Impact of female obesity and assisted reproduction on uncomplicated pregnancies and healthy births: a study of 428 336 births in Flanders.

STUDY QUESTION: What is the impact of BMI on uncomplicated pregnancies and healthy births in women who did or did not have medically assisted reproduction (MAR, i.e. ART or hormonal stimulation without manipulation of eggs or embryos) in the Flanders region (Belgium)? SUMMARY ANSWER: Women with a higher BMI who use MAR are at the highest risk of pregnancy and birth complications. WHAT WE KNOW ALREADY: Medically assisted reproduction (MAR) is used increasingly worldwide and is associated with increased risk of adverse perinatal outcomes. Obesity is also increasing globally and obese women are more likely to seek MAR since obesity is associated with infertility. When obese women undergo MAR, the risk of adverse outcomes may be enhanced but it is not clear to what extent. STUDY DESIGN, SIZE, DURATION: We conducted a registry-based study using the data from the Study Centre for Perinatal epidemiology database for years 2009-2015, region of Flanders, Belgium. This included 428 336 women. PARTICIPANTS/MATERIALS, SETTING, METHODS: The average age was 30.0 years (SD 4.78), 194 061 (45.31%) were nulliparous, and 6.3% (n = 26 971) conceived with MAR. We examined the association of BMI and MAR with the following composite primary outcomes: 'uncomplicated pregnancy and birth' and 'healthy baby'. We conducted Poisson regression and adjusted for maternal age, parity, gestational weight gain, smoking and previous caesarean section. MAIN RESULTS AND THE ROLE OF CHANCE: In our study, 36.80% (n = 157 623) of women had an uncomplicated pregnancy and birth according to the definition used. The predicted probability of having an uncomplicated pregnancy and birth for women with a BMI of 25 kg/m2 who conceived spontaneously was 0.33 (0.32 to 0.35), while it was 0.28 (0.24 to 0.32) for women who used hormonal stimulation and 0.26 (0.22 to 0.29) for women who used IVF/ICSI. This probability reduced with increasing BMI category for both MAR and non-MAR users. For women with a BMI of 30 kg/m2, the predicted probability of having an uncomplicated pregnancy and birth was 0.28 (0.26 to 0.30) for women who conceived spontaneously, and 0.22 (0.16 to 0.29) and 0.20 (0.14 to 0.26) for women who used hormonal stimulation only or IVF/ICSI, respectively. The predicted probability of having a healthy baby for women with a BMI of 25 kg/m2 who conceived spontaneously was 0.92 (0.91 to 0.93), 0.89 (0.87 to 0.92) for women who used hormonal stimulation only and 0.85 (0.84 to 0.87) for women who used IVF/ICSI. LIMITATIONS, REASONS FOR CAUTION: The database did not include data on socio-economic status, pre-pregnancy morbidities and paternal BMI. Subsequently, we could not adjust for these factors in the analysis. WIDER IMPLICATIONS OF THE FINDINGS: Obese women who use MAR are at the highest risk of pregnancy and birth complications. This increase in interventions also has cost and resource implications which is relevant for funding policies. Weight loss interventions prior to MAR seem plausible but their (cost-) effectiveness needs urgent investigation. STUDY FUNDING/COMPETING INTEREST(S): F.W. received an Erasmus Plus training grant to visit A.B., L.A. and R.D. and conducted this study during this visit. The authors have no competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Diseases involving the Golgi calcium pump.

Secretory-pathway Ca2(+)-transport ATPases (SPCA) provide the Golgi apparatus with Ca2+ and Mn2+ needed for the normal functioning of this organelle. Loss of one functional copy of the human SPCA1 gene (ATP2C1) causes Hailey-Hailey disease, a rare skin disorder characterized by recurrent blisters and erosions in the flexural areas. Here, we will review the properties and functional role of the SPCAs. The relationship between Hailey-Hailey disease and its defective gene (ATP2C1) will be adressed as well.

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function.

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.

Replacement of the muscle-specific sarcoplasmic reticulum Ca(2+)-ATPase isoform SERCA2a by the nonmuscle SERCA2b homologue causes mild concentric hypertrophy and impairs contraction-relaxation of the heart.

The cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase gene (ATP2A2) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a(-/-) mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a(-/-) mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a(-/-) mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a approximately 20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a(-/-) animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved beta-adrenergic response. Ca(2+) uptake levels in SERCA2a(-/-) cardiac homogenates were reduced by approximately 50%. In isolated cells, relaxation and Ca(2+) removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.

Cytosolic Ca2+ signals depending on the functional state of the Golgi in HeLa cells.

The Golgi apparatus is, like the endoplasmic reticulum, an inositol-1,4,5-trisphosphate-sensitive Ca2+ store, but its role in setting up Ca2+ signals is not well understood. We have now measured histamine-induced Ca2+ signals in HeLa cells pretreated with brefeldin A, a fungal metabolite that leads to the fragmentation and subsequent disappearance of the Golgi apparatus by its reabsorption within the endoplasmic reticulum. Ca2+ responses in which the free cytoplasmic Ca2+ concentration returned to resting levels during the histamine stimulation (mainly baseline Ca2+ oscillations or a single Ca2+ peak) occurred more often in brefeldin A pretreated cells, resulting in a lower Ca2+ plateau in population measurements. The latencies before the onset of the Ca2+ signals were longer after brefeldin A pretreatment. These results suggest that the integrity of the Golgi apparatus contributes to the shaping of intracellular Ca2+ signals.

Demonstration of a (Ca2+ + Mg2+)-ATPase activity probably related to Ca2+ transport in the microsomal fraction of porcine coronary artery smooth muscle.

A (Ca2+ + Mg2+)-ATPase activity is demonstrated in the microsomal fraction of porcine coronary artery. The characteristics of the ATPase activity are compared with those of the Ca2+ transport, both measured in similar solutions. It is concluded that the (Ca2+ + Mg2+)-ATPase is related to the Ca2+ transport because: 1. Both transport and ATPase have similar low Km values for Ca2+ as well as comparable Hill coefficients. The Km values are respectively 0.34 +/- 0.03 microM [4] and 1.17 +/- 0.15 microM [6]. The Hill coefficients are n = 1.69 +/- 0.09 [4] and n = 1.23 +/- 0.17 [6]. 2. Ionophores A23187 and X537A stimulate (Ca2+ + Mg2+)-ATPase activity while they inhibit net Ca2+ accumulation by increasing the Ca2+ permeability of the membranes. 3. The V values for Ca2+ accumulation and for (Ca2+ + Mg2+)-ATPase are comparable.

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.

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.

Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin.

It is proposed that smooth-muscle endoplasmic reticulum contains calsequestrin and that this protein in smooth muscle resembles cardiac calsequestrin more than the skeletal-muscle form. This proposal is based on seven similarities between the smooth-muscle protein and cardiac calsequestrin. Proteins with an Mr of 55,000 can be extracted from the membranes of smooth muscle and of cardiac muscle using 100 mM Na2CO3. The protein from smooth muscle binds to phenyl-Sepharose in the absence of Ca2+ and is released by 10 mM CaCl2, as has been observed for cardiac calsequestrin. The protein from smooth muscle comigrates with the cardiac calsequestrin on Laemmli-type SDS-polyacrylamide gel electrophoresis. The protein of Mr 55,000 from smooth muscle and cardiac calsequestrin both strain blue with the carbocyanine dye Stains-all. Both proteins present similar one-dimensional Cleveland peptide maps although minor differences might exist. From an analysis of subcellular membranes separated by sucrose gradient centrifugation it is concluded that the protein with Mr 55,000 from the smooth muscle is confined to the endoplasmic reticulum, the same subcellular structure from which, in heart muscle, calsequestrin can be isolated. Antibodies raised against canine cardiac calsequestrin bind to a protein of similar Mr in smooth-muscle endoplasmic reticulum. In addition to the calsequestrin, three other extrinsic proteins with an Mr of 130,000, 100,000 and 63,000, stain blue with Stains-all and occur in the endoplasmic reticulum of smooth muscle.

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes.

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.

The calcium accumulation in a microsomal fraction from porcine coronary artery smooth muscle. A study of the heterogeneity of the fraction.

1. Microsomes prepared from the combined media and intima of pig coronary artery, take up Ca in an ATP-dependent way. This uptake is stimulated by oxalate. 2. Conditions have been determined to optimize the preparation of the microsomes in terms of their Ca accumulation activity. Careful homogenization of the tissue mince in 0.25 M sucrose by means of a Potter-Elvehjem homogenizer gives microsomal preparations with the highest specific activity for Ca accumulation. 3. Arguments are presented to support the hypothesis that, even in the absence of oxalate, Ca accumulation occurs into the lumen of the vesicles, and that these vesicles have a low Ca permeability. 4. Density gradient analysis shows that the microsomal fraction prepared from pig coronary artery media and intima is composed of vesicles that are heterogeneous in enzymatic composition. 5. Adenylate cyclase appears to be a predominantly plasma membrane-bound enzyme. Rotenone-insensitive NADH-cytochrome c reductase and choline phosphotransferase, two putative markers for internal membranes, give distinct banding patterns on on isopycnic centrifugation, indicating different intracellular localization. 6. There is a difference between the density gradient distribution pattern of Ca uptake measured in the presence or absence of oxalate. The latter coincides more closely with plasma membrane markers. The former resembles more the distribution of rotenone-insensitive NADH-cytochrome c reductase.

Demonstration of the phosphorylated intermediates of the Ca2+-transport ATPase in a microsomal fraction and in a (Ca2+ + Mg2+)-ATPase purified from smooth muscle by means of calmodulin affinity chromatography.

Ca2+ -dependent hydroxylamine-sensitive phosphorylated proteins can be demonstrated in a microsomal fraction of porcine antrum (stomach) smooth muscle and in a Ca2+ -transport ATPase ((Ca2+ + Mg2+)-ATPase) purified from this tissue by means of a calmodulin affinity technique. These phosphoproteins represent the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPases. In the (Ca2+ + Mg2+)-ATPase purified from smooth muscle the phosphorylated intermediate has an Mr of 130000 corresponding to the value found for erythrocyte (Ca2+ + Mg2+)-ATPase. In the smooth muscle microsomal fraction this 130 kDa phosphoprotein can also be seen, although its intensity is usually very low compared to a corresponding phosphorylation at Mr 100000. Including La3+ together with Ca2+ during phosphorylation of the microsomes increased selectively the steady state-level of the 130 kDa phosphoprotein over the value of the 100 kDa one. The 100 kDa Ca2+ -dependent phosphoprotein could either indicate the presence of a (Ca2+ + Mg2+)-ATPase of the same type of sarcoplasmic reticulum of skeletal muscle, or it could represent a proteolytic product of the 130 kDa phosphoprotein.

Calcium-induced phosphorylations and [125I]calmodulin binding in renal membrane preparations.

Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from the renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [125I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of 20 microM La2+ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.

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.

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.

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay.

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.

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.

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.

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.

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.