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Biomedical subjects

L Missiaen

Publications and source records attributed to L Missiaen.

At least 109 records · Page 6Linked to original sources
PubMed1992

Luminal Ca2+ controls the activation of the inositol 1,4,5-trisphosphate receptor by cytosolic Ca2+.

Luminal Ca2+ controls the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3-induced Ca2+ release is also controlled by cytosolic Ca2+; low concentrations of Ca2+ stimulate the release. The aim of this work was to investigate whether luminal Ca2+ would affect the stimulation of the Ins(1,4,5)P3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. We also report that the Ins(1,4,5)P3 receptor in A7r5 cells is activated by low concentrations of cytosolic Ca2+. Cytoplasmic Ca2+ increases the Ins(1,4,5)P3 sensitivity without affecting the cooperativity. The increase in Ins(1,4,5)P3 sensitivity becomes relatively more pronounced when the Ca2+ content of the stores decreases. This modulatory effect of luminal Ca2+ on the responsiveness to cytosolic Ca2+ is an intrinsic property of the Ins(1,4,5)P3 receptor.

Animals↗
PubMed1992

Ca2+ release induced by inositol 1,4,5-trisphosphate is a steady-state phenomenon controlled by luminal Ca2+ in permeabilized cells.

Low concentrations of inositol 1,4,5-trisphosphate (InsP3) evoke a very rapid mobilization of intracellular Ca2+ stores in many cell types, which can be followed by a further, much slower efflux. Two explanations have been suggested for this biphasic release. The first proposes that the Ca2+ stores vary in their sensitivity to InsP3, and each store releases either its entire contents or nothing (all-or-none release); the second proposes instead that the stores are uniformly sensitive to the effects of InsP3, but that they can release only a fraction of their Ca2+ before their sensitivity is somehow attenuated (steady-state release). Experiments using purified InsP3 receptor molecules reconstituted into lipid vesicles have shown heterogeneity of the receptors in their response to InsP3 under conditions in which the total Ca2+ level at both sides of the receptor is held constant. We now report that in permeabilized A7r5 smooth-muscle cells incubated in Ca(2+)-free medium, the amount of 45Ca2+ remaining in the stores after the rapid transient phase of release is independent of their initial Ca2+ levels, indicating that partially depleted stores are less sensitive to InsP3. Moreover, if the stores are reloaded with 40Ca2+ after the first stimulus, reapplication of the same low concentration of InsP3 will release further 45Ca2+. This recovery of InsP3 sensitivity is almost complete. Under these conditions, Ca2+ release must thus occur by a steady-state mechanism, in which the decreasing Ca2+ content of the stores slows down further release.

Calcium↗
PubMed1992

Stimulation of the catalytic cycle of the Ca2+ pump of porcine plasma-membranes by negatively charged phospholipids.

The (Ca(2+)+Mg2+)-ATPase of the plasma membrane is activated by negatively charged phospholipids. The mechanism of this activation was investigated by studying the effect of negatively charged phospholipids on the steady-state phosphointermediate level and on the p-nitrophenylphosphatase activity. Both parameters were differentially affected by different acidic phospholipids. The level of phosphoprotein intermediate was not affected by phosphatidylserine (20% of total phospholipid), but it was increased by 60% by phosphatidylinositol 4-phosphate. Phosphatidylserine increased the p-nitrophenylphosphatase activity, whereas phosphatidylinositol 4-phosphate had no significant effect. It is suggested that phosphatidylinositol 4-phosphate mainly affects a reaction step which leads to accelerated formation of the phosphointermediate, whereas the action of phosphatidylserine would affect two reaction steps, one upstream and one downstream of the phosphointermediate.

4-Nitrophenylphosphatase↗
PubMed1992

Calcium ion homeostasis in smooth muscle.

Ca2+ plays an important role in the regulation of smooth-muscle contraction. In this review, we will focus on the various Ca(2+)-transport processes that contribute to the cytosolic Ca2+ concentration. Mainly the functional aspects will be covered. The smooth-muscle inositol 1,4,5-trisphosphate receptor and ryanodine receptor will be extensively discussed. Smooth-muscle contraction also depends on extracellular Ca2+ and both voltage- and Ca(2+)-release-activated plasma-membrane Ca2+ channels will be reviewed. We will finally discuss some functional properties of the Ca2+ pumps that remove Ca2+ from the cytoplasm and of the Ca2+ regulation of the nucleus.

Animals↗
PubMed1992

Luminal Ca2+ promoting spontaneous Ca2+ release from inositol trisphosphate-sensitive stores in rat hepatocytes.

1. Spontaneous Ca2+ release from the inositol 1,4,5-trisphosphate (InsP3)-sensitive stores in permeabilized hepatocytes was monitored using Fluo-3 to measure the free [Ca2+] of the medium bathing the cells. 2. Permeabilized cells rapidly sequestered Ca2+, reducing the [Ca2+] to 103 +/- 5 nM. Under conditions that depended critically upon cell density and the amount of Ca2+ in the medium, this was followed by a slow increase in [Ca2+] culminating in a substantial Ca2+ spike representing synchronous discharge from the InsP3-sensitive stores. 3. During the latency preceding the Ca2+ spike, the stores increased their sensitivity to InsP3. This sensitization seemed to be an all-or-none phenomenon. 4. Oxidized glutathione and thimerosal promoted the spontaneous release by sensitizing the InsP3 receptor. 5. An increase in the [Ca2+] within the stores was required for both the increased sensitivity to InsP3 and the subsequent spike. 6. Caffeine (6 mM) antagonized the effect of very low InsP3 concentrations and abolished the Ca2+ spike, without itself releasing Ca2+. 7. Our results suggesting that luminal Ca2+ may sensitive InsP3-sensitive stores leading to spontaneous Ca2+ mobilization will be discussed in the light of a modified version of the two-pool model for explaining cytosolic Ca2+ oscillations.

Action Potentials↗
PubMed1991

Spontaneous calcium release from inositol trisphosphate-sensitive calcium stores.

Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger to mobilize Ca2+ from intracellular reservoirs. The release mechanism displays all-or-none characteristics, that may account for other observations that the InsP3-induced mobilization of Ca2+ is quantal. Quantal release may depend on the sensitivity of the InsP3 receptor being regulated by the Ca2+ concentration in the lumen of the endoplasmic reticulum. We report here that the InsP3-sensitive store in hepatocytes discharges spontaneously when overloaded with Ca2+. The release, which is blocked by heparin, is preceded by an increasing sensitivity of the InsP3 receptor to endogenous InsP3, and is promoted by those sulphydryl reagents (oxidized glutathione and thimerosal) that induce Ca2+ oscillations in intact cells (ref. 8, and T. A. Rooney, D. C. Renard, E. J. Sass and A. P. Thomas, manuscript in preparation). This novel process could have a role in generating both Ca2+ oscillations and Ca2+ waves.

Animals↗
PubMed1991

Bovine adrenal chromaffin cells contain an inositol 1,4,5-trisphosphate-insensitive but caffeine-sensitive Ca2+ store that can be regulated by intraluminal free Ca2+.

We have characterized some properties of the caffeine-sensitive Ca2+ store in bovine chromaffin cells. Addition of 10 mM-caffeine to permeabilized cells that were allowed to sequester Ca2+ in the presence of the precipitating anion pyrophosphate induced a transient rise in free Ca2+ concentration that was blocked by 10 microM-Ruthenium Red. Caffeine was able to release Ca2+ after the InsP3-sensitive Ca2+ pool had been completely emptied, and 10 microM-InsP3 still released Ca2+ in the presence of a high dose (50 mM) of caffeine, indicating that there are selectively sensitive Ca2+ pools in these cells. The progressive hydrolysis of pyrophosphate by a cytosolic pyrophosphatase induced a spontaneous Ca2+ release after a latency. Caffeine prevented this spontaneous Ca2+ release, indicating that the pyrophosphate-sensitive Ca2+ pool was caffeine-sensitive. On varying the free Ca2+ concentration within the caffeine-sensitive pool (by using methylenediphosphonic acid, pyrophosphate or no precipitating anion), we observed that the Ca(2+)-releasing effect of caffeine was dependent on an elevated intraluminal free Ca2+ concentration. In conclusion, the caffeine-sensitive Ca2+ store in bovine chromaffin cells is largely distinct from the InsP3-sensitive Ca2+ store, and its release mechanism shares characteristics with the ryanodine receptor of muscle cells.

Adrenal Medulla↗
PubMed1991

Isoform switching of the sarco(endo)plasmic reticulum Ca2+ pump during differentiation of BC3H1 myoblasts.

We have studied the expression of the gene 2 for the sarco(endo)plasmic reticulum Ca2+ pump (SERCA2) in BC3H1 cells. Myogenic differentiation not only activated the SERCA2 expression but it also induced an isoform switch. Undifferentiated myoblasts only expressed the SERCA2b isoform (non-muscle) whereas differentiated myocytes predominantly contained the SERCA2a isoform (cardiac/slow skeletal muscle). The isoform switch was documented by immunoblot analysis with isoform-specific antibodies. This observation was confirmed at the mRNA level by using antisense RNA probes specific for class 1 (SERCA2a) or class 2 (SERCA2b) messengers. The expression of the SERCA2a isoform after differentiation was accompanied by a decreased sensitivity of the Ca2+ uptake in permeabilized cells to the Ca2+ pump inhibitor thapsigargin.

Animals↗
PubMed1991

Uptake characteristics of the InsP3-sensitive and -insensitive Ca2+ pools in porcine aortic smooth-muscle cells: different Ca2+ sensitivity of the Ca2(+)-uptake mechanism.

We have investigated the Ca2(+)-uptake characteristics of the InsP3-sensitive and -insensitive non-mitochondrial Ca2+ pools in permeabilized cultured porcine aortic smooth-muscle cells. The InsP3-sensitive Ca2+ pool, which was also GTP sensitive, had a high Ca2+ affinity and was highly oxalate permeable. The InsP3-insensitive Ca2+ store, which was also GTP insensitive, had a much lower Ca2+ affinity and presented a low oxalate permeability. The loading of both pools decreased at high free [Ca2+], although these cells did not have a Ca2(+)-induced Ca2+ release mechanism. This decreased loading of the InsP3-sensitive Ca2+ pool at higher free [Ca2+] must be taken into consideration when investigating a possible Ca2(+)-inhibition of the InsP3-induced Ca2+ release. Part of the Ca2+ uptake into the InsP3-insensitive Ca2+ pool was not affected by the Ca2(+)-pump inhibitors vanadate, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone.

Animals↗
PubMed1991

Changes in the mechanism of Ca2(+) mobilization during the differentiation of BC3H1 muscle cells.

Ca2+ sequestration and release in BC3H1 muscle cells is strongly dependent on the stage of differentiation. In proliferating cells, more than 90% of the sequestered Ca2+ was Ins(1,4,5)P3-sensitive and 25% was caffeine-sensitive. In differentiated cells, the Ca2+ accumulation was 5-fold higher and was InsP3-insensitive, but about 60% of the sequestered Ca2+ was caffeine-sensitive. These changes were reversible upon addition of growth stimuli. Similarly, by measuring the intracellular Ca2+ concentration in single intact BC3H1 cells, it was found that the number of histamine-responsive cells decreased and the number of caffeine-responsive cells increased during muscle cell differentiation. These data indicate that the development of the muscle phenotype in BC3H1 myoblasts induces a major rearrangement of the mechanisms for Ca2+ mobilization.

Animals↗
PubMed1991

AlF4- induces Ca2+ oscillations in guinea-pig ileal smooth muscle.

The effects of different compounds that inhibit the isolated plasma-membrane Ca2+/Mg2(+)-ATPase on the cytosolic free Ca2+ concentration ([Ca2+]i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F-, in the presence of Al3+, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca2+]i while the phasic contractions were accompanied by concomitant oscillations in [Ca2+]i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca2+]i and of force elicited by AlF4- were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca2+ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF4-, vanadate and calmidazolium induce cytoplasmic Ca2+ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca2+/Mg2(+)-ATPase. The generation of membrane-linked Ca2+ oscillations could therefore be related to an inhibition of the plasma-membrane Ca2+ pump resulting in an increase of [Ca2+]i. This change in [Ca2+]i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.

Aluminum↗
PubMed1991

Ca2+ extrusion across plasma membrane and Ca2+ uptake by intracellular stores.

The aim of this review is to summarize the various systems that remove Ca2+ from the cytoplasm. We will initially focus on the Ca2+ pump and the Na(+)-Ca2+ exchanger of the plasma membrane. We will review the functional regulation of these systems and the recent progress obtained with molecular-biology techniques, which pointed to the existence of different isoforms of the Ca2+ pump. The Ca2+ pumps of the sarco(endo)plasmic reticulum will be discussed next, by summarizing the discoveries obtained with molecular-biology techniques, and by reviewing the physiological regulation of these proteins. We will finally briefly review the mitochondrial Ca(2+)-uptake mechanism.

Animals↗
PubMed1990

Ruthenium red and compound 48/80 inhibit the smooth-muscle plasma-membrane Ca2+ pump via interaction with associated polyphosphoinositides.

We will demonstrate the compound 48/80 and ruthenium red inhibit the smooth-muscle plasma-membrane Ca2+ pump by counteracting the stimulant effect of negatively charged phospholipids. Both substances did not affect the purified enzyme re-activated by pure phosphatidylcholine or phosphatidylinositol and measured in the absence of calmodulin, indicating that under these conditions they did not have a direct effect on the ATPase protein. Ruthenium red and compound 48/80 however inhibited the (Ca2(+) + Mg2+)-ATPase in the presence of phosphatidylinositol 4-phosphate and especially phosphatidylinositol 4,5-bisphosphate. The K0.5 for inhibition was 25 microM ruthenium red and 9 micrograms/ml of compound 48/80. The inhibition by ruthenium red developed slowly with half maximal inhibition occurring after about 75 s while that by compound 48/80 developed immediately within the time required for mixing. The efficacy of ruthenium red increased as the concentration of the acidic phospholipid increased, while no such cooperativity was observed for compound 48/80. Ruthenium red reduced the Vmax for Ca2+ without affecting the affinity for Ca2+, while compound 48/80 decreased both parameters. In conclusion, although ruthenium red and compound 48/80 affect the ATPase differently, both substances most likely inhibit the plasma-membrane Ca2+ pumping by counteracting the stimulation by negatively charged phospholipids.

Animals↗
PubMed1990

Transport systems for polyamines in the established renal cell line LLC-PK. Polarized expression of an Na(+)-dependent transporter.

We present evidence for the existence of an Na(+)-dependent transporter and an Na(+)-independent transporter for polyamines in LLC-PK1 cells. Both transporters could be discriminated by their sensitivity to inhibitors, particularly rho-chloromercuriphenyl sulphate and various polycationic molecules. By using cell monolayers grown on a permeable filter support, we have found that the Na(+)-dependent polyamine uptake occurred preferentially from the basolateral side. The Na(+)-independent uptake, on the other hand, occurred to the same extent from either the apical or the basolateral side.

4-Chloromercuribenzenesulfonate↗
PubMed1990

Agonist-dependent Ca2+ and Mn2+ entry dependent on state of filling of Ca2+ stores in aortic smooth muscle cells of the rat.

1. The properties of intracellular Ca2+ stores of intact- and of saponin-skinned A7r5 (an established cell line from embryonic rat aorta) smooth muscle cells were studied by measuring 45Ca2+ and 54Mn2+ fluxes. 2. Application of 5 microM-vasopressin to intact cells increased the fractional loss of 45Ca2+ in Ca2(+)-free solution by a factor of 5.2. This effect was not influenced by a pre-incubation with 10 microM-ryanodine. Caffeine (25 mM) did not stimulate the fractional loss of 45Ca2+ from intact cells. 3. In skinned cells 10 microM-IP3 (inositol 1,4,5-trisphosphate) and 5 microM-A23187 (a calcium ionophore) released the same amount of 45Ca2+. This release did not require GTP and was not affected by a pre-incubation with 10 microM-ryanodine. Caffeine (25 mM) did not release stored Ca2+. 4. NaF (1 mM) plus 10 microM-AlCl3 inhibited by 72% the 45Ca2+ uptake by the IP3-sensitive store of skinned cells at 0.15 microM-Ca2+. Cyclic AMP-dependent protein kinase did not stimulate this ATP-dependent 45Ca2+ uptake, nor could the presence of phospholamban be demonstrated immunologically. 5. The 45Ca2+ uptake by cells which had been depleted of Ca2+ with 5 microM-vasopressin was 69% higher than the uptake obtained without such proceeding depletion. This enhanced 45Ca2+ uptake did not occur through voltage-operated Ca2+ channels, because blockade of these channels with verapamil, or depolarization of the plasma membrane by increasing [K+] from 5.9 to 59 mM in the presence of verapamil, did not modify this uptake. 6. A similar increase of the 54Mn2+ uptake occurred in intact cells with a depleted Ca2+ store. If, however, the cells were first skinned and subsequently exposed to 54Mn2+, the ATP-dependent 54Mn2+ uptake amounted to less than 6% of the ATP-dependent 45Ca2+ uptake. 7. If intact cells were first exposed to a 45Ca2(+)- or 54Mn2(+)-containing solution, and subsequently skinned in a non-radioactive intracellular solution, the addition of 10 microM-A23187 to these cells released stored Ca2+ or Mn2+. The amount of released Ca2+ was only slightly larger than the amount of released Mn2+. If the intracellular store was depleted before loading, the amount of Ca2+ or Mn2+ released by the ionophore increased by 68 and 28%, respectively. 8. It is concluded that A7r5 smooth muscle cells do not express a Ca2(+)-induced Ca2+ release mechanism, but do contain an IP3-induced Ca2+ release mechanism which can release approximately all intracellularly accumulated 45Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗