Plasma Membrane Ca2+-ATPase Activity Research Articles

PMCA activity and membrane tubulin affect deformability of erythrocytes from normal and hypertensive human subjects

Our previous studies demonstrated formation of a complex between acetylated tubulin and brain plasma membrane Ca2+-ATPase (PMCA), and the effect of the lipid environment on structure of this complex and on PMCA activity. Deformability of erythrocytes from hypertensive human subjects was reduced by an increase in membrane tubulin content. In the present study, we examined the regulation of PMCA activity by tubulin in normotensive and hypertensive erythrocytes, and the effect of exogenously added diacylglycerol (DAG) and phosphatidic acid (PA) on erythrocyte deformability. Some of the key findings were that: (i) PMCA was associated with tubulin in normotensive and hypertensive erythrocytes, (ii) PMCA enzyme activity was directly correlated with erythrocyte deformability, and (iii) when tubulin was present in the erythrocyte membrane, treatment with DAG or PA led to increased deformability and associated PMCA activity. Taken together, our findings indicate that PMCA activity is involved in deformability of both normotensive and hypertensive erythrocytes. This rheological property of erythrocytes is affected by acetylated tubulin and its lipid environment because both regulate PMCA activity.

SERCA and PMCA pumps contribute to the deregulation of Ca2 + homeostasis in human CF epithelial cells

Cystic Fibrosis (CF) disease is caused by mutations in the CFTR gene (CF transmembrane conductance regulator). F508 deletion is the most represented mutation, and F508del-CFTR is absent of plasma membrane and accumulates into the endoplasmic reticulum (ER) compartment. Using specific Ca2+ genetics cameleon probes, we showed in the human bronchial CF epithelial cell line CFBE that ER Ca2+ concentration was strongly increased compared to non-CF (16HBE) cells, and normalized by the F508del-CFTR corrector agent, VX-809. We also showed that ER F508del-CFTR retention increases SERCA (Sarcoplasmic/Reticulum Ca2+ ATPase) pump activity whereas PMCA (Plasma Membrane Ca2+ ATPase) activities were reduced in these CF cells compared to corrected CF cells (VX-809) and non-CF cells. We are showing for the first time CFTR/SERCA and CFTR/PMCA interactions that are modulated in CF cells and could explain part of Ca2+ homeostasis deregulation due to mislocalization of F508del-CFTR. Using ER or mitochondria genetics Ca2+ probes, we are showing that ER Ca2+ content, mitochondrial Ca2+ uptake, SERCA and PMCA pump, activities are strongly affected by the localization of F508del-CFTR protein.

Defining Post as a Modulator of STIM1 Function during T Cell Activation

Upon engagement of the T cell receptor, InsP3 is produced, triggering the release of Ca2+ from ER Ca2+ stores. The resulting ER Ca2+ store depletion is sensed by STIM1, which relocalizes to ER-plasma membrane junctions to activate Ca2+ channels and inhibit Plasma Membrane Ca2+/ATPase (PMCA)-mediated Ca2+ extrusion, thereby enabling sustained increases in cytosolic Ca2+ that drive the process of T cell activation. Partner of STIM1 (POST) is a recently identified STIM1 adaptor protein with multiple binding partners, including STIM1, PMCA and several other transporters, pumps and exchangers. POST is a multi-pass transmembrane protein found in the ER and PM predicted to have 10 transmembrane domains. To assess this predicted topology, we used fluorescence protease protection assays, finding that POST is actually a 9 transmembrane-containing protein with a cytosolic N-terminus and a luminal/extracellular C-terminus. To assess the dynamics of STIM1/POST-mediated control of PMCA activity, we utilized colocalization and FRET. Upon T cell activation, POST migrated and co-localized with both STIM1 and PMCA4 to the immunological synapse. Interestingly, FRET between POST and both proteins was observed, yet FRET between STIM1 and PMCA4 could not be detected. Finally, POST knockdown inhibited activation-dependent inhibition of PMCA4-mediated Ca2+ clearance. These studies provide new insight into the topology and dynamics of POST/STIM1/PMCA4 interactions during T cell activation. Given its numerous targets, further investigations into POST function may reveal additional roles for POST in the T cell activation process.

Presynaptic Control of Glycine Transporter 2 (GlyT2) by Physical and Functional Association with Plasma Membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ Exchanger (NCX)

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca(2+)-ATPase (PMCA) isoforms 2 and 3, and Na(+)/Ca(2+)-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2-3·NCX complex would help Na(+)/K(+)-ATPase in controlling local Na(+) increases derived from GlyT2 activity after neurotransmitter release.

The G Protein‐Coupled Estrogen Receptor 1 Regulates Endothelial Ca2+ efflux via the plasma membrane Ca2+‐ATPase. (LB568)

The G protein‐coupled estrogen receptor 1 (GPER) has been demonstrated to have a vast array of cardiovascular effects. Ca2+ extrusion via the plasma membrane Ca2+‐ATPase (PMCA) plays important roles in Ca2+ homeostasis. We tested the hypothesis that GPER is involved in the regulation of calcium efflux in the vasculature. In primary vascular endothelial cells, the specific GPER agonist G‐1 dose‐dependently inhibits PMCA activity. Knockdown of GPER in endothelial cells using antisense directed against GPER is associated with a ~ 40% enhancement of PMCA activity. Heterologous expression of human GPER in fusion with the fluorescent protein DsRed2 in HEK 293 cells results in a 38% reduction of PMCA activity. Co‐IP experiments show that PMCA and GPER form a complex during Ca2+ signals triggered by the SERCA pump inhibitor thapsigargin in endothelial cells. In addition, the fusion proteins PMCA‐DsRed2 colocalizes with GPER‐ECFP heterologously expressed in HEK 293 cells. These data suggest that GPER regulates Ca2+ efflux in endothelial cells by interacting with PMCA.Grant Funding Source: National Institutes of Health

Effects of gangliosides on the activity of the plasma membrane Ca2 +-ATPase

Control of intracellular calcium concentrations ([Ca2+]i) is essential for neuronal function, and the plasma membrane Ca2+-ATPase (PMCA) is crucial for the maintenance of low [Ca2+]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca2+ homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca2+ transporter.

Multifaceted regulation of Ca2+ efflux via the plasma membrane Ca2+‐ATPase by 17β‐estradiol in the vascular endothelium

Many of the beneficial effects of estrogen in the vasculature involve Ca2+‐dependent activities. Ca2+ efflux via the plasma membrane Ca2+‐ATPase (PMCA) represents a major component of the Ca2+ signaling machinery in cells. We observed in vascular endothelial cells that chronic treatment with 17β‐estradiol dose‐dependently decreases PMCA activity without affecting PMCA expression levels. Treatment with 17β‐estradiol promotes tyrosine phosphorylation at both the 135‐kDa (PMCA) and 60‐kDa levels on PMCA immunoprecipitate. We identified this 60‐kDa protein to be a glycosylated form of the novel G protein‐coupled estrogen receptor 1 (GPER). Inhibition of Src kinase activity suppresses estrogen‐induced tyrosine phosphorylation on PMCA and GPER. Functionally, Src inhibition not only restores PMCA activity in cells treated with 17β‐estradiol, but increases it by 50%. Consistent with the 50% increase in PMCA activity upon Src inhibition in estrogen‐treated cells, the interaction between PMCA and calmodulin is increased by 50%, an effect that occurs independently of Src kinase activity. These results indicate that estrogen inhibits PMCA activity by promoting Src‐dependent tyrosine phosphorylation and interaction with GPER, effects that mask the stimulatory effect of enhanced CaM binding.

Regulation of presynaptic calcium in a mammalian synaptic terminal

Ca(2+) signaling in synaptic terminals plays a critical role in neurotransmitter release and short-term synaptic plasticity. In the present study, we examined the role of synaptic Ca(2+) handling mechanisms in the synaptic terminals of mammalian rod bipolar cells, neurons that play a pivotal role in the high-sensitivity vision pathway. We found that mitochondria sequester Ca(2+) under conditions of high Ca(2+) load, maintaining intraterminal Ca(2+) near resting levels. Indeed, the effect of the mitochondria was so powerful that the ability to clamp intraterminal Ca(2+) with a somatically positioned whole cell patch pipette was compromised. The plasma membrane Ca(2+)-ATPase (PMCA), but not the Na(+)/Ca(2+) exchanger (NCX) or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), was an important regulator of resting Ca(2+). Furthermore, PMCA activity, but not NCX or SERCA activity, was essential for the recovery of Ca(2+) levels following depolarization-evoked Ca(2+) entry. Loss of PMCA function was also associated with impaired restoration of membrane surface area following depolarization-evoked exocytosis. Given its roles in the regulation of intraterminal Ca(2+) at rest and after a stimulus-evoked Ca(2+) rise, the PMCA is poised to modulate luminance coding and adaptation to background illumination in the mammalian rod bipolar cell.

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-ATPase Isoform 4b (PMCA4b)

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.

Calmodulin antagonizes amyloid-β peptides-mediated inhibition of brain plasma membrane Ca2+-ATPase

The synaptosomal plasma membrane Ca2+-ATPase (PMCA) plays an essential role in regulating intracellular Ca2+ concentration in brain. We have recently found that PMCA is the only Ca2+ pump in brain which is inhibited by amyloid-β peptide (Aβ), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aβ. Results from kinetic assays indicate that Aβ aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aβ could be blocked by pretreating the purified protein with Ca2+-calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aβ. Dot-overlay experiments indicated a physical association of Aβ with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aβ by burying exposed sites on PMCA, making them inaccessible to Aβ, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca2+ dysregulation by PMCA inhibition induced by Aβ.

Calmodulin Prevents the Inhibitory Effect of Neurotoxic β-Amyloid Peptide on Synaptosomal Plasma Membrane Ca2+-ATPase

Dysregulation of cellular Ca2+ homeostasis and β-amyloid peptide (Aβ) have been linked to cell dysfunction and death in neurodegenerative disorders, such as Alzheimer's disease (AD). We have recently reported that Ca2+-dependence of plasma membrane Ca2+-ATPase (PMCA) activity is altered in AD-affected brains, and in pig brain in the presence of Aβ fragment (25-35) and full-length Aβ1-40 or 1-42 peptides, being inhibited by those peptides. In the present work, performed with Aβ1-42, we found that Ca2+-calmodulin, the main endogenous activator of PMCA, was able to block the inhibitory effect of Aβ on PMCA activity. Besides, the activity of truncated PMCA mutant lacking the C-terminal domain involved in calmodulin binding was not inhibited by Aβ. Dot blot overlay assays further confirmed an interaction of Aβ peptide with both PMCA and calmodulin. Thus, calmodulin may prevent the inhibitory effect of Aβ on PMCA activity by a mechanism that involves its direct binding to the protein, changing PMCA conformation and thus occluding peptide binding to PMCA, and/or its direct binding to the peptide. These finding may reveal a protection role of calmodulin against the Aβ-induced neurotoxicity in AD.This work was supported by Ministerio de Ciencia e Innovacion (MICINN, project BFU2008-00182), Fundacion Marcelino Botin, Junta de Extremadura and FEDER.

Regulation of plasma membrane Ca2 +-ATPase activity by acetylated tubulin: Influence of the lipid environment

We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca2+-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca2+ transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.

Effects of low-level copper exposure on Ca2+-ATPase and carbonic anhydrase in the freshwater bivalve Anodonta anatina

Calcium (Ca2+) in body fluids and cellular compartments is maintained within narrow concentration ranges by various regulatory processes and enzymes including plasma membrane Ca2+-ATPase (PMCA) and carbonic anhydrase (CA). Ca2+ has key regulatory functions in cell physiology and biomineralization, and therefore any disturbance of its homeostasis may have severe pathobiochemical consequences. Some bivalent heavy metals may interfere with Ca2+-homeostasis, one of them being copper. Therefore, in the freshwater bivalve Anodonta anatina the effects of Cu(II) at environmentally relevant concentrations were assessed on PMCA activities in kidney, gills, and digestive gland, and on CA in gills and mantle. During the first day of Cu(II) exposure at 0.35 µmol L−1, PMCA in the kidney became strongly inhibited, recovering during the following days of exposure; at 0.64 µmol L−1, the inhibition persisted for more than 15 days. Recovery of the enzyme function suggests that metal-detoxification is induced. In the digestive gland, inhibition was also observed though non-significantly. CA activities in mantle and gills were not significantly affected, except in the gills upon 15 days of exposure at 0.35 µmol L−1 Cu(II). The high accumulation of Cu(II) in the kidney and the ensuing sensitivity of PMCA in this organ may entail disturbance of the renal electrolyte- and osmo-regulations and Ca2+ re-uptake in A. anatina. This suggests that disruption of Ca2+-homeostasis by Cu(II) may be involved in the general decline of freshwater mussel populations.

Mechanisms of estrogen's effects on calcium signaling in vascular endothelial cells

17β‐estradiol (E2) exerts many cardioprotective effects via actions on endothelial cells (ECs). It has been observed that E2 increases Ca2+ signals produced in ECs in response to agonists. However, the underlying mechanisms are unclear. Here we have examined the main components of Ca2+ signaling in primary ECs in response to long‐term exposure to physiological concentrations of E2. E2 treatment for 48 hrs did not affect thapsigargin‐induced internal Ca2+ store release or store‐operated Ca2+ entry. However, we observed a 35% reduction in the activity of the calmodulin (CaM)‐dependent plasma membrane Ca2+‐ATPase (PMCA), the main Ca2+ efflux mechanism in ECs. In contrast, calmodulin (CaM) binding to the PMCA is increased by 50%, while total PMCA expression is not affected. Inhibition of pp60src‐mediated tyrosine phosphorylation of PMCA reveals the effects of E2‐induced increase in CaM binding to the PMCA, demonstrating a 30% increase in PMCA activity in E2‐treated cells. Our results suggest that via pp60src‐dependent phosphorylation of PMCA, estrogen decreases Ca2+ efflux, which provides an explanation for the enhanced Ca2+ signals in ECs exposed to E2.

A new Mathematical Formulation of the Plasma Membrane Ca 2+-Atpase (PMCA) Provides Stability to Simulated Ca 2+ Signaling in Human T Cells

In lymphocytes Ca2+ signals are essential for diverse cellular functions. After antigen binds to the T cell receptor a series of reactions are initiated that generate IP3 and culminate in an increase in cytosolic Ca2+. Mechanisms that remove Ca2+ also exert an important influence on the net Ca2+ level. The SERCA pump resequesters Ca2+ into the ER and the PMCA transports Ca2+ to the extracellular side. Two key features of the PMCA are its stimulation by Ca2+-calmodulin and by PKA-dependent phosphorylation (Bers, 2001) but in most of the mathematical models this pump is represented with a simple Michaelis-Menten formulation due to its small contribution to the overall Ca2+ fluxes. This is not the case in T cells, Bautista et al (2002) showed that this pump is the primary means of Ca2+ extrusion in T cells and its activity is modulated by Ca2+ enabling the cell to adapt to higher Ca2+ values during T cell activation. Additionally once [Ca2+]i returned to baseline levels the PMCA recovered slowly with a time constant of ∼ 4 min providing a “memory” of previous [Ca2+]i. We created a first order kinetics model of the PMCA that mimics its activation and recovery as function of [Ca2+]i (Vmax increased from 30 to 60 nM s−1, Km decreased from 500 to 400 nM when fully activated) and incorporated into a mathematical description of Ca2+ signaling in T cells (LymphoLAB). Our results indicate that modulation of the PMCA activity improves the stability of Ca2+ signaling by adjusting the pump rate to Ca2+ influx even at high [Ca2+]i levels (preventing a harmful Ca2+ overload). Moreover the delay in modulation permits small Ca2+ fluxes to increase transiently enhancing Ca2+ signaling dynamics.

Regulatory phosphorylation of FXYD2 by PKC and cross interactions between FXYD2, plasmalemmal Ca-ATPase and Na,K-ATPase

FXYD2 is a regulatory peptide associated with the α-subunit of the kidney Na,K-ATPase. FXYD2 can be phosphorylated by PKA, and its phosphorylation activates Na,K-ATPase. Here we show that FXYD2 is phosphorylated by PKC (PKC-FXYD2-P), by PKA (PKA-FXYD2-P) or by PKA and PKC simultaneously (FXYD2-P 2) modulating both the erythrocyte Na,K-ATPase and the plasma membrane Ca 2+-ATPase (PMCA). In erythrocyte ghosts, the addition of PKA-FXYD2-P activated Na,K-ATPase by 80%, while non-phosphorylated FXYD2 (np) activated only 55%. The addition of np FXYD2 did not affect PMCA basal activity, but FXYD2-P 2 increased the basal PMCA activity by up to 200%. Calmodulin-activated PMCA activity was increased by np FXYD2 (3-fold) or FXYD2-P 2 (2.5-fold). However, PKC-FXYD2-P increased PMCA activity only by 50%. In contrast, when PMCA was treated with PKA-FXYD2-P, the ATPase activity was inhibited by 50%. The effect of all forms of FXYD2-P on calcium uptake from PMCA resembled the pattern observed in ATP hydrolysis. Our results suggest that the FXYD2 anchoring site could be conserved among the P-ATPase family permitting cross regulation. The effects of FXYD2 on calcium uptake and calcium-stimulated ATP hydrolysis suggest a novel role for FXYD2 on PMCA.

Calmodulin Wraps around Its Binding Domain in the Plasma Membrane Ca2+ Pump Anchored by a Novel 18-1 Motif

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.C20 complex with this holoCaM.C28 complex supports the idea that the initial binding step is represented by (holoCaM.C20) and the final bound complex by (holoCaM.C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an alpha(D)-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.

Can Endocrine disrupters interfere with Ca2+ homeostasis in invertebrate cells?

A wide range of environmental chemicals have been shown to alter the endocrine system of both wildlife and humans. There is increasing evidence that many of these endocrine disruptors (EDs), in particular estrogenic chemicals, can rapidly affect cellular homeostasis and signaling in mammalian Ca2+ systems. In this work, in vitro and in vivo data are summarised on the effects of different compounds known or suspected as EDs on homeostasis in Ca2+ marine invertebrate, the blue mussel Mytilus spp. Both synthetic estrogens and different EDs (DES, BPA, NP, PCB congeners, etc.) rapidly increased sytosolic [Ca2+] in mussel hemosytes, as evaluated by FURA2 single cell fluorescence microscopy. The observed [Ca2+] increase was unaffected by the antiestrogen Tamoxifen and was due to either increased influx or release from Ca2+ intracellular stores, depending on the compound. Moreover, different ED,s including the brominated flame retardant TBBPA (tetrabromo bisphenol A) induced a dose-dependent inhibition of the plasma membrane Ca2+ -ATPase (PMCA) activity from mussel gills in vitro, this supporting a direct effect on membrane pumps. The in vitro effects of EDs were observed at concentrations generally higher than those of E2. However, in vivo, mussel exposure to environmetal concentrations of Bisphenol A (BPA) and of the polybrominated diphenyl ether TBDE-47 resulted in large inhibition of PMCA activity in the digestive gland. The results indicate that, in invertebrate like in mammalian systems, interference with Ca2+ homeostasis may represent a significant mode of action of a variety of EDs.

Impairment of PMCA Activity by Amyloid β-Peptide in Membranes from Alzheimer's Disease-Affected Brain and from Other Model Systems

High-affinity Ca2+-transport ATPases are key components in the machinery of cytosolic Ca2+ regulation. We have found that the Ca2+-dependence of plasma membrane Ca2+-ATPase (PMCA) activity is constant between pCa values of 6.5 and 4.5 in membranes from Alzheimer's disease (AD) affected-brain whereas in normal brain the activity increases from pCa 6.5 to 5.5 and then decreases from 5.5 to 4.5. However, the Ca2+-dependencies of sarco(endo)plasmic- (SERCA) and secretory pathway- (SPCA) Ca2+-ATPases activity are similar in AD and normal brains.

Cytochemical localization and quantification of plasma membrane Ca2+-ATPase activity in mollusc digestive gland cells.

A cytochemical method allowing the localization and quantification of plasma membrane Ca2+-ATPase (PMCA) in frozen sections obtained from digestive gland cells of Mytilus galloprovincialis, Tapes tapes and Chamelea gallina, is presented. The method utilizes lead as a trapping agent of PO4(2-) ions released by Ca2+-ATPase activity. The amount of lead sulphide precipitate proportionally related to PMCA activity was quantified by a light microscopy digital imaging analysis system. The optimal assay conditions of Ca2+-ATPase activity evaluated at pH 7.4 were: 200 microM free Ca2+, 200 mM KCl, 2 mM ATP, and under such analysis conditions the enzyme showed a linear trend up to 60 min (at 20 degrees C). The PMCA activity was substrate specific: ADP was utilized only at a low rate (24% with respect to an equimolar ATP concentration), while glucose-6-phosphate and beta-glycerophosphate were poorly hydrolyzed. The enzyme activity was strongly inhibited by sodium ortho-vanadate. Our detection of a Ca2-ATPase activity at nanomolar concentrations of free Ca2+ suggests that we have identified a plasma membrane Ca2-ATPase involved in Ca2+ homeostasis. The Ca2+-ATPase was found to be localized in the basal part of the plasma membrane in the digestive gland cells of Mytilus galloprovincialis and Tapes tapes, but in the apical plasma membrane of Chamelea gallina. The possible implications of the different cellular distributions of PMCA activity is discussed.