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R Casteels

Publications and source records attributed to R Casteels.

At least 73 records · Page 4Linked to original sources
PubMed1993

Post-receptor pathway of the ATP-induced relaxation in smooth muscle of the mouse vas deferens.

1. The post-receptor pathway of the ATP relaxant effect in K(+)-precontracted vas deferens smooth muscle (VD) was examined. 2. The relaxation to ATP was not antagonized either by 10 microM methylene blue, a cyclic GMP inhibitor, by 10 microM indomethacin, an inhibitor of prostaglandin synthesis or by 100 microM NG-nitro-L-arginine, an inhibitor of NO production. 3. The Rp-diastereomer of adenosine 3':5'-cyclic monophosphorothioate (Rp-cAMPS) 200 microM, a competitive inhibitor of cyclic AMP significantly diminished the relaxant response to ATP. 4. Isoprenaline 10 microM, a beta-adrenoceptor agonist, produced a sustained relaxation, inhibited by Rp-cAMPS, without a significant change in [Ca2+]i, thereby mimicking the ATP-induced relaxant effect. 5. The level of the phosphorylated myosin light chain in the precontracted VD was significantly lowered by 1000 microM ATP. 6. ATP (1000 microM) and isoprenaline (10 microM) produced the same increase (+ 50%) of [cyclic AMP] when applied to a resting VD. 7. The effect of simultaneous increases of [Ca2+]i and of [cyclic AMP] produced by externally applied ATP are discussed. 8. These results suggest that ATP-induced relaxation in K(+)-precontracted VD is mediated by the activation of adenylyl cyclase.

Adenosine Triphosphate↗
PubMed1993

Staurosporine induced Ca2+ increase in DDT1MF-2 smooth muscle cells.

The free calcium concentrations in nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured DDT1MF-2 smooth muscle cells were estimated using the fluorescent dye indo-1 and laser confocal microscopy. The alkaloid staurosporine mainly increased [Ca2+]c during the initial minutes of stimulation and the nucleocytoplasmic gradient was maintained. Thereafter [Ca2+]n increased further while [Ca2+]c decreased, resulting after 10 min in a reversion of the nucleocytoplasmic gradient. Staurosporine increased the Ca2+ influx but also released intracellular Ca2+. In Ca(2+)-free solution no inversion of the nucleocytoplasmic gradient was seen in most cells. Ca2+ increased more rapidly in the area of the perinuclear sarcoplasmic reticulum (SR) than in the central cytoplasm. This is suggestive of the release of Ca2+ from this region. Ca2+ concentration remained also enhanced near the plasmalemma during this time period. This might indicate that the efflux mechanism is also affected. It is unlikely that these changes are due to inhibition of protein kinase C, since phorbol esters are ineffective and since downregulation by prolonged incubation with phorbol 12-myristate 13-acetate only affected the time periods required to attain the described [Ca2+]c and [Ca2+]n peaks. The slow time course of the staurosporine effects allowed demonstration of the spatiotemporal Ca2+ gradients between the nucleus and the cytoplasm and also inside the cytosol. The different time course of the increases in [Ca2+]c and [Ca2+]n suggests that two different Ca2+ mobilization events are present. The occurrence of an inversion of the nucleocytoplasmic gradient whereby [Ca2+]n increased while [Ca2+]c values decreased indicates that the increase in [Ca2+]n might result partially from release sites inside the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1993

Intracellular Ca2+ signaling induced by vasopressin, ATP, and epidermal growth factor in epithelial LLC-PK1 cells.

The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured renal LLC-PK1 epithelial cells were estimated by confocal laser microscopy. No difference between the resting mean [Ca2+]n and [Ca2+]c was found. During stimulation with maximal effective concentrations of arginine vasopressin (AVP) or the purinergic agonist ATP, the transient Ca2+ rise was followed mostly by a decline to basal levels. A differential rise could be observed when the increase in [Ca2+]n attained higher values than [Ca2+]c. In 50-60% of the cells, epidermal growth factor (EGF) also induced a transient Ca2+ rise, and a differential increase ([Ca2+]n > [Ca2+]c) was found. The G protein-linked stimuli AVP and ATP were however quantitatively much more efficacious at stimulating the [Ca2+]n and [Ca2+]c increases than was EGF. To investigate whether AVP, ATP, and EGF released Ca2+ from distinct or overlapping stores, the agonists were sequentially added. AVP and ATP applied after EGF in Ca(2+)-free medium elicited an increase in [Ca2+]n and [Ca2+]c that was not significantly lower than the release of Ca2+ in control cells without EGF prestimulation. Similarly, the amplitude of the Ca2+ responses attained by EGF in cells prestimulated by ATP or AVP was comparable to the response in naive cells. Neither EGF, ATP, nor AVP evoked a Ca2+ signal after thapsigargin treatment, indicating that the intracellular Ca2+ pools stimulated by all these agonists are part of the thapsigargin-sensitive Ca2+ pools. In contrast, when ATP was applied after AVP in Ca(2+)-containing as well as in Ca(2+)-free solutions, the Ca2+ transients were lower as compared with the response without preincubation. No differential rise could be found in Ca(2+)-free conditions. An explanation could be the use of different phospholipase C isozymes by the different receptor types, which possibly gives rise to the mobilization of different Ca2+ pools.

Adenosine Triphosphate↗
PubMed1992

2,5-Di-(tert-butyl)-1,4-benzohydroquinone and cyclopiazonic acid decrease the Ca2+ permeability of endoplasmic reticulum.

Specific inhibitors of the endoplasmic-reticulum Ca2+ pump will deplete intracellular stores and are therefore useful to study the role of store depletion on plasma-membrane Ca2+ permeability. We now report that the Ca(2+)-pump inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) reduces the passive Ca2+ leak from the internal stores in permeabilized A7r5 vascular smooth-muscle cells. This aspecific effect occurred at concentrations that are normally used to empty the stores in intact cells. Cyclopiazonic acid exerted a similar, although less pronounced effect, while thapsigargin did not affect the passive Ca2+ leak. The inositol 1,4,5-trisphosphate-mediated Ca2+ release was not affected. tBuBHQ and cyclopiazonic acid cannot therefore be used as specific tools to probe the mechanism of receptor-mediated Ca2+ entry.

Benzoquinones↗
PubMed1992

Ca(2+)-transport ATPases and their regulation in muscle and brain.

Eukaryotic cells express one or more isoforms of a sarco(endo)plasmic reticulum (SERCA) and of a plasma membrane (PMCA) Ca2+ pump. Both the SERCA and PMCA gene transcripts are subject to alternative processing in a differentiation stage-dependent and tissue-dependent manner. The Ca2+ pump isoforms thus generated may present different functional properties. This is exemplified by the SERCA2a and SERCA2b isoforms which differ in their Ca2+ sensitivity. Analysis of the cDNA structures for PMCA1 predicts protein isoforms with variant calmodulin- and phospholipid-binding domains. A comparative study of the tissue-specific mechanisms governing SERCA-PMCA transcript processing and a more detailed study of the functional implication of the PMCA pumps isoform diversity will be challenging subjects for future studies.

Alternative Splicing↗
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

Functional difference between SERCA2a and SERCA2b Ca2+ pumps and their modulation by phospholamban.

COS 1 cells were transfected with full-length pig stomach sarcoplasmic/endoplasmic reticulum Ca2+ pump (SERCA)2a or SERCA2b cDNA. Ca2+ uptake by microsomes from transfected cells revealed that the Ca2+ affinity of the SERCA2b Ca2+ pump (K0.5 0.17 +/- 0.01 microM) was higher than that of the SERCA2a Ca2+ pump (K0.5 0.31 +/- 0.02 microM). Thapsigargin-sensitivity was found to be identical for the two isoforms. The Ca2+ affinity of both the SERCA2a and SERCA2b Ca2+ pumps was decreased by a factor of two when they were co-expressed with phospholamban.

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

Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse.

We compared the changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), as measured with the fluorescent Ca2+ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K+ depolarization and by agonists. Resting [Ca2+]i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca2+ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca2+]i increase, the agonist stimulation induced a higher force development than the K+ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca2+]o. Our results indicate that the Ca2+ threshold for force development was about 150% of the resting [Ca2+]i in both cell types. The resting [Ca2+]i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca2+ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca2+ sensitivity during contraction. The desensitization to Ca2+ was specific for phasic muscle, in which it occurred as an early, time- and Ca(2+)-dependent process that was partially reversible.

Animals↗
PubMed1992

Thrombin stimulates L-type calcium channels of guinea pig cardiomyocytes in cell-attached patches but not after intracellular dialysis.

The action of the blood clotting enzyme thrombin on single channel and whole cell Ca(2+)-currents was studied in isolated mammalian cardiac myocytes. Thrombin, at a concentration of 10(-8) mol/l, increased the Ca(2+)-channel activity in cell-attached patches. The mean open probability of the channel was enhanced, while the number of sweeps without openings, which reflects the availability of the channel, was significantly reduced. Neither the single channel conductance nor the activation curve were affected by thrombin. Thrombin was added to the bath solution, and its effect is therefore indirect and probably mediated via a second messenger. However, thrombin did not affect whole-cell Ca(2+)-currents, whereas a beta-adrenergic stimulation in the same cell increased the Ca(2+)-current. It is concluded that thrombin affects an intracellular mechanism for Ca2+ channel current regulation, which is still unknown and which is rapidly lost during conventional whole-cell Ca2+ current measurements.

Animals↗
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

ATP activates P2x-contracting and P2y-relaxing purinoceptors in the smooth muscle of mouse vas deferens.

1. The mechanism for the low potency of exogenous ATP in producing contraction at the P2x-purinoceptors in the smooth muscle of the mouse vas deferens (VD) was examined. 2. The measure of the breakdown of ATP in contact with the VD showed that its degradation was limited and did not account for its weak contractile effect. 3. Externally applied, ATP induced a small and transient contraction but a marked and prolonged increase of the cytosolic Ca2+ concentration ([Ca2+]i), which suggests an efficient binding to the P2x-purinoceptors. Such a calcium-force dissociation was not observed with beta, gamma-methylene ATP (beta, gamma-Me-ATP), a structural ATP analogue. 4. The force response of precontracted VD to ATP was biphasic, consisting of a small initial contraction followed by a sustained marked relaxation. In contrast, beta, gamma-Me-ATP elicited a pronounced contraction without ensuing relaxation. 5. ATP was more potent than adenosine in producing relaxation, and the relaxation was not antagonized by 8-phenyltheophylline, suggesting the activation of P2-purinoceptors. 6. For this relaxation, the rank order of potency was 2-methyl-thio-ATP (2-MeSATP) > ATP > beta, gamma-Me-ATP, which is characteristic for the P2y-purinoceptors. 7. Reactive Blue 2, a P2y-purinoceptor antagonist, was found to reduce the relaxation mediated by ATP. 8. These results indicate that ATP acts in VD not only on contracting but also on relaxing P2-purinoceptors, eliciting thereby overlapping opposite effects. In VD, the classical low potency of ATP or contraction is thus not explained by its low bioavailability or its low binding, but rather by its low specificity for the contracting P2x-purinoceptors, leading to the activation of the relaxing P2y-purinoceptors.

Adenosine Diphosphate↗
PubMed1992

Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells.

The free Ca2+ concentrations in the nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured smooth muscle cells were estimated using the fluorescent dye indo-1 and the ACAS 570 confocal laser microscope. In resting DDT1MF2 smooth muscle cells [Ca2+]n was found to be lower than [Ca2+]c. Both values increased transiently in response to histamine (100 microM), but during this stimulation [Ca2+]n exceeded [Ca2+]c. Maximal increase of [Ca2+]n was observed in the center of the nucleus, and a maximal increase of [Ca2+]c was observed in the immediate vicinity of the plasma membrane. A similar response was obtained with other agonists, such as carbachol or ATP. Comparable results with ATP were obtained in cultured aorta cells. The differential rise of [Ca2+]n over [Ca2+]c in DDT1MF2 cells did not occur during either spontaneous release of Ca2+ or Ca2+ release induced by caffeine (7.5 mM). The differential rise during histamine stimulation was abolished by the presence of the intercalating substance ethidium bromide. Thapsigargin, a presumed specific inhibitor of the endoplasmic reticulum Ca(2+)-Mg(2+)-adenosine-triphosphatase, abolished the Ca2+ gradient between nucleus and cytosol at rest. During subsequent histamine stimulation the Ca2+ increase was largely blocked in both compartments and attained similar levels. We propose that the lower value of [Ca2+]n at rest is dependent on an active Ca2+ extrusion system. The differential rise of [Ca2+]n over [Ca2+]c during agonist stimulation can be explained by an influx of Ca2+ from perinuclear stores and/or by a release of intranuclear Ca2+ possibly mediated by a process dependent on the inositol lipid metabolism.

Animals↗
PubMed1992

Kinetics of nucleocytoplasmic Ca2+ transients in DDT1 MF-2 smooth muscle cells.

The free calcium concentrations in the nucleus ([Ca2+]n) and in the cytoplasm ([Ca2+]c) of cultured DDT1 MF-2 smooth muscle cells were estimated using the fluorescent dye indo-1. With the use of confocal microscopy, line scans were made during the onset and the differential rise of the Ca2+ signal elicited by the agonists histamine and ATP. The results confirm our earlier findings that in these cells [Ca2+]n at rest was lower than [Ca2+]c. The present experiments show that this gradient over the nuclear envelope was also preserved in Ca(2+)-free solution containing 2 mM EGTA, underlining the selective barrier function of the nuclear envelope. During stimulation with histamine, an early Ca2+ rise in the vicinity of the nuclear envelope was found in contrast to the delayed Ca2+ rise 2 microns away on both sides of the envelope. This suggests the release of Ca2+ stored in the envelope and the perinuclear sarcoplasmic reticulum. The time course for reaching a uniform Ca2+ concentration ([Ca2+]u = [Ca2+]n = [Ca2+]c) in the nuclear and cytosolic compartment varied with the agonist used for stimulation and was dependent on the external Ca2+ concentration. The value of this uniform Ca2+ concentration itself was, however, independent of the type of stimulation. After reaching [Ca2+]u, a further rise occurred with [Ca2+]n becoming larger than [Ca2+]c. It is postulated that a critical Ca2+ concentration must be reached to induce this differential Ca2+ rise by releasing Ca2+ from an intranuclear Ca2+ store.

Adenosine Triphosphate↗
PubMed1992

Effects of dimethyl sulfoxide and polycationic neomycin on stimulation of purified plasma membrane Ca(2+)-pump by negatively charged phospholipids.

At least two reaction steps are involved in the activation of purified plasma membrane Ca(2+)-transport ATPase by negatively charged phospholipids depending on the type of phospholipids (Lehotský et al. 1992). The effect of negatively charged phospholipids on Ca(2+)-stimulated ATPase (cycling activity) was compared with that of p-nitrophenylphosphatase (E2-form activity) catalyzed by Ca(2+)-pump. PIP like PS, activated Ca(2+)-ATPase activity by modifying ATP activation curve with increasing Vmax of the high affinity site. Ca(2+)-ATPase activity reconstituted in PC was stimulated by DMSO(10%) by a factor of 1.36. The activity stimulation by DMSO was only weak in PS and activity was inhibited in PIP. Also, phosphatase activity catalyzed by Ca(2+)-pump was strongly stimulated by DMSO and was differentially affected by phospholipid head group. Positively charged neomycin (5 mmol/l) had no effect on Ca(2+)-ATPase activity reactivated in PC or PS, but the stimulatory action of PIP was suppressed. Relative stimulation of phosphatase activity by PS was not influenced. Both hydrolytic activities catalyzed by Ca(2+)-transport ATPase were differentially affected by organic solvents and polycations with respect to the kind of the phospholipid.

Animals↗
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↗