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Biomedical subjects

R Casteels

Publications and source records attributed to R Casteels.

At least 145 records · Page 8Linked to original sources
PubMed1985

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum.

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.

Animals↗
PubMed1985

Excitation-contraction coupling in vascular smooth muscle cells and perivascular nerve stimulation.

Smooth muscle contraction depends largely on the increase of the cytoplasmic Ca2+ concentration. This change can be brought about by the opening of voltage-dependent Ca channels or receptor operated Ca channels. Although in some vascular smooth muscle cells the addition of noradrenaline does not appreciably change the membrane potential, it is observed that stimulation of the perivascular nerve fibres elicits excitatory junction potentials and action potentials. This difference between the action of exogenous and endogenous noradrenaline could be due to either the existence of intrajunctional gamma-receptors, which differ from the extrajunctional alpha-receptors or to the release from the nerve fibres of cotransmitters together with noradrenaline. It can be concluded that the clear distinction between electromechanical coupling and pharmacomechanical coupling only applies to some experimental conditions in vitro, but that the distinction cannot be used for conditions in vivo.

Adenosine Triphosphate↗
PubMed1985

Dependence on calcium of potassium- and agonist-induced changes in potassium permeability of rabbit ear artery.

The effect of K+ depolarization and agonists on the 86Rb+ efflux from rabbit ear artery has been investigated. K+ depolarization with 59 mM-K+ induces an increase of the 86Rb+ efflux rate, which is dependent on [Ca2+]o and is correlated with the concomitant force development. This effect is largely reduced by Ca2+ antagonists, such as D-600 and Mn2+. The residual increase of the 86Rb+ efflux rate is much smaller than that predicted by the constant-field equations. Stimulation with 10(-5) M-noradrenaline or 10(-4) M-histamine induces a biphasic increase of the efflux rate. The initial transient effect is reduced in low [Ca2+]o solutions, whereas the maintained component is largely independent of [Ca2+]o. Stimulation with noradrenaline during depolarization of the tissues with K+ induces, after a transient increase of the efflux rate, an inhibition of the K+-induced increase of the efflux rate. Both phases of the noradrenaline action are due to activation of alpha-adrenoreceptors. Exposure to Ca2+-free medium induces a progressive increase of the 86Rb+ efflux rate, which reaches a new steady-state value after about 60 min. Stimulation with noradrenaline after this 60 min exposure to Ca2+-free solution no longer induces a significant effect. Stimulation with noradrenaline after shorter exposures to Ca2+-free solution immediately increases the 86Rb+ efflux to a value close to the steady-state value obtained after prolonged exposure to Ca2+-free medium. Washing out the agonist has no effect on the rate constant. It will only return to its control value after exposure to solutions containing Ca2+. This recovery of the rate constant by external Ca2+ also occurs in the presence of 1 mM-Mn2+ in the perfusion fluid. On re-exposure of the tissues in the presence of 1 mM-Mn2+ to Ca2+-free solution the rate constant of the 86Rb+ efflux increases at once to the steady-state value observed in Ca2+-free solution. This increase proceeds gradually if the tissues have been re-exposed in the absence of Mn2+. It is concluded that K+ permeability might be regulated by [Ca2+]i and that this relationship can be affected by agonists. In order to explain the effects of Ca2+-free medium on the 86Rb+ efflux we have to assume that at very low values of [Ca2+]o and [Ca2+]i the membrane permeability for K+ is modified by a different mechanism.

Action Potentials↗
PubMed1985

Na+-K+ ATPase, Na-Ca exchange, and excitation-contraction coupling in smooth muscle.

The evidence in favor of a direct role of active Na transport in the regulation of excitation-contraction coupling in vascular smooth muscle has been examined. The observations in vivo and those obtained in isolated tissues do not always lead to the same conclusions. The changes of the membrane potential obtained in vitro by slight reductions in, or increases of [K]o do not modify the resting potential of the cells sufficiently to make them contract. Applying K-free or Na-free medium on isolated tissues is a much more vigorous procedure than the limited changes of [K]o that can occur in vascular beds in situ. The Na-Ca exchange-mechanism does not seem to play a major role in those smooth-muscle cells that have been analyzed in detail, but even here the experimental procedures have neither given precise information about the composition of the intracellular compartment nor allowed sufficient control of the parameters studied. The comparison of membrane vesicles from smooth muscle and from cardiac muscle indicates that important differences exist in Na-Ca exchange and in activities of Na+-K+ ATPase and Ca2+-Mg2+ ATPase. These findings suggest a poor development of Na-Ca exchange in smooth muscle as compared to cardiac muscle. Finally, the changes in the Na metabolism of erythrocytes from hypertensives are mentioned, and the present difficulties of linking those changes to an increased reactivity of vascular smooth-muscle cells are briefly discussed.

Animals↗
PubMed1984

Evidence for the presence in smooth muscle of two types of Ca2+-transport ATPase.

Membrane fractions prepared from smooth muscle of the pig stomach (antral part) contain two Ca2+-dependent phosphoprotein intermediates belonging to different Ca2+-transport ATPases. These alkali-labile phosphoproteins can be separated by electrophoresis in acid medium. The 130 kDa phosphoprotein resembles a corresponding protein in the erythrocyte membrane, whereas the 100 kDa protein resembles that of the Ca2+-transport ATPase in sarcoplasmic reticulum from skeletal muscle. These resemblances are expressed in terms of Mr, reaction to La3+ and in a similar proteolytic degradation pattern. The presence of the calmodulin-stimulated ATPase in mixed membranes from smooth muscle is confirmed by its binding of calmodulin and antibodies against erythrocyte Ca2+-transport ATPase, whereas such binding does not occur with proteins present in the presumed endoplasmic reticulum from smooth muscle.

Animals↗
PubMed1984

Tissue levels and purification by affinity chromatography of the calmodulin-stimulated Ca2+ -transport ATPase in pig antrum smooth muscle.

The Ca2+ -transport ATPase [Ca2+ + Mg2+)-ATPase) in a plasma membrane-rich fraction of porcine antrum (stomach) smooth muscle, is stimulated 2.9-times by calmodulin in the presence of 0.2 mg/ml saponin and reaches a value of 12.0 +/- 2.0 (4) mumol/100 mg protein (equivalent to 110 g wet tissue) per min at 37 degrees C and 10(-5) M [Ca2+]. Saponin was found to specifically potentiate the calmodulin-(Ca2+ + Mg2+)-ATPase interaction, even in the Triton X-100 solubilized enzyme. The conditions for purification of the (Ca2+ + Mg2+)-ATPase by affinity chromatography on a calmodulin-Sepharose 4B gel were optimized. The purified enzyme has a specific activity of 11.9 mumol/mg protein per min at 37 degrees C, 10(-5) M [Ca2+], 0.6 microM calmodulin, and shows a double polypeptide band at 140 and 150 kDa. The (Ca2+ + Mg2+)-ATPase can be incorporated in artificial liposomes that thereupon show an ATP-dependent Ca2+ uptake (Ca:ATP = 1.0). The magnitude of the calmodulin stimulation of the isolated enzyme depends on its phospholipid environment. When isolated in the presence of phosphatidylserine no calmodulin stimulation is observed. After reconstitution in phosphatidylcholine the calmodulin stimulation amounts to 4.05 +/- 0.63 (n = 12) times.

Animals↗
PubMed1984

The calcium uptake in smooth muscle microsomal vesicles is reduced by centrifugation.

A membrane fraction was isolated from the smooth muscle of the pig stomach by density gradient centrifugation. It was observed that the ATP-dependent Ca uptake in this fraction was diminished if the microsomes were pelleted by differential centrifugation. The decrease of the oxalate-independent Ca uptake was relatively small, but the oxalate-stimulated Ca uptake was reduced dramatically. Evidence is presented which indicates that the selective decrease of the oxalate-stimulated Ca uptake is mainly caused by mechanical damage of the vesicles. Since the oxalate-stimulated Ca uptake can be largely preserved by avoiding pelleting during the membrane fractionation, this observation may be very useful for the further study of Ca transport in subcellular fractions of smooth muscle.

Animals↗
PubMed1984

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle.

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.

Animals↗
PubMed1984

Cell membrane responsiveness and excitation-contraction coupling in smooth muscle.

In this study of the excitation-contraction coupling in arterial smooth-muscle cells, the relations between changes of the membrane potential, force development, and 45Ca exchange have been investigated. It is proposed that the cell membrane plays a primary role by regulating the Ca entry into smooth-muscle cells and by extruding the excess cytoplasmic Ca. The possible relation between the receptor-operated channels and the sarcoplasmic reticulum has been discussed, and the peculiar differences between exogenous noradrenaline and noradrenaline released from the nerve terminals have been described.

Animals↗
PubMed1984

Effects of 2-nicotinamidoethyl nitrate (Nicorandil) on excitation-contraction coupling in the smooth muscle cells of rabbit ear artery.

The effect of SG-75 (Nicorandil) on the neuromuscular transmission and smooth muscle membrane of the rabbit ear artery was investigated by means of intracellular microelectrodes, isometric tension recording and flux experiments. This agent caused a pronounced hyperpolarization of the cell membrane and a significant increase of the K-permeability. During perivascular nerve stimulation, the amplitude and the time constant of the falling phase of the excitatory junction potentials were reduced, causing a suppression of spike generation and of the concomitant contractile responses, but the release of [3H]noradrenaline was not affected. At high doses this agent inhibited significantly submaximal contractile responses induced by various stimuli. However, these effects could not be correlated with changes in 45Ca fluxes. It is suggested that although the effect of SG-75 could be due mainly to an increase of the K-permeability and the concomitant hyperpolarization, it might also reduce the force development by a mechanism which does not depend on a reduction of the concentration of ionized Ca in the cytoplasm.

Animals↗
PubMed1983

Antibodies to the calmodulin-binding Ca2+-transport ATPase from smooth muscle.

Antibodies were raised against a calmodulin-binding CaMg-ATPase (Ca2+-transport ATPase) from smooth muscle. The binding of these antibodies to a number of related Ca2+-transport ATPases was studied. Antibodies to the calmodulin-binding ATPase from porcine antrum (stomach) smooth muscle do not only bind to this CaMg-ATPase, but also to the corresponding enzyme in porcine erythrocytes. However, they do not bind to the CaMg-ATPase from sarcoplasmic reticulum of porcine skeletal muscle. The binding of these antibodies to the CaMg-ATPase of smooth muscle, does not inhibit the enzyme activity.

Animals↗
PubMed1983

Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum.

1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.

Animals↗
PubMed1983

Effects of Ca-antagonists on neuromuscular transmission in the rabbit ear artery.

1. The effects of three Ca-antagonists: diltiazem, nicardipine and flunarizine have been studied on excitatory junction potentials (e.j.p.s), force development and efflux of transmitter during stimulation of perivascular nerves in the rabbit ear artery. 2. Stimulation of these perivascular nerves produces excitatory junction potentials and repetitive stimulation causes facilitation. Increasing the frequency or the number of stimuli initiates an action potential and a large contraction. Both phenomena are completely suppressed by 3 X 10(-7) M TTX. 3. Ca-antagonists at 10(-5) M do not affect the resting membrane potential, but flunarizine and nicardipine at concentrations exceeding 10(-5) M reduce the amplitude of e.j.p.s and of the action potentials and also the concomitant contraction induced by nerve stimulation. Diltiazem at concentrations below 3 X 10(-5) M has no effect on e.j.p.s and action potentials while at 10(-4) M, it largely suppresses e.j.p.s, spikes and contraction. This inhibitory effect of the Ca-antagonists increases with prolonged exposure. 4. All these Ca-antagonists induce an increased release of 3H-DOPEG from the nerve terminals, but in our experiments in vitro they do not reduce the efflux of 3H-noradrenaline induced by nerve stimulation. 5. The results indicate that Ca-antagonists might affect the excitation-contraction coupling in vivo by inhibiting the Ca influx activated by endogenous noradrenaline. They do not exert an acute effect on the noradrenaline release induced by stimulation. The increased DOPEG release by Ca-antagonists remains unexplained.

Animals↗
PubMed1983

Reserpine has a direct action as a calcium antagonist on mammalian smooth muscle cells.

The effects of reserpine on excitation-contraction coupling and 45Ca exchange of smooth muscle cells of the rabbit ear artery and the guinea-pig taenia coli have been studied. Reserpine inhibited the spontaneous mechanical activity of the taenia coli and the force development induced by 59 mM-external K or 10(-5) M-carbachol. In the ear artery reserpine blocked the K-induced contraction but its effect on the contraction elicited by noradrenaline was smaller. At 0.2 mM-Ca, the inhibition of the tonic component of the noradrenaline-induced contraction was more pronounced than that of the phasic component. This reserpine action was fully reversible for the noradrenaline stimulus in the ear artery but less so for K-induced contractions. The inhibitory action on contractions induced in taenia coli by K-rich solution and by carbachol was even less reversible. The analysis of the effect of reserpine on the 45Ca exchange in the ear artery has revealed that it inhibits the increase of the fractional loss induced by K depolarization, but that it does not exert a significant effect on the increased fractional loss induced by 10(-5) M-noradrenaline. Reserpine slows down the filling with 45Ca of the agonist-sensitive store without affecting the steady-state amount of Ca taken up by the store. A study of the degree of filling of the store by measuring the force development and the 45Ca release elicited by noradrenaline in Ca-free medium, reveals that the force development after loading in a reserpine-containing medium remains less than the control, although the same amount of Ca is released from the store. It was shown by using tetrabenazine that the inhibitory action of reserpine on the Ca exchange and the force development is not due to an interaction of reserpine with the receptor molecules that are responsible for its depleting action on aminergic granules. These results strongly suggest that reserpine exerts a Ca antagonistic action on smooth muscle whereby it blocks the potential-dependent channels. However, reserpine also affects the receptor-operated channels to some extent and in addition at a high concentration it seems to exert an unspecific inhibitory action on the contractile system.

Animals↗