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F Wuytack

Publications and source records attributed to F Wuytack.

At least 73 records · Page 4Linked to original sources
PubMed1993

The sarco(endo)plasmic reticulum Ca(2+)-ATPase mRNA isoform, SERCA 3, is expressed in endothelial and epithelial cells in various organs.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase mRNA isoform, SERCA 3, was previously shown to be expressed in a great variety of muscle and non-muscle tissues [(1989) J. Biol. Chem. 264, 18568] but its cellular localization within these organs was unknown. We have used in situ hybridization and RNase protection techniques to demonstrate that SERCA 3 mRNA is expressed in specific cell types, namely the endothelial and epithelial cells.

Animals↗
PubMed1993

Alternative processing of the gene transcripts encoding a plasma-membrane and a sarco/endoplasmic reticulum Ca2+ pump during differentiation of BC3H1 muscle cells.

The effect of differentiation on the RNA processing of the PMCA1 gene encoding a plasma-membrane Ca2+ pump and of the SERCA2 gene encoding a sarco(endo)plasmic reticulum Ca2+ pump was studied in the myogenic BC3H1 cell line. A differentiation stage-dependent change in the RNA processing was observed for both genes. Proliferating myoblasts only expressed the non-muscle mRNA isoform whereas in differentiated cells muscle-specific processing became activated. The switch to muscle-specific RNA processing for both the PMCA1 and SERCA2 genes was found to be linked to the myogenic conversion of the BC3H1 cells. Our results furthermore indicated that the myogenic RNA processing could be reversed for both types of Ca2+ pumps since the expression of the PMCA1 and SERCA2 muscle-specific messengers was rapidly down-regulated by cycloheximide treatment.

Alternative Splicing↗
PubMed1993

Differential distribution of the alternative forms of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, SERCA2b and SERCA2a, in the avian brain.

Cellular distribution of the two forms of SERCA2 was examined in adult chicken brain. Four regions of the brain were analyzed with three immunological reagents: a monoclonal antibody that recognizes both forms of SERCA2, and two antisera which are specific for the two alternative forms, SERCA2b or SERCA2a. Cerebellar Purkinje cells express predominantly SERCA2b but also low levels of SERCA2a, as has been reported for mammals. The nucleus isthmo-opticus, nucleus magnocellularis cochlearis, and nucleus laminaris all express high levels of SERCA2 but with different ratios of SERCA2b and SERCA2a. These immunohistochemical results were supported by in situ hybridization analysis. Therefore, it appears that regions within the brain have specific requirements for the two forms of SERCA2. This suggests functional significance for the alternative forms SERCA2b and SERCA2a, and possible functions are discussed.

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↗
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

Simultaneous presence of two distinct endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization by radio-immunoblotting and inhibition by 2,5-di-(t-butyl)-1,4-benzohydroquinone.

Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to characterize the sarcoendoplasmic-reticulum Ca2+ ATPases in a variety of human cell types. In platelets, several megakaryoblastoid and lymphoblastoid cell lines two distinct autophosphorylated forms of these ATPases with molecular mass of 100 and 97 kDa could be observed, whereas in several other cell types the 97 kDa form was absent. On immunoblots the 97 kDa species was specifically recognized by an inhibitory monoclonal antibody raised against the Ca2+ pump of platelet internal membranes, yielded on trypsinolysis a major fragment of 80 kDa, exhibited a distinct electrophoretic migration pattern as compared with the skeletal-, cardiac- and smooth-muscle Ca2+ pumps, and its autophosphorylation was strongly inhibited by the Ca(2+)-mobilizing agent 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ). The 100 kDa species reacted with an antibody specific for the cardiac- and smooth-muscle Ca2+ pumps, yielded on trypsinolysis fragments of 55 and 35 kDa, and its autophosphorylation was much less sensitive to tBHQ inhibition. These findings indicate the simultaneous presence of two different endoplasmic-reticulum Ca2+ pumps in a variety of human cell types, and may explain the previously observed differences in the Ca(2+)-handling characteristics of different intracellular Ca2+ pools and cell types.

Antibodies, Monoclonal↗
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

Human platelets express the SERCA2-b isoform of Ca(2+)-transport ATPase.

Previous biochemical studies suggested that the human platelet Ca2+ATPase system may be cell-specific. To test this hypothesis, we first undertook the molecular cloning of Ca2+ATPase from human erythroleukaemia (HEL) cells, because this human cell line exhibits megakaryocytic features and expresses a Ca2+ATPase that cross-reacts with platelet Ca(2+)-ATPase. For this cloning, an HEL-cell cDNA library was screened with a rat cardiac Ca2+ATPase cDNA probe. The insert of the longest clone isolated was 3.9 kb and its sequence displayed a 100% identity with that of the non-muscle human Ca2+ATPase 2-b isoform, termed SERCA2-b (sarco-endoplasmic-reticulum Ca2+ATPase). The 3.9 kb cDNA covered a subtotal coding region and part of the 3' non-coding end of the SERCA2-b mRNA. It cross-hybridized with the 4 kb transcript species of cardiac SERCA2-a and with non-muscle SERCA2-b mRNAs, but not with fast-skeletal-muscle SERCA1 mRNA. We next confirmed that SERCA2-b was a component of the platelet Ca2+ATPase system because (1) the platelet clones isolated from a platelet cDNA library exhibited a 100% homology with HEL-cell cDNA; (2) SERCA2-b mRNA was amplified by PCR on total platelet RNA and (3) platelet Ca2+ATPase cross-reacted with a polyclonal SERCA2-b-specific antiserum. Platelets therefore contain a Ca2+ATPase definitely identified as the SERCA2-b isoform of Ca2+ATPase, thus eliminating the possibility that they only contain a single specific Ca2+ATPase.

Animals↗
PubMed1992

Regulation of sarco-endoplasmic reticulum Ca(2+)-ATPases during platelet-derived growth factor-induced smooth muscle cell proliferation.

The role of the sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA) in the regulation of cell proliferation by Ca2+ was investigated by testing the effect of platelet-derived growth factor (PDGF) on cultured pig aorta smooth muscle cells. For this purpose, the PDGF-mediated rise in the Ca2+ concentration was first examined for its ability to induce the formation of prostaglandins from the specific membrane enzyme, cyclooxygenase. In parallel experiments, similar conditions (10 ng/ml PDGF for 24 h) were used to investigate the smooth muscle cell membrane SERCA2 isoforms. Total SERCA2 activity rose by 472% as reflected by their specific formation of phosphorylated intermediate (E approximately P). This rise correlated with an increase in the amount of SERCA2 proteins (100 kDa) as shown by Western blotting. With isoform-specific anti-SERCA2-a and anti-SERCA2-b antibodies, we demonstrated that the increase in total SERCA2 proteins concerned the minor isoform SERCA2-a, which rose 10-fold, whereas SERCA2-b proteins were not affected. Lastly, Northern blotting using riboprobes showed that PDGF treatment increased the SERCA2-a mRNA species by 82%, and concomitantly decreased the SERCA2-b mRNA by 28%, as a result of isoform switching. We conclude that up-regulation of the SERCA2-type Ca(2+)-ATPases occurs in PDGF-treated smooth muscle cells, which suggests that this enzymatic system plays an essential part in cell proliferation.

6-Ketoprostaglandin F1 alpha↗
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↗
PubMed1991

Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles.

In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100- and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes.

Antibodies↗
PubMed1991

Isoform switching of the sarco(endo)plasmic reticulum Ca2+ pump during differentiation of BC3H1 myoblasts.

We have studied the expression of the gene 2 for the sarco(endo)plasmic reticulum Ca2+ pump (SERCA2) in BC3H1 cells. Myogenic differentiation not only activated the SERCA2 expression but it also induced an isoform switch. Undifferentiated myoblasts only expressed the SERCA2b isoform (non-muscle) whereas differentiated myocytes predominantly contained the SERCA2a isoform (cardiac/slow skeletal muscle). The isoform switch was documented by immunoblot analysis with isoform-specific antibodies. This observation was confirmed at the mRNA level by using antisense RNA probes specific for class 1 (SERCA2a) or class 2 (SERCA2b) messengers. The expression of the SERCA2a isoform after differentiation was accompanied by a decreased sensitivity of the Ca2+ uptake in permeabilized cells to the Ca2+ pump inhibitor thapsigargin.

Animals↗
PubMed1991

Characterization of the 3' end of the pig sarcoplasmic/endoplasmic-reticulum Ca2+ pump gene 2.

The gene 2 for the sarcoplasmic/endoplasmic-reticulum Ca2+ pump (SERCA2) is expressed both in muscle and non-muscle tissues and four distinct SERCA2 mRNAs which differ only in their 3' end have been described. In order to understand the differential 3' end processing of the SERCA2 transcripts, we have now cloned a 12.6 kb pig genomic clone which contains the 3' end of the pig SERCA2 gene. Part of this clone (7510 nucleotides) was sequenced and it was found to contain four constitutive exons followed by four optional exons which underlie the 3' end diversity of the SERCA2 mRNAs. The inclusion or the exclusion of these optional exons is explained by the presence of four optional processing sites: two polyadenylation sites and two optional donor splice sites.

Amino Acid Sequence↗
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↗