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Biomedical subjects

R Casteels

Publications and source records attributed to R Casteels.

At least 127 records · Page 7Linked to original sources
PubMed1988

Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.

Adenosine Triphosphate↗
PubMed1988

Characterization of the Mg2+-activated ATPase activity in smooth-muscle membranes. NADH oxidase and adenylate kinase interfere with the NADH-coupled enzyme assay.

The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (microsomal fractions) from myometrium, aorta and taenia coli declines non-linearly during the assay. Its initial high rate gradually diminishes over 15-60 min, depending on the type of smooth muscle, to a constant value. This decline depends on the presence of ATP and can be partially prevented by concanavalin A. The non-linearity is limited in microsomes from rabbit ear artery. In microsomes from antrum the apparent Mg2+-ATPase activity actually increases with time, albeit gradually. Storage on ice of the microsomes of the aorta, and especially of myometrium of pregnant rats and of taenia coli, is accompanied over a few hours after their preparation by a gradual suppression of the component of the Mg2+-ATPase activity that is inhibited by ATP. The Mg2+-ATPase activity in microsomes from antrum remains constant. NADH oxidase activity accounts for 10% of the Mg2+-ATPase activity in microsomes from stomach smooth muscle. The apparent initial non-linearity of the Mg2+-ATPase activity in that tissue is due to a time-dependent decrease of a rotenone-sensitive NADH oxidase activity. The adenylate kinase activity, as deduced from the effect of the adenylate kinase inhibitor P1,P5-di(adenosine-5') pentaphosphate, could account for 45.0, 35.0 and 31.0% respectively of the Mg2+-ATPase activity in microsomes from stomach, myometrium and aorta. No adenylate kinase activity could be detected in microsomes from ear artery and taenia coli. When microsomes from stomach smooth muscle were separated on a sucrose gradient, the contribution of adenylate kinase and NADH oxidase to the Mg2+-ATPase activity was most pronounced in the higher-density fractions. Part of the NADH oxidase activity and of the Mg2+-ATPase activity, and most of the adenylate kinase activity, are not sedimented at 224000 gmax. for 30 min and may therefore be present as soluble enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗
PubMed1988

Carbachol partially inhibits the plasma-membrane Ca2+-pump in microsomes from pig stomach smooth muscle.

A plasmalemmal enriched membrane fraction, prepared from pig stomach smooth-muscle, contains a calmodulin-stimulated (Ca2+ + Mg2+)-ATPase and presents an ATP-dependent 45Ca-uptake. If these smooth-muscle strips are preincubated with 10(-3) M-carbachol, this Ca2+ + Mg2+)-ATPase and the 45Ca-uptake are reduced by 21.4% and 13.5%, respectively, as compared to controls. This inhibitory effect of carbachol can be completely blocked by atropine. Carbachol does neither affect the passive permeability of the microsomes to 45Ca, nor the passive 45Ca-binding to the vesicles. Neither does it exert an effect on the proportion of closed inside-out plasma-membrane vesicles. Likewise, preincubation of rat myometrium with 90 nM-oxytocin induces a 20.4% inhibition of the ATP-dependent 45Ca-uptake, without having an effect on the passive 45Ca-binding, the permeability to 45Ca or the sideness of the vesicles. From these results, it is concluded that some agonists as carbachol and oxytocin induce a decrease in the activity of the plasmalemmal Ca2+-pump.

Animals↗
PubMed1988

Ryanodine reduces the amount of calcium in intracellular stores of smooth-muscle cells of the rabbit ear artery.

We have investigated the mechanism of action of ryanodine on intact and skinned smooth-muscle cells of the rabbit ear artery. The amplitude of the phasic response induced by low noradrenaline (NA) concentrations (less than 30 nM) was inhibited by 10 microM ryanodine, while that elicited by high NA concentrations (greater than 100 nM) was not affected. The phasic contractions induced by both low and high NA concentrations in Ca2+-free solution containing 2 mM EGTA were suppressed by 10 microM ryanodine. The rate of 45Ca efflux in Krebs solution was enhanced by 10 microM ryanodine, while the increased 45Ca efflux induced by 10 microM NA was inhibited by ryanodine. 10 microM ryanodine did not affect the contractile proteins in saponin-treated smooth-muscle cells. The intracellular Ca2+ stores of these skinned cells could be filled by exposing these cells to a solution containing 0.6 microM Ca2+. After a wash in a Ca2+-free solution, a contraction due to a release of the accumulated Ca2+ could be induced by either 25 mM caffeine or 20 microM inositol 1,4,5-trisphosphate (InsP3) or 10 microM A23187. These contractions did not occur if 10 microM ryanodine was present during Ca2+ loading. The addition of ryanodine during the Ca2+-free wash did not affect the subsequent force development. These observations indicate that ryanodine, in the presence of Ca2+, depletes the intracellular Ca2+ stores, and that this depletion is responsible for the inhibition of the component of the NA-induced contraction which depends on the release of Ca2+ from intracellular stores.

Alkaloids↗
PubMed1988

Properties of intracellular calcium stores in pregnant rat myometrium.

1. The properties of the Ca2+ stores in myometrium of 21-day pregnant rats were studied by recording the contractile responses of saponin-treated skinned muscles. 2. After accumulation of Ca2+ into the stores in the presence of 5 mM NaN3, inositol 1,4,5-trisphosphate (InsP3) at concentrations exceeding 3 microM produced a contraction. The amplitude of this contraction was maximal at about 20 microM. A second application of 20 microM InsP3 produced a smaller contraction than the first one. However after reloading the stores with Ca2+, 20 microM InsP3 produced a contraction of the same amplitude as the initial one. 3. After application of 20 microM InsP3, 1 microM A23187 still evoked a large contraction. If A23187 was applied first, the subsequent application of InsP3 or A23187 no longer induced a contraction, even after Ca2+ loading. 4. Guanosine triphosphate (GTP) or arachidonic acid, both 100 microM neither evoked a contraction nor enhanced the subsequent contraction elicited by 20 microM InsP3. 5. Caffeine 25 mM did not induce a contraction nor did it affect the contraction elicited by 20 microM InsP3. 6. The results indicate that in pregnant rat myometrium InsP3 releases Ca2+ from intracellular stores as has been proposed in vascular smooth muscles.

Animals↗
PubMed1988

Ca2+-transport ATPases of vascular smooth muscle.

To characterize the Ca2+-transport properties of the plasma membrane and of the endoplasmic reticulum of bovine pulmonary artery, membrane vesicles are subfractionated by a procedure of density-gradient centrifugation that takes advantage of the selective effect of digitonin on the density of plasma-membrane vesicles. The obtained endoplasmic-reticulum fraction contains hardly any plasma-membrane vesicles, whereas the plasma-membrane fraction is still contaminated by a substantial amount of endoplasmic-reticulum vesicles. An adenosine 5'-triphosphate (ATP) energized Ca2+-transport system and a Ca2+-stimulated ATPase activity are present in both subcellular fractions. The Ca2+ transport by the plasma membrane is catalyzed by a (Ca2+,Mg2+)-ATPase of Mr 130,000. It binds calmodulin and it has a low steady-state phosphoprotein intermediate level. The endoplasmic-reticulum vesicles contain a Ca2+-transport ATPase of Mr 100,000 that is characterized by a high steady-state phosphointermediate level. It is antigenically related to the Ca2+-pump protein of cardiac sarcoplasmic reticulum. Phospholamban, the regulatory protein of the Ca2+-transport enzyme of cardiac sarcoplasmic reticulum, is also present in the endoplasmic reticulum of the pulmonary artery. A comparison of these fractions with the previously characterized fractions from porcine gastric smooth muscle reveals important differences in the basal Mg2-ATPase activity, in the ratio of the (Ca2+,Mg2+)-ATPase of the plasmalemma to that of the endoplasmic reticulum, and in the ratio of the (Na+,K+)-ATPase activity to the plasmalemmal (Ca2+,Mg2+)-ATPase activity. These differences can be ascribed in part to the species and in part to the tissue. These data suggest that in the bovine pulmonary artery the Ca2+ extrusion via the ATP-dependent Ca2+ pump may have a less predominant role, and that the Ca2+ uptake by the endoplasmic reticulum, and possibly also the Ca2+ extrusion via the Na+-Ca2+ exchanger could be more important in this tissue than in the porcine stomach.

Adenosine Triphosphatases↗
PubMed1988

The (Ca2+-Mg2+)-ATPases of the plasma membrane and of the endoplasmic reticulum in smooth muscle cells and their regulation.

Smooth muscle cells contain two distinct Ca2+-transport ATpases with a different subcellular localization. The plasmalemmal Ca2+ pump has a relative molecular weight (Mr) of 140k and its phospho-intermediate level is increased by La3+. Its resemblance to the erythrocyte Ca2+ pump is further confirmed by its calmodulin-binding capacity and its antigenic properties. A 100k Ca2+-transport ATPase is localized in the endoplasmic reticulum. Its phospho-intermediate level is decreased by La3+, and it is antigenically related to the cardiac sarcoplasmic reticulum Ca2+-transport ATPase. These two different Ca2+-transport ATPases are present in both visceral and vascular smooth muscle, but tissue- and species-dependent differences in their relative amount have been observed. The endoplasmic-reticulum Ca2+-transport ATPase is regulated via phospholamban. Phosphorylation of this regulatory protein by cAMP-dependent as well as by cGMP-dependent protein kinase stimulates the endoplasmic-reticulum Ca2+ pump. The activity of the plasmalemmal Ca2+-transport ATPase can be modulated by calmodulin, negatively charged phospholipids, and by receptor-binding agonists. cGMP-dependent protein kinase also exerts a stimulatory effect on the plasmalemmal Ca2+ pump, but this effect is not mediated via a direct phosphorylation of the Ca2+ pump.

Animals↗
PubMed1987

Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin.

It is proposed that smooth-muscle endoplasmic reticulum contains calsequestrin and that this protein in smooth muscle resembles cardiac calsequestrin more than the skeletal-muscle form. This proposal is based on seven similarities between the smooth-muscle protein and cardiac calsequestrin. Proteins with an Mr of 55,000 can be extracted from the membranes of smooth muscle and of cardiac muscle using 100 mM Na2CO3. The protein from smooth muscle binds to phenyl-Sepharose in the absence of Ca2+ and is released by 10 mM CaCl2, as has been observed for cardiac calsequestrin. The protein from smooth muscle comigrates with the cardiac calsequestrin on Laemmli-type SDS-polyacrylamide gel electrophoresis. The protein of Mr 55,000 from smooth muscle and cardiac calsequestrin both strain blue with the carbocyanine dye Stains-all. Both proteins present similar one-dimensional Cleveland peptide maps although minor differences might exist. From an analysis of subcellular membranes separated by sucrose gradient centrifugation it is concluded that the protein with Mr 55,000 from the smooth muscle is confined to the endoplasmic reticulum, the same subcellular structure from which, in heart muscle, calsequestrin can be isolated. Antibodies raised against canine cardiac calsequestrin bind to a protein of similar Mr in smooth-muscle endoplasmic reticulum. In addition to the calsequestrin, three other extrinsic proteins with an Mr of 130,000, 100,000 and 63,000, stain blue with Stains-all and occur in the endoplasmic reticulum of smooth muscle.

Animals↗
PubMed1987

Measurement by Quin2 of changes of the intracellular calcium concentration in strips of the rabbit ear artery and of the guinea-pig ileum.

Ca2+ transient and force development were investigated in smooth muscle strips of the rabbit ear artery and the longitudinal layer of the guinea-pig ileum by using the fluorescent indicator Quin2. Agonists only transiently increased the fluorescence intensity despite the enhanced contraction while excess potassium resulted in a maintained light signal. In Ca2+ free solutions the release by an agonist of Ca2+ from an intracellular store can be demonstrated. These observations illustrate the usefulness of the Ca2+ indicator Quin2 in the study of the excitation-contraction coupling in smooth muscle under various conditions.

Aminoquinolines↗
PubMed1987

Effect of adrenergic agonists on Ca2+-channel currents in single vascular smooth muscle cells.

Ca2+-channel currents have been measured in enzymatically dispersed single smooth muscle cells of the rabbit ear artery using the whole-cell patch clamp technique. Inward currents were elicited by depolarizing test pulses from a holding potential of -50 mV. These currents were activated from -30 mV onward and reached full activation around 0 mV. alpha-Adrenergic agonists did not affect the background current measured at the holding potential, but markedly reduced the peak amplitude of the voltage-activated Ca2+-channel currents. This alpha-adrenergic inhibition also occurred in cells which were internally perfused with solutions containing either 10 microM cAMP, 10 microM cGMP or 0.1 mM GTP, but became irreversible when the pipette solution contained a non-hydrolyzable GTP-analog. The action of beta-agonists on the voltage-activated Ca2+-channel currents was variable, and ranged from no effect at all to a 50% reduction of the current. It is concluded that alpha-agonists do not open receptor-operated Ca2+-channels in these smooth muscle cells. The inhibition of the voltage-activated Ca2+-currents does not seem to be mediated through changes in cyclic nucleotide levels, but might be mediated through G-proteins. Its physiological relevance remains however unclear. The action of beta-agonists is consistent with their relaxing effect, but the reason for the non-uniform response has not been elucidated.

Adrenergic Agonists↗
PubMed1987

Effect of nicorandil on the excitation-contraction coupling in smooth muscle cells of the rabbit ear artery.

The effects of nicorandil on excitation-contraction coupling in the ear artery of the rabbit have been investigated. Perivascular nerve stimulation induces excitatory junction potentials (e.j.p.'s) and action potentials, which can be inhibited by nicorandil. The inhibition of the nerve-evoked responses by nicorandil cannot be due to an effect of nicorandil on neurotransmitter metabolism because of its failure to inhibit the release of 3H-noradrenaline from the nerve terminals. The decreased excitability of the smooth muscle cells can be partially explained by the increase of K permeability, as measured by 87Rb efflux, and by the concomitant hyperpolarization of the cell membrane. Because the inhibitory action of nicorandil on the contractile responses also occurs in K-depolarized tissues in which nicorandil no longer affects the membrane potential, additional mechanisms of action have to be sought. A study of 45Ca exchange under these conditions does not suggest that this relaxing effect is mediated by changes in cytoplasmic Ca.

Action Potentials↗
PubMed1986

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1986

Regulatory systems for the cytoplasmic calcium concentration in smooth muscle.

Changes of the intracellular Ca concentrations play a predominant though not unique role in the regulation of the force development of smooth muscle cells. Contraction is initiated by an influx of Ca2+ through the cell membrane or by the release of Ca2+ from intracellular storage sites. Ca influx occurs via voltage operated channels and receptor operated channels. The intracellular Ca release induced by agonists probably originates from the endoplasmic reticulum. The removal of Ca2+ from the cytoplasm occurs by extrusion across the plasmalemma and by reaccumulation in the endoplasmic reticulum. These active Ca2+ transport systems are catalysed by (Ca2+ +Mg2+) ATPases. Na-Ca exchange across the sarcolemma of smooth muscle is probably of minor importance since the (Ca2+ +Mg2+)ATPase activity of plasma membranes is higher than the activity of the Na+K+ ATPase, the ultimate energy source for Na+-dependent Ca2+ extrusion. The (Ca2+ +Mg2+)ATPase of the plasmalemma has a Mr of 130.000 and it is stimulated by calmodulin. It resembles the Ca2+ transport ATPase of erythrocyte membranes, including immunological cross-reactivity. The Ca2+ transport enzyme of the endoplasmic reticulum has a Mr of 100.000, is insensitive to calmodulin and resembles the Ca2+ pump of sarcoplasmic reticulum of skeletal muscle. However, antibodies against the Ca2+ pump of skeletal muscle do not cross react with the enzyme of smooth muscle. Subcellular fractionation of pig stomach smooth muscle indicates that in this tissue the large fraction of the (Ca2+ +Mg2+)ATPase activity is present in the plasma membrane while less activity is found in the endoplasmic reticulum.

Animals↗
PubMed1985

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

Animals↗
PubMed1985

Isolation of calelectrin-like proteins associated with smooth muscle plasma membranes.

Plasma membranes prepared from pig stomach smooth muscle (antral part) were extracted with Triton X-100 to isolate insoluble cytoskeletal components. Reextraction of the insoluble material in EGTA yielded a protein complex which resembled the family of proteins that has been designated as 'mammalian calelectrins' [Südhof, T.C. et al. (1984) Biochemistry 23, 1103-1109]. Plasma membranes prepared in the presence and in the absence of 0.6 M KCl differed by the amount of these proteins, but in both preparations the EGTA-extractable proteins were quantitatively important constituents. Two of these proteins were further purified by means of ion exchange chromatography to apparent homogeneity as judged from sodium dodecyl sulfate gel electrophoresis.

Animals↗
PubMed1985

Action of pinaverium bromide on calmodulin-regulated functions.

Pinaverium bromide at concentrations below 10(-5) M did not inhibit calmodulin-dependent enzymes such as phosphodiesterase and the Ca transport ATPase of the plasma membrane. At higher concentrations the compound interacted with the stimulation of those enzymes by calmodulin and also inhibited the calmodulin-independent activity. A similar inhibitory action was observed for the NaK ATPase. It is concluded that the inhibitory action of pinaverium bromide on smooth muscle concentration at concentrations below 10(-5) M was due to its interaction with the voltage-dependent Ca channels and not to its interference with the calmodulin-dependent activation of the contractile proteins.

Animals↗
PubMed1985

The Ca2+-transport ATPases in smooth muscle.

A calmodulin stimulated Ca2+-transport ATPase which has many of the characteristics of the erythrocyte type Ca2+-transport ATPase has been purified from smooth muscle. In particular, the effect of calmodulin on these transport enzymes is mimicked by partial proteolysis and antibodies against erythrocyte Ca2+-transport ATPase also bind to the smooth muscle (Ca2+ + Mg2+)ATPase. A correlation between the distribution of the calmodulin stimulated (Ca2+ + Mg2+)ATPase and (Na+ + K+)ATPase activities in smooth muscle membranes separated by density gradient centrifugation suggests a plasmalemmal distribution of this (Ca2+ + Mg2+)ATPase. A phosphoprotein intermediate in smooth muscle which strongly resembles the corresponding phosphoprotein in sarcoplasmic reticulum of skeletal muscle may indicate the presence in smooth muscle of a similar type of Ca2+-transport ATPase.

Animals↗