Biomedical subjects

R Casteels

Publications and source records attributed to R Casteels.

At least 91 records · Page 5Linked to original sources

PubMed1991

Characterization of the 3' end of the pig sarcoplasmic/endoplasmic-reticulum Ca2+ pump gene 2.

The gene 2 for the sarcoplasmic/endoplasmic-reticulum Ca2+ pump (SERCA2) is expressed both in muscle and non-muscle tissues and four distinct SERCA2 mRNAs which differ only in their 3' end have been described. In order to understand the differential 3' end processing of the SERCA2 transcripts, we have now cloned a 12.6 kb pig genomic clone which contains the 3' end of the pig SERCA2 gene. Part of this clone (7510 nucleotides) was sequenced and it was found to contain four constitutive exons followed by four optional exons which underlie the 3' end diversity of the SERCA2 mRNAs. The inclusion or the exclusion of these optional exons is explained by the presence of four optional processing sites: two polyadenylation sites and two optional donor splice sites.

Amino Acid Sequence↗

PubMed1991

Phosphoinositide-protein interactions of the plasma-membrane Ca2(+)-transport ATPase as revealed by fluorescence energy transfer.

Fluorescence energy transfer has been used to study the interaction of various phospholipids with the erythrocyte (Ca2+ + Mg2+)-ATPase. The fluorescence energy transfer between tryptophan residues of the (Ca2+ + Mg2+)-ATPase purified from erythrocytes and pyrene-labelled analogues of phosphatidylcholine (Pyr-PC), phosphatidylinositol (Pyr-PI), phosphatidylinositol 4-phosphate (Pyr-PIP), phosphatidylinositol 4,5-bisphosphate (Pyr-PIP2), phosphatidylglycerol (Pyr-PG) and phosphatidic acid (Pyr-PA) was measured. A positive correlation was found between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA greater than PI = PG greater than PC) and the potency of their pyrene-labelled analogues to act as quantum acceptors in fluorescence energy transfer from the tryptophan residues of the (Ca2+ + Mg2+)-ATPase. This is the first time that a physical interaction between PIP/PIP2 and an intrinsic membrane protein has been demonstrated. The dependence of the energy transfer on the number of negative charges of the phospholipids closely resembles the previously demonstrated charge dependence of the enzymatic activity of the (Ca2+ + Mg2+)-ATPase (Missiaen, L., Raeymaekers, L., Wuytack, F., Vrolix, M., Desmet, H. and Casteels, R. (1989) Biochem. J. 263, 687-694). It is concluded that the stimulation of the (Ca2+ + Mg2+)-ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the (Ca2+ + Mg2+)-ATPase and that the negative charges are a major modulatory factor for this interaction.

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

Carbachol-induced nonspecific desensitization in guinea-pig ileum.

The effects of repeated stimulation by carbachol on force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. Carbachol was applied at 20 degrees C for 5 min. Each application was followed by a 25-min washout period and the desensitization was expressed by the decline of the maximal force development. Three hours after the first carbachol-induced contraction the peak amplitude was about 40% of the initial value. Increasing the frequency of application, thereby decreasing the washout time, enhanced the desensitization, while the presence of the competitive blocker atropine reduced the phenomenon. At 35 degrees C no desensitization could be observed. Blocking the Na+/K+ pump by ouabain or by K(+)-free solution reduced the force development to less than 20%. Increasing [K+]0 in the washout solution at 20 degrees C reduced the desensitization phenomenon, while decreasing [K+]0 resulted in an enhanced desensitization as expressed by a decline of the force development. The total cellular Na+ content after various stimulation sequences was determined at 20 degrees and 35 degrees C from the 22Na+ effluxes. At 35 degrees C the cellular Na+ content did not change significantly during stimulation for 10 min with 10(-4) mol/l carbachol. At 20 degrees C the resting Na+ content was significantly increased, and it doubled during carbachol stimulation for 10 min. Furthermore, the recovery of the cellular Na+ content after washout proceeded extremely slowly at that temperature. The appearance of desensitization was increased by 10 mumol/l ryanodine, while it was reduced by adding the Ca2+ agonist Bay K 8644.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

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↗

PubMed1991

ATP-induced Ca2+ release and Cl- current in cultured smooth muscle cells from pig aorta.

1. The effect of exogenous ATP on transmembrane currents and on the cytoplasmic Ca2+ has been investigated in single cultured smooth muscle cells of pig aorta. 2. ATP applied to cells held at a potential of -50 mV evoked a transient inward current and a transient rise in [Ca2+]i. At a potential of +20 mV the ATP-induced increase in [Ca2+]i was accompanied by an outward current. 3. At a potential of -50 mV, ATP evoked in Ca(2+)-free solution an inward current which was similar to that in the presence of external Ca2+. A second application of ATP in Ca(2+)-free solution induced a much smaller current. 4. ATP induced in Ca(2+)-free solution a pronounced transient stimulation of the 45Ca2+ efflux from confluent smooth muscle monolayers. 5. The I-V curve of the ATP-activated current has a reversal potential close to 0 mV. A reduction of external Cl- shifts this reversal potential in accordance with the change of the Cl- equilibrium potential. 6. It is concluded that ATP causes a release of calcium from intracellular stores. The ensuing increase of [Ca2+]i activates a Cl- current, which can depolarize the cell membrane and thereby promote a voltage-gated Ca2+ entry.

Adenosine Triphosphate↗

PubMed1991

Ca(2+)-transport ATPases and Ca(2+)-compartments in smooth muscle cells.

The Ca(2+)-pump ATPases of the plasma membrane and of the endoplasmic reticulum play an important role in controlling the intracellular Ca(2+)-concentration. In this perspective it is not unexpected that these enzymes are modulated by different factors. The activity of the plasmalemmal (Ca2+ +Mg2+)ATPase is modified by the amount of negatively charged phospholipids surrounding the enzyme. Some evidence is presented indicating that in stomach and myometrium smooth muscle agonists inhibit the extrusion of Ca2+ by reducing the negatively charged phospholipids surrounding the plasmalemmal Ca(2+)-pump, while c-GMP dependent protein kinase would activate this Ca(2+)-pump by increasing this amount. The regulation of the Ca(2+)-pump of the endoplasmic reticulum depends on the phosphorylation of phospholamban by cAMP- and cGMP-dependent protein kinase. In the second part of this review, the heterogeneity of the intracellular Ca2+ compartments and a possible connection between the intracellular compartment and the extracellular solution are discussed. In addition, some data on the regulation of Ca2+ inside the nucleus are presented.

Animals↗

PubMed1991

A study of the organellar Ca2(+)-transport ATPase isozymes in pig cerebellar Purkinje neurons.

Pig cerebellar Purkinje neurons express a high level of Ca2(+)-transport ATPases in their intracellular Ca2+ stores. This was shown at the mRNA level by Northern blotting and in situ hybridization and at the protein level by Western blotting and immunocytochemistry. The majority of the Ca2(+)-transport ATPases in these neurons belongs to the SERCA2b type (i.e., the Ca2(+)-pump isoform found in most nonmuscle cells). The SERCA2a (cardiac/slow-twitch skeletal/smooth muscle) Ca2(+)-pump isoform is expressed only at very low levels. The main Ca2(+)-pump messenger is 6.0 kilobases long and belongs to a class 4-type processing of SERCA2, which is exclusively confined to the cerebrum and cerebellum. Phospholamban, a regulator of the SERCA2 Ca2(+)-transport ATPase in cardiac/slow-twitch skeletal/smooth muscle, could not be detected in Purkinje neurons.

Animals↗

PubMed1990

Expression of endoplasmic-reticulum Ca2(+)-pump isoforms and of phospholamban in pig smooth-muscle tissues.

The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.

Animals↗

PubMed1990

Molecular cloning and sequencing of the plasma-membrane Ca2+ pump of pig smooth muscle.

cDNAs coding for the plasma-membrane Ca2+ pump have been isolated from a pig smooth-muscle cDNA library and sequenced. The open reading frame encodes a protein of 1220 amino acids, which corresponds to the one already described in a human teratoma cell line. We demonstrate here that this cDNA probably represents the only isoform of the plasma-membrane Ca2(+)-transport ATPase expressed in this smooth muscle. There is no evidence for the expression of any other plasma-membrane Ca2(+)-pump gene, or for the presence of other alternatively spliced isoforms. These results are in apparent contradiction to those obtained on protein levels which demonstrate the reaction of at least two different polypeptides with a panel of antibodies against the plasma-membrane ATPase. It is suggested that these two polypeptides could result from a post-translational modification of one single enzyme.

Amino Acid Sequence↗

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

Characterization of the mRNAs encoding the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2+ pump in pig smooth muscle.

The gene 2 sarcoplasmic/endoplasmic-reticulum (SR/ER) Ca2+ pump is expressed in slow skeletal and cardiac muscle, smooth muscle and non-muscle tissues. We have analysed the gene 2 Ca2+ pump mRNAs using a panel of anti-sense RNA probes which recognize either the muscle (class 1) or the non-muscle (class 2) transcript, or both. In pig smooth muscle, we confirmed the presence of the class 1 and class 2 mRNAs of 4.4 kb length and we also detected a third mRNA of 8.0 kb which reacted with both the class 1 and class 2 riboprobes. A 4.2 kb cDNA corresponding to the 3' part of the 8.0 kb mRNA was cloned from a pig gastric smooth muscle cDNA library. Nucleotide sequence analysis of this clone revealed that the 8.0 kb mRNA (class 3 transcript) contained both the non-muscle-specific and the muscle-specific exons separated by a 2.4 kb intron which has not been removed. The class 3-mRNA-encoded SR/ER Ca2+ pump is identical to the class 2-encoded non-muscle isoform. Northern blot analysis demonstrated that, in cardiac muscle, the class 1 mRNA (encoding the muscle isoform) is the predominant messenger, whereas in non-muscle tissues the class 2 and 3 mRNAs (encoding the non-muscle isoform) predominate. In smooth muscle all three mRNA types are present. The tissue distribution of the mRNA types suggests a tissue-dependent processing of the primary transcript of the sarcoplasmic/endoplasmic reticulum Ca2+ pump gene 2.

Animals↗

PubMed1990

Different effects of depolarization and muscarinic stimulation on the Ca2+/force relationship during the contraction-relaxation cycle in the guinea pig ileum.

The effects of K+ depolarization and of the muscarinic agonist carbachol on [Ca2+]i and force were investigated in smooth muscle sheets of the longitudinal layer of the ileum loaded with Fura-2. K(+)-rich solutions increased [Ca2+]i and force to an initial peak value, which was determined by the concentration of [K+]o. Thereafter, [Ca2+]i and force declined to a lower maintained level. The Ca2+/force relationship observed during this contraction-relaxation cycle is represented by a clockwise hysteresis loop. At 140 mM [K+]o, this loop consisted of three components while at lower [K+]o a two-component loop was observed. The stimulation with 0.1 mM carbachol resulted in a transient increase of [Ca2+]i and force followed by a continuous decline of these parameters despite the presence of the drug. Its EC50 of relaxation was around 270 nM [Ca2+]i. The Ca2+/force relationship proceeded along a counterclockwise hysteresis loop during the contraction-relaxation cycle. The extent of this loop decreased but remained unaltered in its direction during repeated stimulation with carbachol. These results suggest that (a) both agonists increase force and [Ca2+]i during stimulation; (b) during depolarization with K+, desensitization to CA2+ occurs resulting in a clockwise hysteresis loop; (c) during carbachol stimulation, a counterclockwise hysteresis is observed. This could be due to an increased sensitivity to Ca2+ mainly in tonic smooth muscle. These observations might be explained by a modulation of the Ca2+ sensitivity by sensitizing and desensitizing mechanisms. These modulations during different stimuli could be due to different myosin light-chain kinase/myosin light-chain phosphatase ratios.

Animals↗

PubMed1990

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium.

The free intracellular calcium ion concentration ([Ca2+]i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5-15 min) between spontaneous contractions [Ca2+]i continues to decrease after full mechanical relaxation to reach a minimal level of 86 +/- 8 nM (n = 9) just before the start of the next contraction. During the spontaneous contractions (5-15 min) [Ca2+]i reached a maximum of 211 +/- 19 nM and then oscillated between 155 +/- 16 nM and 194 +/- 9 nM. Increased extracellular Ca2+ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca2+]i both in the relaxed and contracted states. In 10 mM extracellular Ca2+, addition of AlF4-, as 1 mM NaF + 10 microM AlCl3, caused a sustained increase in [Ca2+]i and maintained force. Addition of verapamil (10 microM) in this situation decreased [Ca2+]i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca2+]i, possibly via inactivation of Ca2(+)-dependent K+ channels.

Action Potentials↗

PubMed1990

The alpha 1-agonist phenylephrine inhibits voltage-gated Ca2(+)-channels in vascular smooth muscle cells of rabbit ear artery.

The effects of the alpha 1-agonist phenylephrine on the voltage-gated Ca2(+)-entry in vascular smooth muscle cells has been studied by measuring the agonist-induced changes of [Ca2+]i in K(+)-depolarized tissues. These changes have been estimated from the changes in fluorescence of the Ca2(+)-indicator fura-2, or have been assessed from the changes in 86Rb-efflux rate through Ca2(+)-activated K(+)-channels. Phenylephrine increases the force development in K(+)-depolarized tissues, but reduces [Ca2+]i and inhibits the 86Rb-efflux rate. However, in the presence of the Ca2(+)-entry blocker verapamil, phenylephrine increases both force development and [Ca2+]i. It is concluded that phenylephrine inhibits voltage-gated Ca2(+)-channels, and also induces an influx of calcium by activating a verapamil-insensitive pathway.

Animals↗

PubMed1990

Effects of cyclic nucleotide dependent protein kinases on the endoplasmic reticulum Ca2+ pump of bovine pulmonary artery.

This paper describes the stimulation by cyclic nucleotide dependent protein kinases on the Ca2+ uptake by isolated endoplasmic reticulum (ER) vesicles from the bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake. As previously observed for the phosphorylation of phospholamban, the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with cAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2(+)-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed.

Albumins↗

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↗