Biomedical subjects

G Droogmans

Publications and source records attributed to G Droogmans.

At least 19 recordsLinked to original sources

PubMed1992

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

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

Benzoquinones↗

PubMed1992

Luminal Ca2+ controls the activation of the inositol 1,4,5-trisphosphate receptor by cytosolic Ca2+.

Luminal Ca2+ controls the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3-induced Ca2+ release is also controlled by cytosolic Ca2+; low concentrations of Ca2+ stimulate the release. The aim of this work was to investigate whether luminal Ca2+ would affect the stimulation of the Ins(1,4,5)P3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. We also report that the Ins(1,4,5)P3 receptor in A7r5 cells is activated by low concentrations of cytosolic Ca2+. Cytoplasmic Ca2+ increases the Ins(1,4,5)P3 sensitivity without affecting the cooperativity. The increase in Ins(1,4,5)P3 sensitivity becomes relatively more pronounced when the Ca2+ content of the stores decreases. This modulatory effect of luminal Ca2+ on the responsiveness to cytosolic Ca2+ is an intrinsic property of the Ins(1,4,5)P3 receptor.

Animals↗

PubMed1992

Ca2+ release induced by inositol 1,4,5-trisphosphate is a steady-state phenomenon controlled by luminal Ca2+ in permeabilized cells.

Low concentrations of inositol 1,4,5-trisphosphate (InsP3) evoke a very rapid mobilization of intracellular Ca2+ stores in many cell types, which can be followed by a further, much slower efflux. Two explanations have been suggested for this biphasic release. The first proposes that the Ca2+ stores vary in their sensitivity to InsP3, and each store releases either its entire contents or nothing (all-or-none release); the second proposes instead that the stores are uniformly sensitive to the effects of InsP3, but that they can release only a fraction of their Ca2+ before their sensitivity is somehow attenuated (steady-state release). Experiments using purified InsP3 receptor molecules reconstituted into lipid vesicles have shown heterogeneity of the receptors in their response to InsP3 under conditions in which the total Ca2+ level at both sides of the receptor is held constant. We now report that in permeabilized A7r5 smooth-muscle cells incubated in Ca(2+)-free medium, the amount of 45Ca2+ remaining in the stores after the rapid transient phase of release is independent of their initial Ca2+ levels, indicating that partially depleted stores are less sensitive to InsP3. Moreover, if the stores are reloaded with 40Ca2+ after the first stimulus, reapplication of the same low concentration of InsP3 will release further 45Ca2+. This recovery of InsP3 sensitivity is almost complete. Under these conditions, Ca2+ release must thus occur by a steady-state mechanism, in which the decreasing Ca2+ content of the stores slows down further release.

Calcium↗

PubMed1992

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells.

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca2+]o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca2+ ([Ca2+]i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential Erev shifted from -2.3 +/- 0.8 mV (n = 4) in a nominally Ca2+ free solution to +1.5 +/- 1.6 mV at 1.5 mM [Ca2+]o (n = 4) and to +21.9 +/- 4.4 mV (n = 7) at 10 mM [Ca2+]o. From our data we conclude that shear stress opens an ion channel that is 12.5 +/- 2.9 (n = 7) times more permeable for calcium than for sodium or cesium.

Calcium↗

PubMed1992

Paradoxical decrease in cytosolic calcium with increasing depolarization by potassium in guinea-pig mesotubarium smooth muscle.

The free intracellular Ca2+ concentration ([Ca2+]i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. [Ca2+]i and force were maximal at a relatively low (30 mM) concentration of extracellular K+ ([K+]o), and declined at 90 and 140 mM K+. Plateau values of both [Ca2+]i and force were higher in the presence of 5.10(-6) M ryanodine, indicating that the sarcoplasmic reticulum (SR) contributes to the decline with depolarization. Force and [Ca2+]i at 90 mM K+ were both lower then the high-K+ solution was applied after a period in 30 mM K+ than after a period in normal solution (5.9 mM K+), consistent with inactivation of Ca2+ channels during prolonged depolarization. Addition of carbachol to the depolarized muscle caused a maintained increase in force without maintained increase in [Ca2+]i. We conclude that the decrease in force at increased [K+]o (the "calcium-potassium paradox") is due to a membrane-potential-mediated decrease in [Ca2+]i and, to a lesser extent, to desensitization of the contractile-regulatory apparatus to Ca2+.

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

PubMed1992

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

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

Animals↗

PubMed1992

Calcium ion homeostasis in smooth muscle.

Ca2+ plays an important role in the regulation of smooth-muscle contraction. In this review, we will focus on the various Ca(2+)-transport processes that contribute to the cytosolic Ca2+ concentration. Mainly the functional aspects will be covered. The smooth-muscle inositol 1,4,5-trisphosphate receptor and ryanodine receptor will be extensively discussed. Smooth-muscle contraction also depends on extracellular Ca2+ and both voltage- and Ca(2+)-release-activated plasma-membrane Ca2+ channels will be reviewed. We will finally discuss some functional properties of the Ca2+ pumps that remove Ca2+ from the cytoplasm and of the Ca2+ regulation of the nucleus.

Animals↗

PubMed1992

Shear stress-induced calcium transients in endothelial cells from human umbilical cord veins.

1. Changes of the free cytosolic Ca2+ concentration induced by shear stress were measured in Fura-2 acetoxymethyl ester-loaded endothelial cells from human umbilical cord veins. 2. We were able to induce Ca2+ transients in almost every cell by blowing a stream of physiological solution onto a single endothelial cell thereby inducing shear stress between 0 and 50 dyn cm-2. The Ca2+ response could be graded by varying the shear stress, and reached a half-maximal value at a shear stress of 30 dyn cm-2. 3. The shear stress responses critically depended on the extracellular Ca2+ concentration and were absent in a Ca(2+)-free solution. Repetitive application of short pulses of shear stress induced cumulative effects because of the slow decay of the shear stress Ca2+ responses (time constants 82.3 +/- 17.8 s from twenty-five cells). Application of a depolarizing high potassium solution to reduce the driving force for Ca2+ entry decreased the Ca2+ transients in some of the cells. 4. Application of shear stress in the presence of other divalent cations, such as nickel, cobalt or barium, always produced substantial changes in the ratio of the 390/360 nm fluorescence signal, indicating influx of these cations and subsequent quenching of the Fura-2 fluorescence. 5. Shear stress responses in the presence of 10 mM Ca2+ were completely blocked by application of 1 mM La3+. 6. Incubation of the cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not alter the shear stress response, but completely blocked histamine-induced Ca2+ transients. 7. Small submaximal shear stress potentiated the Ca2+ transients induced by histamine. 8. We conclude that shear stress-dependent Ca2+ signals are induced by an influx of calcium that is not modulated via protein kinase C and not activated by membrane depolarization. The influx pathway is also permeable to divalent cations such as Ni2+, Co2+ and Ba2+, but is blocked by La3+.

Barium↗

PubMed1992

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

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

Animals↗

PubMed1991

The conductance of single cardiac sodium channels from guinea pig depends on the intracellular sodium concentration.

Currents through DPI 201-106 modified single sodium channels have been measured in cell-free inside-out patches from guinea-pig ventricular myocytes. Single-channel conductance and reversal potential of the sodium channel have been calculated at different intracellular sodium concentrations [( Na+]i) from microscopic I-V curves, which were obtained by application of linear voltage ramps. The relation between the reversal potential and [Na+]i could be fitted with a modified Goldman-Hodgkin-Katz equation with a relative permeability for K+ over Na+ ions of 0.054. The zero-current conductance of the Na channel as a function of [Na+]i shows a plateau value at low Na concentrations, and increases in a sigmoidal manner at higher concentrations. It is concluded that the Na channel can carry outward currents and that its conductance depends on [Na+]i.

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↗

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

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

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↗