  
L M Blayney ◽  
A C Newby
Keyword(s):  
Endoplasmic Reticulum ◽  
Steady State ◽  
Guanine Nucleotide ◽  
Intact Cells ◽  
Receptor Sites ◽  
H2 Antagonist ◽  
Dependent Inhibition ◽  
Calcium Permeability ◽  
H1 Antagonist ◽  
Stimulation Of

ATP-dependent Ca2+ accumulation was measured in pig aortic microsomal fractions containing plasmalemma and endoplasmic reticulum. In vesicles sonicated with histamine, to allow access to internally located receptor sites, guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG), added to activate externally located guanine-nucleotide-transducing proteins, caused a concentration-dependent decrease in steady-state Ca2+ accumulation that was reversed by guanosine 5′-[beta-thio]diphosphate. In the presence of p[NH]ppG, sonication with histamine produced a concentration-dependent inhibition of Ca2+ accumulation that could be antagonized by the H1 antagonist mepyramine, but not by the H2 antagonist cimetidine. The inhibition of steady-state Ca2+ accumulation could have resulted from an inhibition of ATP-dependent Ca2+ uptake or a stimulation of Ca2+ release. We observed, however, that p[NH]ppG plus histamine stimulated, rather than inhibited, Ca2(+)-ATPase activity. We concluded that p[NH]ppG and histamine acted together to increase Ca2+ permeability. In support of this, p[NH]ppG accelerated efflux of Ca2+ from passively loaded vesicles sonicated with, but not without, histamine. The effect of p[NH]ppG was unlikely to be due to Ins(1,4,5)P3 (and hence release from endoplasmic-reticulum vesicles), since addition of Ins(1,4,5)P3 to vesicles sonicated with histamine did not alter steady-state Ca2+ accumulation. Our results therefore suggest that histamine and p[NH]ppG increased the permeability of the plasmalemma vesicles and may thus model the process of receptor-mediated Ca2+ entry into intact cells.

scholarly journals myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

1986 ◽  
Vol 236 (1) ◽  
pp. 37-44 ◽  
Author(s):  
D M Delfert ◽  
S Hill ◽  
H A Pershadsingh ◽  
W R Sherman ◽  
J M McDonald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Initial Rate ◽  
Membrane Vesicles ◽  
Heavy Fraction ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Inositol 1,4,5 Trisphosphate ◽  
Uptake And Release

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ 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 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 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 Regulation of inositol 1,4,5-trisphosphate metabolism in insulin-secreting RINm5F cells

1988 ◽  
Vol 251 (2) ◽  
pp. 435-440 ◽  
Author(s):  
T J Biden ◽  
L Vallar ◽  
C B Wollheim
Keyword(s):  
Rinm5f Cells ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Tetradecanoyl Phorbol Acetate ◽  
Inositol 1,4,5 Trisphosphate ◽  
Muscarinic Stimulation ◽  
Cellular Stimulation ◽  
Stimulation Of ◽  
Rate Of Accumulation

Factors underlying the transience of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation following muscarinic stimulation of RINm5F cells were examined. Transience was not due to a protein kinase C-mediated stimulation of Ins(1,4,5)P3 dephosphorylation, since pretreatment of cells with tetradecanoyl-phorbol acetate (TPA) did not alter the rate of this conversion. However, preincubation with TPA did inhibit carbamoylcholine-stimulated Ins(1,4,5)P3 formation. In permeabilized cells, the conversion of Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was slightly enhanced in the presence of TPA or cyclic AMP, but much more markedly by raising the Ca2+ concentration from 10(-7) M to 10(-6) or 10(-5) M. In intact cells the most rapid rate of accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 occurred in the first 2 s following stimulation, whereas the levels of inositol 1,4-bisphosphate were not increased until after 5 s. This suggests that Ins(1,4,5)P3 kinase is chiefly responsible for the early disposal of Ins(1,4,5)P3 following cellular stimulation. The results are consistent with the proposal that the transient accumulation of Ins(1,4,5)P3 is due both to its enhanced metabolism via the Ca2+-calmodulin-sensitive Ins(1,4,5)P3 kinase, as well as a down-regulation of phosphatidylinositol 4,5-bisphosphate hydrolysis.

The Fine Structure Of Photoreceptors In Mitopus Morio (Phalangida)

1969 ◽  
Vol 4 (2) ◽  
pp. 327-351
Author(s):  
D. J. CURTIS
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Acetylcholinesterase Activity ◽  
Structural Studies ◽  
Hexagonal Array ◽  
Laminated Structure ◽  
Dense Bodies ◽  
Single Lens ◽  
Dioptric Apparatus ◽  
Stimulation Of

Fine structural studies on the eyes of the harvestman Mitopus morio revealed the presence of microvilli in the rhabdom. The microvilli vary in length between 1 µ and 2 µ, are about 800 Å wide, and curved or straight. They derive from the plasma membranes of the four retinula cells which surround the rhabdom. Approximately cylindrical in shape, the rhabdoms are about 40 µ long by about 4-6 µ in cross-diameter. Each rhabdom is situated at the centre of a retinula, and these retinulae are packed in a hexagonal array to form the retina. Distally, rhabdom fusion occurs to form a rhabdom network. The retina lies beneath the dioptric apparatus which consists of a single lens, surmounting a glassy body composed of lentigen cells. The cytoplasmic organelles of the retinula cells include mitochondria, lysosomes, sparse elements of endoplasmic reticulum, vesicular components, prominent Golgi complexes and pigment granules which possess a laminated structure. An important feature of the retinula cell is the presence of many small vesicles, about 0.1 µ in diameter, clustered beneath the rhabdom. Incubation of glutaraldehyde-fixed eyes in a Gomori medium with acetylthiocholine as substrate, coupled with inhibition of controls by 62C47, indicates the presence of a presumed acetylcholinesterase in these vesicles. Similar vesicles also occur in the proximal cytoplasm of the retinula cells. Other larger vesicles, often with a core of whorled membranes, as well as dense bodies, also show acetylthiocholine-splitting activity. This latter activity is not inhibited by 62C47 and is probably the effect of lysosomal non-specific esterase. These bodies also exhibit acid phosphatase activity when incubated in a Gomori medium with β-glycerophosphate as substrate. The presence of acetylcholinesterase activity, as distinct from non-specific esterase, in vesicles closely associated with the rhabdom and in more proximally situated vesicles is significant. It would point to the presence of an acetylcholine/acetylcholinesterase system involved in the generation and/or propagation of the sensory impulse arising from photo-stimulation of the rhabdom.

Regulation of Na-K-Cl cotransport in cultured canine airway epithelia: a [3H]bumetanide binding study

1990 ◽  
Vol 259 (4) ◽  
pp. C557-C569 ◽  
Author(s):  
M. Haas ◽  
L. G. Johnson ◽  
R. C. Boucher
Keyword(s):  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Cell Protein ◽  
Adrenergic Stimulation ◽  
Additional Increase ◽  
Airway Epithelia ◽  
Basolateral Side ◽  
Solution Bathing ◽  
Stimulation Of

We examined [3H]bumetanide binding to membranes isolated from canine tracheal and bronchial epithelia and to confluent primary cultures of these cells. Crude plasma membranes from trachea and bronchus bind [3H]bumetanide in a saturable manner; tracheal membranes have a higher affinity but lower maximal binding (K1/2 approximately equal to 0.7 microM; Bmax approximately equal to 2.5 pmol/mg protein) than do bronchial membranes (K1/2 approximately equal to 3.5 microM; B(max) approximately equal to 7.5 pmol/mg). In both cases, saturable binding is reduced by greater than 65% when either Na, K, or Cl is removed from the medium. In primary cultures, saturable [3H]bumetanide binding (inhibited by a 30-fold excess of unlabeled bumetanide) occurs when [3H]bumetanide (1.0 microM) is added to the solution bathing the basolateral side of tracheal (1.20 +/- 0.10 pmol bound/mg total cell protein) and bronchial (1.79 +/- 0.52 pmol/mg) cultures; minimal binding is seen with apical [3H]bumetanide. Isoproterenol (10(-5) M; basolateral exposure) produces approximately 100% increase in saturable basolateral [3H]bumetanide binding to tracheal cultures and approximately 30% increase in bronchial cultures. Similar augmentation of binding is seen when apical Cl is reduced from 134 to 4 mM and when both apical and basolateral media are made hypertonic by addition of 100 mM sucrose. Under these latter two conditions, isoproterenol produces little or no additional increase in binding. Our results indicate that the increase in basolateral Cl influx via Na-K-Cl cotransport that must occur during beta-adrenergic stimulation of net salt secretion in canine airway epithelia is related to an actual increase in the number of functioning cotransporters in the basolateral membrane and is not simply due to a change in ion gradients. The increase in cotransport sites, however, may be secondary to initial stimulation of apical Cl channels, with resultant cell shrinkage.

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