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B Himpens

Publications and source records attributed to B Himpens.

At least 19 recordsLinked to original sources
PubMed1992

Functional difference between SERCA2a and SERCA2b Ca2+ pumps and their modulation by phospholamban.

COS 1 cells were transfected with full-length pig stomach sarcoplasmic/endoplasmic reticulum Ca2+ pump (SERCA)2a or SERCA2b cDNA. Ca2+ uptake by microsomes from transfected cells revealed that the Ca2+ affinity of the SERCA2b Ca2+ pump (K0.5 0.17 +/- 0.01 microM) was higher than that of the SERCA2a Ca2+ pump (K0.5 0.31 +/- 0.02 microM). Thapsigargin-sensitivity was found to be identical for the two isoforms. The Ca2+ affinity of both the SERCA2a and SERCA2b Ca2+ pumps was decreased by a factor of two when they were co-expressed with phospholamban.

Animals↗
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

Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse.

We compared the changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), as measured with the fluorescent Ca2+ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K+ depolarization and by agonists. Resting [Ca2+]i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca2+ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca2+]i increase, the agonist stimulation induced a higher force development than the K+ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca2+]o. Our results indicate that the Ca2+ threshold for force development was about 150% of the resting [Ca2+]i in both cell types. The resting [Ca2+]i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca2+ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca2+ sensitivity during contraction. The desensitization to Ca2+ was specific for phasic muscle, in which it occurred as an early, time- and Ca(2+)-dependent process that was partially reversible.

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

ATP activates P2x-contracting and P2y-relaxing purinoceptors in the smooth muscle of mouse vas deferens.

1. The mechanism for the low potency of exogenous ATP in producing contraction at the P2x-purinoceptors in the smooth muscle of the mouse vas deferens (VD) was examined. 2. The measure of the breakdown of ATP in contact with the VD showed that its degradation was limited and did not account for its weak contractile effect. 3. Externally applied, ATP induced a small and transient contraction but a marked and prolonged increase of the cytosolic Ca2+ concentration ([Ca2+]i), which suggests an efficient binding to the P2x-purinoceptors. Such a calcium-force dissociation was not observed with beta, gamma-methylene ATP (beta, gamma-Me-ATP), a structural ATP analogue. 4. The force response of precontracted VD to ATP was biphasic, consisting of a small initial contraction followed by a sustained marked relaxation. In contrast, beta, gamma-Me-ATP elicited a pronounced contraction without ensuing relaxation. 5. ATP was more potent than adenosine in producing relaxation, and the relaxation was not antagonized by 8-phenyltheophylline, suggesting the activation of P2-purinoceptors. 6. For this relaxation, the rank order of potency was 2-methyl-thio-ATP (2-MeSATP) > ATP > beta, gamma-Me-ATP, which is characteristic for the P2y-purinoceptors. 7. Reactive Blue 2, a P2y-purinoceptor antagonist, was found to reduce the relaxation mediated by ATP. 8. These results indicate that ATP acts in VD not only on contracting but also on relaxing P2-purinoceptors, eliciting thereby overlapping opposite effects. In VD, the classical low potency of ATP or contraction is thus not explained by its low bioavailability or its low binding, but rather by its low specificity for the contracting P2x-purinoceptors, leading to the activation of the relaxing P2y-purinoceptors.

Adenosine Diphosphate↗
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↗
PubMed1992

Kinetics of nucleocytoplasmic Ca2+ transients in DDT1 MF-2 smooth muscle cells.

The free calcium concentrations in the nucleus ([Ca2+]n) and in the cytoplasm ([Ca2+]c) of cultured DDT1 MF-2 smooth muscle cells were estimated using the fluorescent dye indo-1. With the use of confocal microscopy, line scans were made during the onset and the differential rise of the Ca2+ signal elicited by the agonists histamine and ATP. The results confirm our earlier findings that in these cells [Ca2+]n at rest was lower than [Ca2+]c. The present experiments show that this gradient over the nuclear envelope was also preserved in Ca(2+)-free solution containing 2 mM EGTA, underlining the selective barrier function of the nuclear envelope. During stimulation with histamine, an early Ca2+ rise in the vicinity of the nuclear envelope was found in contrast to the delayed Ca2+ rise 2 microns away on both sides of the envelope. This suggests the release of Ca2+ stored in the envelope and the perinuclear sarcoplasmic reticulum. The time course for reaching a uniform Ca2+ concentration ([Ca2+]u = [Ca2+]n = [Ca2+]c) in the nuclear and cytosolic compartment varied with the agonist used for stimulation and was dependent on the external Ca2+ concentration. The value of this uniform Ca2+ concentration itself was, however, independent of the type of stimulation. After reaching [Ca2+]u, a further rise occurred with [Ca2+]n becoming larger than [Ca2+]c. It is postulated that a critical Ca2+ concentration must be reached to induce this differential Ca2+ rise by releasing Ca2+ from an intranuclear Ca2+ store.

Adenosine Triphosphate↗
PubMed1992

Modulation of the Ca(2+)-sensitivity in phasic and tonic smooth muscle.

This work describes the relationship between the cytoplasmic free calcium concentration ([Ca2+]i) measured by the fluorescent Ca(2+)-indicator fura-2, the phosphorylation of the myosin light chain and the force development in the phasic longitudinal smooth muscle layer of guinea-pig ileum and the tonic rabbit pulmonary artery. The close temporal relationship between the rise in cytoplasmic Ca2+ and the initiation of force development as well as the rather good correlation between cytoplasmic Ca2+ and force maintenance leaves little doubt about cytoplasmic Ca2+ being the primary regulator of force. However the present experimental evidence indicate that [Ca2+]i and force are not invariably tightly coupled in smooth muscle. A dissociation between the time course of [Ca2+]i and force was found in the tonic rabbit pulmonary artery but not in the phasic ileum of the guinea-pig. In contrast, there was a pronounced decline in the Ca(2+)-sensitivity of the contractile apparatus (desensitization to Ca2+) in the guinea-pig ileum during prolonged depolarization, an observation not found in the pulmonary artery. Such desensitization could reflect the activation of highly active myosin light chain phosphatase(s) and the different Ca(2+)-sensitivities of tonic and phasic smooth muscles can, at least in part, be due to differences in myosin light chain kinase/phosphatase activity ratios. The sensitivity of the regulatory/contractile apparatus to Ca2+ was increased by agonists in intact and in permeabilized preparations. Furthermore a different sensitizing potentiation between different agonists was observed. The mechanism of the "sensitization" of the contractile response to Ca2+ could act through the activation of the phosphorylation of a protein phosphatase inhibitor, thereby inhibiting the myosin light chain phosphatase. The experiments therefore show that different levels of tension may be present at the same [Ca2+]i and indicate that the Ca(2+)-sensitivity can be modulated in smooth muscle.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Changes in the mechanism of Ca2(+) mobilization during the differentiation of BC3H1 muscle cells.

Ca2+ sequestration and release in BC3H1 muscle cells is strongly dependent on the stage of differentiation. In proliferating cells, more than 90% of the sequestered Ca2+ was Ins(1,4,5)P3-sensitive and 25% was caffeine-sensitive. In differentiated cells, the Ca2+ accumulation was 5-fold higher and was InsP3-insensitive, but about 60% of the sequestered Ca2+ was caffeine-sensitive. These changes were reversible upon addition of growth stimuli. Similarly, by measuring the intracellular Ca2+ concentration in single intact BC3H1 cells, it was found that the number of histamine-responsive cells decreased and the number of caffeine-responsive cells increased during muscle cell differentiation. These data indicate that the development of the muscle phenotype in BC3H1 myoblasts induces a major rearrangement of the mechanisms for Ca2+ mobilization.

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

Sensitive, reproducible and convenient fluorometric assay for the in vitro evaluation of anti-cytomegalovirus agents.

Fluorescein diacetate (FDA), a non-fluorescent diacetyl fluorescein ester that becomes fluorescent upon hydrolysis by cytoplasmic esterases, permitted the easy distinction by fluorometry between non-infected and human cytomegalovirus (CMV)-infected HEL cell cultures. As a result of enhanced cytoplasmic esterase activity after CMV infection, FDA-derived fluorescence intensity was brighter for infected than non-infected HEL cells. A similar increase in fluorescence intensity was observed after loading the cells with Indo-1/AM, a non-fluorescent ester of Indo-1 that becomes fluorescent upon cleavage by cytoplasmic esterases. The 50% effective concentrations of a number of anti-CMV agents as determined by the fluorometric assay were very similar to those obtained by the conventional and more time-consuming microscopic evaluation. The fluorometric assay appears very suitable for an automated evaluation of anti-CMV compounds, and also allows rapid determination of the cytotoxicity of potential antiviral compounds.

Acyclovir↗
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↗
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

Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle.

The effects of the stable thromboxane analogue U46619, the alpha 1-adrenergic agent phenylephrine and depolarization with high K+ on cytoplasmic Ca2+ ([Ca2+]i) and force development were determined in rabbit pulmonary artery smooth muscle. Following stimulation with each of the excitatory agents, the time course of the [Ca2+]i/force relationship described counter-clockwise hysteresis loops with the rise and fall in [Ca2+]i leading, respectively, contraction and relaxation. The rank order of the force/[Ca2+]i ratios evoked by the different methods of stimulation was: U46619 greater than phenylephrine high K+. The difference between the actions of U46619 and phenylephrine was due to the lesser Ca2(+)-releasing and greater Ca2(+)-sensitizing action of U46619. Both U46619 and phenylephrine also released intracellular Ca2+ in intact (non-permeabilized) preparations. The effects of the two agonists on force, at constant free cytoplasmic [Ca2+] maintained with EGTA, were also determined in preparations permeabilized with staphylococcal alpha-toxin, in which intracellularly stored Ca2+ was eliminated with A23187. Sensitization of the contractile response to Ca2+ by agonists was indicated by the contractile responses of permeabilized muscles to U46619 and to phenylephrine, in the presence of constant, highly buffered [Ca2+]i. These contractions were inhibited by GDP [beta S] and could also be elicited by GTP. We conclude that, in addition to changing [Ca2+]i, pharmacomechanical coupling can also modulate contraction by altering the sensitivity of the regulatory/contractile apparatus of smooth muscle to [Ca2+]i, through a G-protein-coupled mechanism.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
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↗