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Biomedical subjects

L Raeymaekers

Publications and source records attributed to L Raeymaekers.

At least 55 records · Page 3Linked to original sources
PubMed1994

Volume-activated Cl- currents in different mammalian non-excitable cell types.

The existence and properties of volume-activated Cl- currents were studied in 15 different cell types (endothelium: human umbilical vein, human aorta, bovine pulmonary artery; fibroblasts: Swiss 3T3, L, C3H 10T1/2 and COS-1; epithelium: KB3, HeLa and A6; blood cells: RBL-2H3 and Jurkat; endothelioma cells derived from both subcutaneous and thymic hemangiomas; skin: IGR1 melanoma). Volume-activated Cl- currents with common characteristics, i.e. small conductance, outward rectification, higher permeability for iodide than for chloride and sensitivity to block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) could be elicited in all cells. The block of this current by tamoxifen and dideoxyforskolin is different for the various cell types, as well as the time course and the amplitude of the responses induced by repetitive applications of hypotonicity. Volume-activated Cl- channels with similar biophysical properties are therefore wide-spread among mammalian cells. This may reflect either a single Cl- channel that is ubiquitously expressed or a family of functionally related Cl- channels with cell specific expression patterns.

Animals↗
PubMed1993

Expression of Ca2+ binding proteins of the sarcoplasmic reticulum of striated muscle in the endoplasmic reticulum of pig smooth muscles.

The Ca2+ binding proteins in the lumen of intracellular Ca2+ stores differ between muscle and non-muscle cells, indicating a specific role of these proteins in intracellular Ca2+ regulation. Since smooth muscle cells possess both muscle and non-muscle characteristics, we have studied the presence and the differential expression of the muscle-type Ca2+ binding proteins--calsequestrin, sarcalumenin, and the histidine-rich Ca2+ binding protein (HCP)--in several smooth muscle tissues from the pig. Western blot analysis showed that among the smooth muscles studied, the cardiac isoform of calsequestrin is expressed at the highest levels in the stomach. Calsequestrin was present at lower levels in ileum and trachea, whereas this protein was undetectable in aorta and main pulmonary artery. The total amount of calsequestrin in the stomach was estimated to be 20-30-times lower than in the pig heart. Whereas calsequestrin from pig presented the same apparent M(r) in sodium dodecyl sulphate polyacrylamide gels as the well characterized protein from rabbit, the apparent M(r) of both sarcalumenin and HCP was lower in pig than in rabbit. The presence of HCP was demonstrated in pig stomach and ileum, while sarcalumenin was detected only in the stomach. These results demonstrate further biochemical differences between smooth muscle cells of large blood vessels and those of the digestive tract. The present findings on the differential distribution of muscle-type Ca2+ binding proteins are discussed in relation to biochemical and functional differences between these smooth muscle cells.

Animals↗
PubMed1993

Regulation of glycolytically fueled Ca2+ uptake in smooth muscle plasmalemmal vesicles by phosphorylation.

A highly purified plasma membrane vesicular preparation from porcine antrum had an endogenous protein kinase activity with substrates of molecular weights of 11, 15, 20.5, 25, 35, 44, 155, and 230 x 10(3). Phosphorylation of the plasma membranes by the endogenous protein kinase activity resulted in a stimulation of initial rates of Ca2+ uptake into inside-out vesicles, which was associated with an increase in the maximum velocity of the Ca2+ pump with no apparent changes in the half-maximal effective concentration for calcium. Because we have previously reported that a membrane-associated glycolytic system may preferentially provide ATP to fuel the Ca2+ pump (9), we examined the effects of phosphorylation on Ca2+ uptake when glycolysis was the sole source of ATP for the pump. We found that the stimulation of Ca2+ uptake by phosphorylation was more pronounced when Ca2+ uptake was supported by glycolysis rather than 2 mM ATP. When ATP was added at a level similar to that produced by endogenous glycolysis, the stimulation of Ca2+ uptake by phosphorylation was comparable to when glycolysis supported the Ca2+ pump. Our observations suggest that the dynamic range (up to threefold) for regulation of the plasmalemmal Ca2+ pump by phosphorylation is considerably larger than previously reported and thus likely to be of physiological significance.

Adenosine Triphosphate↗
PubMed1993

Distribution of Ca(2+)-modulating proteins in sarcoplasmic reticulum membranes after denervation.

The early response to the loss of motor innervation to the muscle is connected with an altered Ca(2+)-homeostasis. Our study, based on Western blotting, indicates that denervation influenced expression of some sarcoplasmic Ca(2+)-modulating proteins. Evidence has been brought for an increase of the level of calsequestrin and of the putative ryanodine receptor paralleled with a slight decrease of the total amount of Ca(2+)-pump protein. The expression of unchanged Ca(2+)-pump isoform and unaltered quantities of other non-junctional Ca(2+)-binding proteins support the hypothesis that changed cellular Ca2+ homeostasis include also an alteration of Ca(2+)-modulating systems, mainly from the junctional region of sarcoplasmic membranes.

Animals↗
PubMed1992

Ca(2+)-transport ATPases and their regulation in muscle and brain.

Eukaryotic cells express one or more isoforms of a sarco(endo)plasmic reticulum (SERCA) and of a plasma membrane (PMCA) Ca2+ pump. Both the SERCA and PMCA gene transcripts are subject to alternative processing in a differentiation stage-dependent and tissue-dependent manner. The Ca2+ pump isoforms thus generated may present different functional properties. This is exemplified by the SERCA2a and SERCA2b isoforms which differ in their Ca2+ sensitivity. Analysis of the cDNA structures for PMCA1 predicts protein isoforms with variant calmodulin- and phospholipid-binding domains. A comparative study of the tissue-specific mechanisms governing SERCA-PMCA transcript processing and a more detailed study of the functional implication of the PMCA pumps isoform diversity will be challenging subjects for future studies.

Alternative Splicing↗
PubMed1992

Stimulation of the catalytic cycle of the Ca2+ pump of porcine plasma-membranes by negatively charged phospholipids.

The (Ca(2+)+Mg2+)-ATPase of the plasma membrane is activated by negatively charged phospholipids. The mechanism of this activation was investigated by studying the effect of negatively charged phospholipids on the steady-state phosphointermediate level and on the p-nitrophenylphosphatase activity. Both parameters were differentially affected by different acidic phospholipids. The level of phosphoprotein intermediate was not affected by phosphatidylserine (20% of total phospholipid), but it was increased by 60% by phosphatidylinositol 4-phosphate. Phosphatidylserine increased the p-nitrophenylphosphatase activity, whereas phosphatidylinositol 4-phosphate had no significant effect. It is suggested that phosphatidylinositol 4-phosphate mainly affects a reaction step which leads to accelerated formation of the phosphointermediate, whereas the action of phosphatidylserine would affect two reaction steps, one upstream and one downstream of the phosphointermediate.

4-Nitrophenylphosphatase↗
PubMed1992

The Ca(2+)-transport ATPases from the plasma membrane.

The initial studies on the plasma membrane (PM) Ca(2+)-transport ATPases were made in the erythrocyte, a structure that can not be taken as representing a typical eukaryotic cell. In other cell types however, the study of the PM Ca(2+)-transport ATPase is complicated by the simultaneous expression of related Ca(2+)-pumps in intracellular stores. Whereas there are as yet no known specific inhibitors for the PM Ca(2+)-transport ATPase, a number of selective inhibitors for the endo(sarco)plasmic reticulum Ca2+ pumps have been described: thapsigargin, cyclopiazonic acid and 2,5-di-(tert-butyl)-1,4-benzohydroquinone. With the recent introduction of the molecular biological approach, it became quickly obvious that a family of at least 5 different PM Ca(2+)-transport ATPase genes govern the tissue-dependent expression of PM Ca2+ pumps. Moreover alternative splicing of the primary gene transcripts was found to further enhance the number of pump variants. The PM Ca(2+)-transport ATPase are subject to modulatory control by calmodulin, by acidic phospholipids, and by the known families of protein kinases. Each of the ensuing effects are mutually related and interdependent. The wide variety PM Ca2+ pump isoforms and their regulation by such an intricate modulatory network allows the distinct tissues to adapt most adequately to the prevailing tissue and stimulus specific requirements.

Animals↗
PubMed1992

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

Adenosine Triphosphate↗
PubMed1992

Effects of dimethyl sulfoxide and polycationic neomycin on stimulation of purified plasma membrane Ca(2+)-pump by negatively charged phospholipids.

At least two reaction steps are involved in the activation of purified plasma membrane Ca(2+)-transport ATPase by negatively charged phospholipids depending on the type of phospholipids (Lehotský et al. 1992). The effect of negatively charged phospholipids on Ca(2+)-stimulated ATPase (cycling activity) was compared with that of p-nitrophenylphosphatase (E2-form activity) catalyzed by Ca(2+)-pump. PIP like PS, activated Ca(2+)-ATPase activity by modifying ATP activation curve with increasing Vmax of the high affinity site. Ca(2+)-ATPase activity reconstituted in PC was stimulated by DMSO(10%) by a factor of 1.36. The activity stimulation by DMSO was only weak in PS and activity was inhibited in PIP. Also, phosphatase activity catalyzed by Ca(2+)-pump was strongly stimulated by DMSO and was differentially affected by phospholipid head group. Positively charged neomycin (5 mmol/l) had no effect on Ca(2+)-ATPase activity reactivated in PC or PS, but the stimulatory action of PIP was suppressed. Relative stimulation of phosphatase activity by PS was not influenced. Both hydrolytic activities catalyzed by Ca(2+)-transport ATPase were differentially affected by organic solvents and polycations with respect to the kind of the phospholipid.

Animals↗
PubMed1991

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

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

Animals↗
PubMed1991

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

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

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

Animals↗
PubMed1991

[The Ca(2+)-transport ATPases of smooth muscle].

The cytoplasmic Ca2+ concentration plays a central role in the contraction of smooth muscle cells. The concentration of cytoplasmic Ca2+ is determined for an important part by the operation of Ca(2+)-transport ATPases which extrude Ca2+ from the cell or accumulate the ion in the endoplasmic reticulum. The present work concerns the characterization of the Ca(2+)-transport ATPases of smooth muscle by biochemical, immunochemical and recombinant DNA techniques. This study also includes the investigation of the regulation of the Ca(2+)-transport ATPases and of the expression of associated Ca(2+)-binding proteins. Methods were developed for the purification of endoplasmic reticulum and plasma membranes from smooth-muscle cells. From the study of the phosphorylated transport intermediates and the proteolytic breakdown products, and by using polyclonal and monoclonal antibodies we could conclude that two different Ca(2+)-transport ATPases are expressed in smooth-muscle cells. Of each of these types of Ca(2+)-transport ATPases different isoforms exist. These isoforms were further characterized at the cDNA level and by generating isoform-specific antibodies. One isoform of the plasma-membrane Ca(2+)-pump and two different organellar-type Ca(2+)-pumps have been cloned and sequenced. In smooth-muscle cells, the primary RNA transcripts of the gene of the SERCA2 Ca(2+)-transport ATPase are alternatively processed in three different ways. In neural tissues even a fourth mode of splicing occurs. These different splice modes can be explained by the analysis of the exon/intron structure of the SERCA2 gene. The regulation of the alternative RNA splicing was studied on the stable muscle-cell line BC3H1 during induced myogenic differentiation. From this study we could conclude that the mechanisms responsible for active Ca(2+)-transport in smooth-muscle cells partially resemble those of non-muscle cells, and partially resemble the corresponding system in cardiac cells, but not those in skeletal muscle. A similar conclusion was reached concerning the regulation of the Ca(2+)-transport ATPase of the endoplasmic reticulum via the phosphorylation of phospholamban, and for the expression of the Ca(2+)-binding protein calsequestrin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
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

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

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

Albumins↗