Biomedical subjects

R Casteels

Publications and source records attributed to R Casteels.

At least 55 records · Page 3Linked to original sources

PubMed1994

Inhibition of inositol trisphosphate-induced calcium release by caffeine is prevented by ATP.

We have investigated the effect of various methylxanthines on the basal and InsP3-stimulated unidirectional 45Ca2+ efflux from permeabilized A7r5 cells under different experimental conditions. We report that caffeine and theophylline inhibit the InsP3-induced Ca2+ release, whereas the basal Ca2+ leak remained largely unaffected. The effect on InsP3-induced Ca2+ release was specific, since isocaffeine was not effective. The inhibition occurred similarly in the absence or presence of extravesicular Ca2+ and was not associated with a decrease in the [3H]InsP3 binding to the receptor. ATP and MgATP (5 mM) prevented the inhibition, suggesting that caffeine may interact with an ATP-binding site on the InsP3 receptor or some associated protein.

Adenosine Triphosphate↗

PubMed1994

Co-activation of inositol trisphosphate-induced Ca2+ release by cytosolic Ca2+ is loading-dependent.

Luminal and cytosolic Ca2+ control the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3). In this work, we have characterized how luminal Ca2+ interfered with the stimulation of the InsP3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. InsP3-induced Ca2+ release from stores containing 34 pmol of Ca2+/10(6) cells absolutely depended on a simultaneous rise in cytosolic Ca2+ concentration in the submicromolar range. This stimulation by cytosolic Ca2+ was more pronounced when the stores were preincubated with Ca2+. In contrast, fully loaded stores containing 3400 pmol of Ca2+/10(6) cells already responded to InsP3 in the absence of cytosolic Ca2+ and the release from these stores was much less stimulated by increasing the cytosolic Ca2+ concentration. The loading dependence of the release was not due to a higher cytosolic Ca2+ concentration around more loaded stores sensitizing the InsP3 receptor by binding at a cytoplasmic site. Luminal Ca2+ was therefore found to functionally substitute for cytosolic Ca2+ in triggering Ca2+ release in the presence of a constant InsP3 concentration. These findings are relevant for explaining base-line Ca2+ spiking in non-excitable cells.

Animals↗

PubMed1994

Kinetics of empty store-activated Ca2+ influx in HeLa cells.

The intracellular Ca2+ indicator Indo-1 was used to monitor changes in cytosolic [Ca2+] ([Ca2+]i) in single HeLa cells upon readmission of external Ca2+ after a short incubation in Ca(2+)-free solution. HeLa cells were responsive to histamine but not to caffeine, and their histamine-sensitive store was totally depleted by a 60-min exposure to 2 microM thapsigargin. The resting [Ca2+]i in thapsigargin-treated cells was higher than in control cells and low amplitude [Ca2+]i oscillations were observed in about 20% of the cells. Readmission of external Ca2+ after a brief withdrawal of extracellular Ca2+ resulted in a transient [Ca2+]i rise, which then decayed to the same elevated [Ca2+]i measured before the Ca2+ withdrawal period. The [Ca2+]i rise was associated with an increased rate of Mn2+ entry, measured as the rate of quenching of intracellular Fura-2. The same procedure did not affect the [Ca2+]i in control cells not pretreated with thapsigargin. The amplitude of this [Ca2+]i transient in thapsigargin pretreated cells depended on the duration of prior incubation in Ca(2+)-free medium. The [Ca2+]i rise induced by elevating the extracellular [Ca2+] from 1.5 to 10 mM was more pronounced if the [Ca2+]i during the initial incubation in 1.5 mM Ca2+ was first lowered by depolarizing the cells. We conclude that an empty store stimulates a Ca2+ entry pathway consisting of two components: a continuously elevated basal leak and a second component that is transient due to the high [Ca2+]i-induced inhibition of the Ca2+ entry pathway. This inhibition and the subsequent recovery from it as the [Ca2+]i is brought to resting levels could cause the oscillatory Ca2+ entry that we recorded in a fraction of the thapsigargin-treated cells.

Biological Transport↗

PubMed1994

A sarco/endoplasmic reticulum Ca(2+)-ATPase 3-type Ca2+ pump is expressed in platelets, in lymphoid cells, and in mast cells.

An organellar-type of Ca2+ pump formerly detected by means of its phosphoprotein intermediate in platelets and in lymphoid cells, and which runs in acid gels at 97 kDa, is now characterized as sarco/endoplasmic reticulum Ca2+ATPase 3 (SERCA3). SERCA3 is co-expressed in these cells along with the housekeeping SERCA2b. This conclusion is based on the following observations. 1) Tryptic digestion the phosphoprotein intermediate of SERCA3 expressed in COS cells yields a phosphorylated fragment of about 80 kDa, which can be clearly distinguished from the 57-kDa fragments formed in the SERCA1 and SERCA2 pumps. This 80-kDa fragment comigrates with a similar phosphoprotein fragment previously observed in human platelets (Papp, B., Enyedi, A., Pászty, K., Kovács, T., Sarkadi, B., Gárdos, G., Wuytack, F., and Enouf, J. (1992) Biochem. J. 288, 297-302). 2) An antiserum directed against an NH2-terminal SERCA3-specific peptide (N89) reacts with SERCA3 expressed in COS cells and with the 97-kDa protein in rat platelets and the corresponding protein in human platelets. Likewise an antiserum against the rat SERCA3 terminus (C90) binds to SERCA3 expressed in COS cells and to the 97-kDa band in rat platelets, but it does not recognize the human platelet pump. In conformity with the predicted absence of the T1 tryptic cleavage site in SERCA3, the autophosphorylated aspartyl residue and the COOH-terminal epitope were co-localized on the 80-kDa fragment. 3) The co-expression of nearly equal levels of SERCA3 and SERCA2b messengers in human lymphoblastoid Jurkat cells and in proliferating rat mucosal mast cells was also demonstrated by reverse transcriptase polymerase chain reaction.

Amino Acid Sequence↗

PubMed1994

Vasopressin responses in electrically coupled A7r5 cells.

Changes in cytosolic Ca2+ concentration ([Ca2+]i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca2+ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca2+]i rise that was about 5 times higher than the amplitude of the spontaneous Ca2+ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca2+ spiking, which persisted after [Ca2+]i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca2+ waves within one cell propagated much more slowly than spontaneous [Ca2+]i rises. We conclude that agonist-induced [Ca2+]i increases in an electrically coupled cell monolayer can be asynchronous.

Animals↗

PubMed1994

Relationship between [Ca2+] changes in nucleus and cytosol.

The free calcium concentration in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of single cells were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. It is shown that in various cell types a nucleo-cytosolic Ca(2+)-gradient is present at rest and during stimulation. The direction and the extent of the nucleo-cytosolic Ca(2+)-gradient may vary with the cell type, differentiation status, phosphorylation conditions and also with the type of agonist. Evidence is given for the role of extra- and intranuclear storage sites as well as for Ca(2+)-influx. Finally potential artefactual interference with the measurements is discussed.

Animals↗

PubMed1994

Subcellular Ca(2+)-gradients in A7r5 vascular smooth muscle.

The free calcium concentrations in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of cultured A7r5 smooth muscle were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. Upon stimulation with 5 microM vasopressin (AVP) a cytosolic Ca2+ gradient was observed whereby the highest increase was observed in the subplasmalemmal region. The maximal nuclear Ca2+ concentration ([Ca2+]n) attained a lower level than that in the cytoplasm ([Ca2+]c > [Ca2+]n). After the initial rise, a second sustained change of the Ca2+ level was found and the initial gradient ([Ca2+]c > [Ca2+]n) was preserved. In Ca(2+)-free solution containing 2 mM EGTA the maximal [Ca2+]c value after AVP stimulation was significantly lower than in the Ca(2+)-containing solution, but it remained higher than [Ca2+]n which was the same in both conditions. The initial Ca2+ rise was followed by a monoexponential decline. When the influx of Ca2+ through voltage-sensitive Ca2+ channels was blocked, the maximal and steady state values of [Ca2+]c but not of [Ca2+]n were lower as compared to the values in non-treated cells. Preincubation with 10 microM verapamil and 2 mM Ni2+ resulted in initial [Ca2+]c and [Ca2+]n rises which were not significantly different from the levels found in the absence of Ni2+, but the sustained phase was absent in both compartments. The differential effect with [Ca2+]c > [Ca2+]n was not observed if 1 nM AVP was applied instead of 5 microM AVP. The results indicate that cytosolic and nuclear Ca2+ stores behave differently with respect to their dependence on the agonist concentration and also with respect to the effect of Ca(2+)-entry mechanisms.

Animals↗

PubMed1994

Partial calcium release in response to submaximal inositol 1,4,5-trisphosphate receptor activation.

Even a prolonged application of a submaximal dose of Ins(1,4,5)P3 is unable to release the same amount of Ca2+ from the Ins(1,4,5)P3-sensitive store as a higher dose of Ins(1,4,5)P3. Low doses of Ins(1,4,5)P3 therefore only induce a partial release of the stored Ca2+. In this review, we will focus on the mechanisms that may contribute to this behaviour. Molecular heterogeneity of the Ins(1,4,5)P3 receptor can contribute to such behaviour if all the gene products and alternatively spliced isoforms would have different functional properties and be located in different store units. We will show that the control of the Ins(1,4,5)P3 receptor by by luminal Ca2+ also contributes to the partial release behaviour; it can set the sensitivity of the Ins(1,4,5)P3 receptor and the decreasing luminal Ca2+ concentration may inhibit further release while some Ca2+ is still left in the store. It is finally possible that the Ins(1,4,5)P3 receptor may adapt to a maintained stimulus.

Animals↗

PubMed1993

Loading dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in the clonal cell line A7r5. Implications for the mechanism of quantal Ca2+ release.

The Ca2+ content of the intracellular Ca2+ stores controls the inositol 1,4,5-trisphosphate receptor (InsP3R) in the clonal cell line A7r5. This regulation was characterized with respect to the understanding of the "quantal" release phenomenon. Independent of the loading protocol used, increasing the Ca2+ content of the stores increased the sensitivity of the inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release until a Ca2+ content of about 30% of the steady-state value was reached. Loading of the stores to higher levels had only a marginal effect on the Ca2+ release. The effects of luminal Ca2+ were still observed in the presence of 10 mM BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), excluding the possibility that luminal Ca2+ acted indirectly via cytoplasmic binding sites. Conditions were developed to simultaneously measure [3H]InsP3 binding to the InsP3R and the 45Ca2+ content of the stores in the same cells. [3H]InsP3 binding to a high affinity binding site was potentiated by luminal Ca2+. Analysis at the molecular level revealed the simultaneous expression of different splice variants of InsP3R-I, as well as the expression of InsP3R-III, and of the putative InsP3R-IV. We conclude that the control of the InsP3R by luminal Ca2+ could account for quantal release and that the observed heterogeneity of the InsP3R may also contribute to this behavior, especially at high levels of store loading.

Animals↗

PubMed1993

Ins(1,4,5)P3 and glutathione increase the passive Ca2+ leak in permeabilized A7r5 cells.

Thapsigargin depletes intracellular Ca2+ stores by its inhibitory effect on the Ca2+ pumps, which unmasks an aspecific Ca2+ leak from the stores. This aspecific Ca2+ permeability of the stores was further investigated using 45Ca2+ fluxes on intact and permeabilized A7r5 smooth-muscle cells. Stores in intact cells were found to be more leaky for Ca2+ than those in saponin-permeabilized or Staphylococcus aureus alpha-toxin-permeabilized cells, which suggests that a cytosolic factor may be involved. Supplementing the medium bathing the permeabilized cells with a submaximal Ins(1,4,5)P3 concentration increased the leakiness of the stores. Glutathione also increased the aspecific Ca2+ leak. This effect occurred with both the reduced and the oxidized form but reduced glutathione was more effective. Our data show that basal Ins(1,4,5)P3 levels and glutathione can contribute to the relatively high Ca2+ leak in intact cells. The washing out of these substances during permeabilization can reduce the aspecific leakiness of the stores.

Bacterial Toxins↗

PubMed1993

Alternative processing of the gene transcripts encoding a plasma-membrane and a sarco/endoplasmic reticulum Ca2+ pump during differentiation of BC3H1 muscle cells.

The effect of differentiation on the RNA processing of the PMCA1 gene encoding a plasma-membrane Ca2+ pump and of the SERCA2 gene encoding a sarco(endo)plasmic reticulum Ca2+ pump was studied in the myogenic BC3H1 cell line. A differentiation stage-dependent change in the RNA processing was observed for both genes. Proliferating myoblasts only expressed the non-muscle mRNA isoform whereas in differentiated cells muscle-specific processing became activated. The switch to muscle-specific RNA processing for both the PMCA1 and SERCA2 genes was found to be linked to the myogenic conversion of the BC3H1 cells. Our results furthermore indicated that the myogenic RNA processing could be reversed for both types of Ca2+ pumps since the expression of the PMCA1 and SERCA2 muscle-specific messengers was rapidly down-regulated by cycloheximide treatment.

Alternative Splicing↗

PubMed1993

Lack of dystrophin but normal calcium homeostasis in smooth muscle from dystrophic mdx mice.

The free cytosolic Ca2+ concentration ([Ca2+]i) in the dystrophin-lacking smooth muscle from mdx mice was studied to gain new insights into the relation between dystrophin and cytoplasmic Ca2+ homeostasis, which was reported to be impaired in the mdx skeletal muscle. We observed that [Ca2+]i, as measured with the fluorescent Ca2+ indicator fura-2, was not elevated in resting smooth muscle of the vas deferens from mdx mice, in comparison with control C57 mice. Changes of the external Ca2+ concentration evoked similar changes of [Ca2+]i in mdx and control vas deferens. During contraction, cytosolic Ca2+ transients were identical, both in amplitude and in kinetics, whether or not dystrophin was present. Stretches evoked similar Ca2+ increases in muscles from both strains. Intracellular Ca2+ homeostasis appears to be unimpaired in mdx smooth muscle. Thus, the lack of dystrophin per se does not automatically induce a perturbation of Ca metabolism in muscle cells.

Animals↗

PubMed1993

Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells.

There is no consensus about the different types of Ca2+ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca2+ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional 45Ca2+ flux technique. Thimerosal up to a concentration of 32 microM did not have an effect on the passive 45Ca2+ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca2+ pump with an EC50 of 9 microM. Thimerosal exerted a biphasic effect on the Ca2+ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with a stimulation of the release at thimerosal concentrations below 10 microM, and an inhibitory effect at higher concentrations. Thimerosal (2.5-250 microM) did not exert an effect on the specific binding of [3H]Ins(1,4,5)P3 to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [3H]Ins(1,4,5)P3 binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 microM can stimulate the Ins(1,4,5)P3-induced Ca2+ release without inhibiting the Ca2+ pumps or without increasing the passive Ca2+ permeability of the endoplasmic reticulum.

Animals↗

PubMed1993

Normal Ca2+ signalling in glutathione-depleted and dithiothreitol-treated HeLa cells.

We have investigated whether reducing agents and substances that interfere with glutathione metabolism would affect the histamine-induced rises in internal Ca2+ concentration ([Ca2+]i) in indo-1-loaded HeLa cells. Individual cells responded to 1 microM histamine with either baseline or sinusoidal Ca2+ oscillations, a single Ca2+ peak or a maintained elevation of the [Ca2+]i. Only a few cells did not respond. The sulphydryl reducing agent dithiothreitol (5 mM) did not affect these responses to histamine. A 24-h preincubation with 1 mM DL-buthionine (SR)-sulphoximine, which reduces the cellular glutathione content to less than 20% of its control value, affected neither these histamine responses, nor the [Ca2+]i rises after application of 2 microM thapsigargin. We conclude that oxidation of critical sulphydryl groups is not required for the normal response to histamine and also that glutathione plays no role in agonist-induced Ca2+ signalling in HeLa cells.

Antimetabolites↗

PubMed1993

Electrogenic Na+/K(+)-transport in human endothelial cells.

Na+/K+ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca2+]i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na+] did not induce significant changes of [Ca2+]i. Superfusing the cells with K(+)-free solutions also did not significantly affect [Ca2+]i. Reapplication of K+ after superfusion of the cells with K(+)-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mumol/l dihydroouabain (DHO) and was therefore identified as a Na+/K+ pump current. During block and reactivation of the Na+/K+ pump no changes in [Ca2+]i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mumol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10-1000 U/l) did not affect the pump currents. An increase of the intracellular Na+ concentration ([Na+]i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na+]i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K+ was 2.4 +/- 1.2 mmol/l, and 0.4 +/- 0.1 and 8.7 +/- 0.7 mmol/l for Tl+ and NH4+ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than -150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1993

Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice.

The fluorescent probe Fura-2/AM was used to determine cytosolic free calcium concentration in soleus muscle and in isolated flexor digitorum brevis fibres. This required a precise calibration; therefore, each calibration parameter was studied in situ. The influence of the dye concentration on calcium measurements was also examined. This precise calibration technique was used to compare absolute free calcium concentration in resting preparations from dystrophic (mdx) and control (C57) mice. We showed that the behavior of the dye was not similar in C57 and in mdx muscles. For this reason, we did not confirm the previous results that cytosolic free calcium concentration is increased in mdx muscles.

Animals↗

PubMed1993

Expression of Ca2+ binding proteins of the sarcoplasmic reticulum of striated muscle in the endoplasmic reticulum of pig smooth muscles.

The Ca2+ binding proteins in the lumen of intracellular Ca2+ stores differ between muscle and non-muscle cells, indicating a specific role of these proteins in intracellular Ca2+ regulation. Since smooth muscle cells possess both muscle and non-muscle characteristics, we have studied the presence and the differential expression of the muscle-type Ca2+ binding proteins--calsequestrin, sarcalumenin, and the histidine-rich Ca2+ binding protein (HCP)--in several smooth muscle tissues from the pig. Western blot analysis showed that among the smooth muscles studied, the cardiac isoform of calsequestrin is expressed at the highest levels in the stomach. Calsequestrin was present at lower levels in ileum and trachea, whereas this protein was undetectable in aorta and main pulmonary artery. The total amount of calsequestrin in the stomach was estimated to be 20-30-times lower than in the pig heart. Whereas calsequestrin from pig presented the same apparent M(r) in sodium dodecyl sulphate polyacrylamide gels as the well characterized protein from rabbit, the apparent M(r) of both sarcalumenin and HCP was lower in pig than in rabbit. The presence of HCP was demonstrated in pig stomach and ileum, while sarcalumenin was detected only in the stomach. These results demonstrate further biochemical differences between smooth muscle cells of large blood vessels and those of the digestive tract. The present findings on the differential distribution of muscle-type Ca2+ binding proteins are discussed in relation to biochemical and functional differences between these smooth muscle cells.

Animals↗

PubMed1993

ATP induced-relaxation in the mouse bladder smooth muscle.

1. The effect of adenosine 5'-triphosphate (ATP) on the free cytosolic Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca(2+)-indicator fura-2, and on force was investigated in the intact smooth muscle strips of the mouse urinary bladder. 2. ATP elicited, when exogenously applied, a large increase of [Ca2+]i with limited force development resulting in a marked Ca(2+)-force dissociation. 3. Release of endogenous neurotransmitters by transmural electrical stimulation (TES) for 30 s induced a steady increase of [Ca2+]i and a peak contraction, followed within 15 s by a relaxation. 4. In carbachol-prestimulated preparations, ATP elicited an initial rise of [Ca2+]i followed by a return to the initial precontraction Ca(2+)-level. Force in contrast presented a biphasic pattern, i.e. an initial contraction was followed by a sustained relaxation. 5. In the K(+)-depolarized precontracted preparation, ATP elicited a slight initial rise of [Ca2+]i. The partial relaxation of the force during depolarization was not preceded by a transient contraction. 6. The ATP-induced relaxation of the K(+)-prestimulated preparations was not inhibited by 8-phenyltheophylline, a potent P1-purinoceptor antagonist. 7. The order of potency for relaxation of the ATP analogues was 2-MeSATP > ATP > beta gamma Me-ATP, which is characteristic for P2y-purinoceptors. 8. These results indicate that, besides its activating effect, ATP also relaxes the mouse urinary bladder. It is suggested that the relaxant effect, mediated through P2y-purinoceptors, is mainly responsible for the low contractile potency of ATP in the bladder.

Adenosine Triphosphate↗