Plasma Membrane Ca2+-ATPase Activity Research Articles

Allyl Isothiocianate Induces Ca2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species Production in the Human Cerebrovascular Endothelial Cell Line hCMEC/D3.

Nitric oxide (NO) represents a crucial mediator to regulate cerebral blood flow (CBF) in the human brain both under basal conditions and in response to somatosensory stimulation. An increase in intracellular Ca2+ concentrations ([Ca2+]i) stimulates the endothelial NO synthase to produce NO in human cerebrovascular endothelial cells. Therefore, targeting the endothelial ion channel machinery could represent a promising strategy to rescue endothelial NO signalling in traumatic brain injury and neurodegenerative disorders. Allyl isothiocyanate (AITC), a major active constituent of cruciferous vegetables, was found to increase CBF in non-human preclinical models, but it is still unknown whether it stimulates NO release in human brain capillary endothelial cells. In the present investigation, we showed that AITC evoked a Ca2+-dependent NO release in the human cerebrovascular endothelial cell line, hCMEC/D3. The Ca2+ response to AITC was shaped by both intra- and extracellular Ca2+ sources, although it was insensitive to the pharmacological blockade of transient receptor potential ankyrin 1, which is regarded to be among the main molecular targets of AITC. In accord, AITC failed to induce transmembrane currents or to elicit membrane hyperpolarization, although NS309, a selective opener of the small- and intermediate-conductance Ca2+-activated K+ channels, induced a significant membrane hyperpolarization. The AITC-evoked Ca2+ signal was triggered by the production of cytosolic, but not mitochondrial, reactive oxygen species (ROS), and was supported by store-operated Ca2+ entry (SOCE). Conversely, the Ca2+ response to AITC did not require Ca2+ mobilization from the endoplasmic reticulum, lysosomes or mitochondria. However, pharmacological manipulation revealed that AITC-dependent ROS generation inhibited plasma membrane Ca2+-ATPase (PMCA) activity, thereby attenuating Ca2+ removal across the plasma membrane and resulting in a sustained increase in [Ca2+]i. In accord, the AITC-evoked NO release was driven by ROS generation and required ROS-dependent inhibition of PMCA activity. These data suggest that AITC could be exploited to restore NO signalling and restore CBF in brain disorders that feature neurovascular dysfunction.

Ca2+ signaling in postsynaptic neurons: Neuroplastin-65 regulates the interplay between plasma membrane Ca2+ ATPases and ionotropic glutamate receptors

Upon postsynaptic glutamate receptor activation, the cytosolic Ca2+ concentration rises and initiates signaling and plasticity in spines. The plasma membrane Ca2+ ATPase (PMCA) is a major player to limit the duration of cytosolic Ca2+ signals. It forms complexes with the glycoprotein neuroplastin (Np) isoforms Np55 and Np65 and functionally interplays with N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptors (iGluNRs). Moreover, binding of the Np65-specific extracellular domain to Ca2+-permeable GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type ionotropic glutamate receptors (iGluA1Rs) was found to be required for long-term potentiation (LTP). However, the link between PMCA and iGluRs function to regulate cytosolic Ca2+ signals remained unclear. Here, we report that Np65 coordinates PMCA and iGluRs’ functions to modulate the duration and amplitude of cytosolic Ca2+ transients in dendrites and spines of hippocampal neurons. Using live-cell Ca2+ imaging, acute pharmacological treatments, and GCaMP5G-expressing hippocampal neurons, we discovered that endogenous or Np65-promoted PMCA activity contributes to the restoration of basal Ca2+ levels and that this effect is dependent on iGluR activation. Super-resolution STED and confocal microscopy revealed that electrical stimulation increases the abundance of synaptic neuroplastin-PMCA complexes depending on iGluR activation and that low-rate overexpression of Np65 doubled PMCA levels and decreased cell surface levels of GluN2A and GluA1 in dendrites and Shank2-positive glutamatergic synapses. In neuroplastin-deficient hippocampi, we observed reduced PMCA and unchanged GluN2B levels, while GluN2A and GluA1 levels were imbalanced. Our electrophysiological data from hippocampal slices argues for an essential interplay of PMCA with GluN2A- but not with GluN2B-containing receptors upon induction of synaptic plasticity. Accordingly, we conclude that Np65 may interconnect PMCA with core players of glutamatergic neurotransmission to fine-tune the Ca2+ signal regulation in basal synaptic function and plasticity.

Lipopolysaccharide increases exosomes secretion from endothelial progenitor cells by toll-like receptor 4 dependent mechanism.

Endothelial progenitor cells (EPCs) can exert angiogenic effects by a paracrine mechanism, where exosomes work as an important mediator. Recent studies reported functional expression of toll-like receptor (TLR) 4 on human EPCs and dose-dependent effects of lipopolysaccharide (LPS) on EPC angiogenic properties. To study the effects of TLR4/LPS signaling on EPC-derived exosomes (Exo) and clarify the mechanism, we investigated the role of LPS on exosomes secretion from human EPCs and tested their anti-oxidation/senescence functions. We employed the inhibitors of the plasma membrane Ca2+ -ATPase (PMCA), endoplasmic reticulum Ca2+ -ATPase (ERCA), PLC-IP3 pathway and store-operated calcium entry to assess the effects of LPS on EPC intracellular calcium signalings which critical for exosome secretion. LPS induced the release of Exo in a TLR4-dependent manner in vitro, which effect can be partly abrogated by an membrane-permeable IP 3 R antagonist, 2-aminoethyl diphenylborinate (2-APB), but not PLC inhibitor, U-73122. The LPS can significantly delay the fallback of [Ca2+ ]i after isolating the cellular PMCA activity, and disturb PMCA 1/4 expression. The distribution of elevated intracellular calcium seemed coincident with the development of the multivesicular bodies (MVBs). furthermore, the anti-oxidation/senescence properties of LPS-induced Exo were validated by the senescence-associated β-galactosidase activity assay and reactive oxygen species (ROS) related H2 DCF-DA assay. The mechanism of PMCA downregulation and IP3 R-dependent ER Ca2+ release may contribute to the pro-exosomal effects of LPS on EPCs. This study provides new insights into the potential role of LPS/TLR4 pathway in regulating EPC-derived exosomes, which may help to develop some feasible approach to manipulate the Exo secretion and promote the clinical application of EPCs therapy in future.

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules.

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

Epigallocatechin 3-gallate inhibits the plasma membrane Ca2+-ATPase: effects on calcium homeostasis

Flavonoids are natural compounds responsible for the health benefits of green tea. Some of the flavonoids present in green tea are catechins, among which are: epigallocatechin, epicatechin-3-gallate, epicatechin, catechin and epigallocatechin-3-gallate (EGCG). The latter was found to induce apoptosis, reduce reactive oxygen species, in some conditions though in others it acts as an oxidizing agent, induce cell cycle arrest, and inhibit carcinogenesis. EGCG also was found to be involved in calcium (Ca2+) homeostasis in excitable and in non-excitable cells. In this study, we investigate the effect of catechins on plasma membrane Ca2+-ATPase (PMCA), which is one of the main mechanisms that extrude Ca2+ out of the cell. Our studies comprised experiments on the isolated PMCA and on cells overexpressing the pump. Among catechins that inhibited PMCA activity, the most potent inhibitor was EGCG. EGCG inhibited PMCA activity in a reversible way favoring E1P conformation. EGCG inhibition also occurred in the presence of calmodulin, the main pump activator. Finally, the effect of EGCG on PMCA activity was studied in human embryonic kidney cells (HEK293T) that transiently overexpress hPMCA4. Results show that EGCG inhibited PMCA activity in HEK293T cells, suggesting that the effects observed on isolated PMCA occur in living cells.

In Vitro Action of Matrix Metalloproteinases 2 and 9 Inhibitors on Na + /K + -ATPase, H + /K + -ATPase and PMCA Activities in the Osmoregulatory Epithelia of Climbing Perch ( Anabas testudineus Bloch)

Matrix metalloproteinases 2 and 9 (MMP2 and MMP9) are involved in the extracellular matrix (ECM) remodeling. We tested the short-term in vitro action of inhibitors of MMP2 and MMP9 on P-type ion transporter function in organ explants of climbing perch ( Anabas testudineus ) to understand how these ECM remodeling components influence the ion transporter function in the osmoregulatory epithelia of fish. Graded doses (10 -8 , 10 -7 and 10 -6 M) of inhibitors of MMP2 and MMP9 were administered in vitro to explants of gills, kidney and intestine, kept for either 15 or 30 min and the activities of P-type ATPase such as Na + /K + -ATPase (NKA), H + /K + -ATPase (HKA) and plasma membrane Ca 2+ -ATPase (PMCA) were quantified. We found that the inhibitors of MMP2 and MMP9 produced dose- and time-dependent modulation in the activities of NKA, HKA and PMCA in the tested tissue explants. Incubation of MMP2 and 9 inhibitors at the highest dose (10 -6 M) for 15 and 30 min produced substantial rise in NKA activity. Likewise, HKA activity that showed significant rise after incubation of 10 -7 and 10 -8 M inhibitors in gills and kidney explants, decreased at the lowest dose (10-8 M) of inhibitors. The lower doses of both inhibitors, while increasing PMCA activity in kidney and intestinal explants inhibited its activity in gill explant. These differential tissue-responsive actions of MMP2 and MMP9 inhibitors indicate that these ECM remodeling components can modify the function of the membrane-bound P-type ion transporters in the osmoregulatory tissues of fish.

Partial Inhibition of Na+/K+-ATPase and Plasma Membrane Ca2+-ATPase from the Rat Cerebral Cortex by S-Nitrosoglutathione

S-nitrosoglutathione (GSNO) is an endogenous S-nitrosylated glutathione derivative (GSH) involved in NO signaling. GSNO acts as a carrier of nitrosyl groups to cysteine (both free amino acid and its residues in proteins), a glutathionylation agent, and an NO donor. We investigated the effects of GSNO, GSH, and oxidized glutathione (GSSG) on the activity of two main ion pumps of the plasma membrane, Na+/K+-ATPase (NKA) and plasma membrane Ca2+-ATPase (PMCA). Plasma membranes were obtained from the rat cerebral cortex by differential centrifugation. Preincubation of the saponinpermeabilized plasma membranes at 37°C (but not at 0°C) with various concentrations of GSNO led to partial inhibition of ATPase activities of NKA and PMCA (on average, by 19.4 ± 2.2 and 42.1 ± 3% at 10 mM GSNO, respectively). The addition of GSH instead of GSNO did not affect the enzyme activities, while GSSG inhibited these enzymes much weaker than GSNO. The inhibited enzyme activities after the action of GSNO were partially restored after incubation of the membranes with a thiol-reducing agent, 2-mercaptoethanol. GSNO-dependent inhibition of NKA was not observed in the presence of ouabain (>1.0 μM), when only isozymes having an α1-subunit were active. GSNO inhibited both high- and lowsensitive to Ca2+ (calmodulin-dependent and calmodulin-independent) PMCA activities by 51 and 33%, respectively, with no changes in Km for Ca2+. It is assumed that GSNO or its degradation product(s), by modifying the thiol groups in NKA and PMCA molecules, partly inhibit their activity. Only NKA isozymes with α2 and α3 subunits, which are highly sensitive to ouabain, are likely to manifest such sensitivity. Inhibition of PMCA is not associated with a change in its sensitivity to Ca2+.

Methylene Blue Blocks and Reverses the Inhibitory Effect of Tau on PMCA Function.

Methylene blue (MB) is a synthetic phenothiazine dye that, in the last years, has generated much debate about whether it could be a useful therapeutic drug for tau-related pathologies, such as Alzheimer’s disease (AD). However, the molecular mechanism of action is far from clear. Recently we reported that MB activates the plasma membrane Ca2+-ATPase (PMCA) in membranes from human and pig tissues and from cells cultures, and that it could protect against inactivation of PMCA by amyloid β-peptide (Aβ). The purpose of the present study is to further examine whether the MB could also modulate the inhibitory effect of tau, another key molecular marker of AD, on PMCA activity. By using kinetic assays in membranes from several tissues and cell cultures, we found that this phenothiazine was able to block and even to completely reverse the inhibitory effect of tau on PMCA. The results of this work point out that MB could mediate the toxic effect of tau related to the deregulation of calcium homeostasis by blocking the impairment of PMCA activity by tau. We then could conclude that MB could interfere with the toxic effects of tau by restoring the function of PMCA pump as a fine tuner of calcium homeostasis.

Natural flavonoids inhibit the plasma membrane Ca2+-ATPase

Research on flavonoids from plant sources has recently sparked increasing interest because of their beneficial health properties. Different studies have shown that flavonoids change the intracellular Ca2+ homeostasis linked to alterations in the function of mitochondria, Ca2+ channels and Ca2+ pumps. These findings hint at plasma membrane Ca2+-ATPase (PMCA) involvement, as it transports Ca2+ actively to the extracellular medium coupled to ATP hydrolysis, thus maintaining ion cellular homeostasis. The present study aims to investigate the effect of several natural flavonoids on PMCA both in isolated protein systems and in living cells, and to establish the relationship between flavonoid structure and inhibitory activity on PMCA.Our results show that natural flavonoids inhibited purified and membranous PMCA with different effectiveness: quercetin and gossypin were the most potent and their inhibition mechanisms seem to be different, as quercetin does not prevent ATP binding whereas gossypin does. Moreover, PMCA activity was inhibited in human embryonic kidney cells which transiently overexpress PMCA, suggesting that the effects observed on isolated systems could occur in a complex structure like a living cell. In conclusion, this work reveals a novel molecular mechanism through which flavonoids inhibit PMCA, which leads to Ca2+ homeostasis and signaling alterations in the cell.

Ca2+ efflux via plasma membrane Ca2+-ATPase mediates chemotaxis in ascidian sperm

When a spermatozoon shows chemotactic behavior, transient [Ca2+]i increases in the spermatozoon are induced by an attractant gradient. The [Ca2+]i increase triggers a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming. However, the molecular mechanism of [Ca2+]i modulation controlled by the attractants is not well defined. Here, we examined receptive mechanisms for the sperm attractant, SAAF, in the ascidian, Ciona intestinalis, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF-binding protein. PMCA is localized in sperm flagella membranes and seems to interact with SAAF through basic amino acids located in the second and third extracellular loops. ATPase activity of PMCA was enhanced by SAAF, and PMCA inhibitors, 5(6)-Carboxyeosin diacetate and Caloxin 2A1, inhibited chemotactic behavior of the sperm. Furthermore, Caloxin 2A1 seemed to inhibit efflux of [Ca2+]i in the sperm, and SAAF seemed to competitively reduce the effect of Caloxin 2A1. On the other hand, chemotactic behavior of the sperm was disordered not only at low-Ca2+, but also at high-Ca2+ conditions. Thus, PMCA is a potent candidate for the SAAF receptor, and direct control of Ca2+ efflux via PMCA is a fundamental mechanism to mediate chemotactic behavior in the ascidian spermatozoa.

Methylene blue activates the PMCA activity and cross-interacts with amyloid β-peptide, blocking Aβ-mediated PMCA inhibition

The phenothiazine methylene blue (MB) is attracting increasing attention because it seems to have beneficial effects in the pathogenesis of Alzheimer's disease (AD). Among other factors, the presence of neuritic plaques of amyloid-β peptide (Aβ) aggregates, neurofibrilar tangles of tau and perturbation of cytosolic Ca2+ are important players of the disease. It has been proposed that MB decreases the formation of neuritic plaques due to Aβ aggregation. However, the molecular mechanism underlying this effect is far from clear. In this work, we show that MB stimulates the Ca2+-ATPase activity of the plasma membrane Ca2+-ATPase (PMCA) in human tissues from AD-affected brain and age-matched controls and also from pig brain and cell cultures. In addition, MB prevents and even blocks the inhibitory effect of Aβ on PMCA activity. Functional analysis with mutants and fluorescence experiments strongly suggest that MB binds to PMCA, at the C-terminal tail, in a site located close to the last transmembrane helix and also that MB binds to the peptide. Besides, Aβ increases PMCA affinity for MB. These results point out a novel molecular basis of MB action on Aβ and PMCA as mediator of its beneficial effect on AD.

Chlorpromazine and dimethyl sulfoxide modulate the catalytic activity of the plasma membrane Ca2+-ATPase from human erythrocyte.

The plasma membrane Ca2+-ATPase (PMCA) removes Ca2+ from the cytosol into the extracellular space. Its catalytic activity can be stimulated by calmodulin (CaM) or by limited proteolysis. We evaluated the effect of chlorpromazine (CPZ) and dimethyl sulfoxide (DMSO) over the hydrolytic activity of PMCA. Activity was monitored in three different forms: native, CaM-activated and proteolyzed by trypsin. CPZ appears to inhibit PMCA without directly interfering with the C-terminal site, since it is affected by CaM and proteolysis. Although the treatment of PMCA with trypsin and CaM produces an activation, it also produces an enzymatic form that is more sensitive to inhibition by CPZ. The same case was observed in the DMSO inhibition experiments. In the absence of CPZ, DMSO produces a progressive loss of activity, but in the presence of CPZ the profile of activity against DMSO changes and produces a recovery of activity, indicating a possible partition of CPZ by the solvent. Increasing Ca2+ concentrations indicated that CPZ interacts with PMCA rather than with CaM. This observation is supported by docking analysis that suggests that the CPZ-PMCA interaction is non-competitive. We propose that CPZ interacts with the state of lower affinity for Ca2 +.

The plasma membrane calcium pumps-The old and the new.

The plasma membrane Ca2+-ATPase (PMCA) pumps play a critical role in the maintenance of calcium (Ca2+) homeostasis, crucial for optimal neuronal function and cell survival. Loss of Ca2+ homeostasis is a key precursor in neuronal dysfunction associated with brain aging and in the pathogenesis of neurodegenerative disorders. In this article, we review evidence showing age-related changes in the PMCAs in synaptic plasma membranes (SPMs) and lipid raft microdomains isolated from rat brain. Both PMCA activity and protein levels decline progressively with increasing age. However, the loss of activity is disproportionate to the reduction of protein levels suggesting the presence of dysfunctional PMCA molecules in aged brain. PMCA activity is also diminished in post-mortem human brain samples from Alzheimer's disease and Parkinson's disease patients and in cell models of these neurodegenerative disorders. Experimental reduction of the PMCAs not only alter Ca2+ homeostasis but also have diverse effects on neurons such as reduced neuritic network, impaired release of neurotransmitter and increased susceptibility to stressful stimuli, particularly to agents that elevate intracellular Ca2+ [Ca2+]i. Loss of PMCA is likely to contribute to neuronal dysfunction observed in the aging brain and in the development of age-dependent neurodegenerative disorders. Therapeutic (pharmacological and/or non-pharmacological) approaches that can enhance PMCA activity and stabilize [Ca2+]i homeostasis may be capable of preventing, slowing, and/or reversing neuronal degeneration.

Long-term effect of a chronic low-protein multideficient diet on the heart: Hypertension and heart failure in chronically malnourished young adult rats

BackgroundWe investigated whether a chronic low-protein multideficient diet (BRD) from weaning turns on cardiovascular adaptive responses that could culminate in hypertension and heart failure. Methods and resultsSystolic pressure (SP) and heart rate (HR) were determined in CTRL (normal diet) and BRD rats. Plasma albumin, plasma urea and urinary urea excretion decreased in BRD rats. In this group, echocardiography and the Langendorff technique showed: (i) increased HR and hypertension; (ii) decreased LVDP, dP/dtmax, dP/dtmin, cardiac output, ejection fraction, stroke volume and left ventricular diameter. BRD rats were less sensitive to isoproterenol (ISO) in LVDP and dP/dtmax, with unchanged dP/dtmin; Pressure-volume relationships indicated left-oriented shifts in LVDP, SP and DP, and decreased capacitance compared to CTRL. BRD rats had higher cardiac and lung indexes, accompanied by muscle atrophy and recent ventricular-infarcted areas, higher ventricular β1-AR content, and decreased β2-AR and α1-AR. Propranolol treatment gave similar ISO responses in both groups, disappearance of the infarcted regions and, except for β2-AR, recovery of normal receptor expression. BRD rats had intense stimulation of plasma membrane Ca2+-ATPase (PMCA) activity, with increased Ca2+ affinity and inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Ventricular phospholamban increased and Na+/Ca2+ exchanger decreased. PMCA activity correlated with an increase in its PKC-mediated phosphorylation, overlying a decrease in PKA-catalyzed phosphorylation. Propranolol normalized PKC and PKA activities with recovery of PMCA but not SERCA. ConclusionBRD triggers sympathetic exacerbation and dysfunction in Ca2+ handling, accompanied by early onset of hypertension and left ventricle congestive heart failure.

Increased thrombospondin-4 after nerve injury mediates disruption of intracellular calcium signaling in primary sensory neurons

Painful nerve injury disrupts Ca2+ signaling in primary sensory neurons by elevating plasma membrane Ca2+-ATPase (PMCA) function and depressing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) function, which decreases endoplasmic reticulum (ER) Ca2+ stores and stimulates store-operated Ca2+ entry (SOCE). The extracellular matrix glycoprotein thrombospondin-4 (TSP4), which is increased after painful nerve injury, decreases Ca2+ current (ICa) through high-voltage–activated Ca2+ channels and increases ICa through low-voltage–activated Ca2+ channels in dorsal root ganglion neurons, which are events similar to the effect of nerve injury. We therefore examined whether TSP4 plays a critical role in injury-induced disruption of intracellular Ca2+ signaling. We found that TSP4 increases PMCA activity, inhibits SERCA, depletes ER Ca2+ stores, and enhances store-operated Ca2+ influx. Injury-induced changes of SERCA and PMCA function are attenuated in TSP4 knock-out mice. Effects of TSP4 on intracellular Ca2+ signaling are attenuated in voltage-gated Ca2+ channel α2δ1 subunit (Cavα2δ1) conditional knock-out mice and are also Protein Kinase C (PKC) signaling dependent. These findings suggest that TSP4 elevation may contribute to the pathogenesis of chronic pain following nerve injury by disrupting intracellular Ca2+ signaling via interacting with the Cavα2δ1 and the subsequent PKC signaling pathway. Controlling TSP4 mediated intracellular Ca2+ signaling in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.

Phospholipids and calmodulin modulate the inhibition of PMCA activity by tau

The disruption of Ca2+ signaling in neurons, together with a failure to keep optimal intracellular Ca2+ concentrations, have been proposed as significant factors for neuronal dysfunction in the Ca2+ hypothesis of Alzheimer's disease (AD). Tau is a protein that plays an essential role in axonal transport and can form abnormal structures such as neurofibrillary tangles that constitute one of the hallmarks of AD. We have recently shown that plasma membrane Ca2+-ATPase (PMCA), a key enzyme in the maintenance of optimal cytosolic Ca2+ levels in cells, is inhibited by tau in membrane vesicles. In the present study we show that tau inhibits synaptosomal PMCA purified from pig cerebrum, and reconstituted in phosphatidylserine-containing lipid bilayers, with a Ki value of 1.5±0.2nM tau. Noteworthy, the inhibitory effect of tau is dependent on the charge of the phospholipid used for PMCA reconstitution. In addition, nanomolar concentrations of calmodulin, the major endogenous activator of PMCA, protects against inhibition of the Ca2+-ATPase activity by tau. Our results in a cellular model such as SH-SY5Y human neuroblastoma cells yielded an inhibition of PMCA by nanomolar tau concentrations and protection by calmodulin against this inhibition similar to those obtained with purified synaptosomal PMCA. Functional studies were also performed with native and truncated versions of human cerebral PMCA4b, an isoform that has been showed to be functionally regulated by amyloid peptides, whose aggregates constitutes another hallmark of AD. Kinetic assays point out that tau binds to the C-terminal tail of PMCA, at a site distinct but close to the calmodulin binding domain. In conclusion, PMCA can be seen as a molecular target for tau-induced cytosolic calcium dysregulation in synaptic terminals.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca2+-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca2+]i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca2+]i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.

Interleukin-1β activates an Src family kinase to stimulate the plasma membrane Ca2+ pump in hippocampal neurons.

The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in clearing Ca(2+) from the neuronal cytoplasm. The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, synaptic plasticity, and neurotransmission. Here, we examined the modulation of PMCA activity by PTKs in hippocampal neurons. PMCA-mediated Ca(2+) clearance slowed in the presence of pyrazolopyrimidine 2, an inhibitor of Src family kinases (SFKs), and accelerated in the presence of C2-ceramide, an activator of PTKs. Ca(2+) clearance kinetics were attenuated in cells expressing a dominant-negative Src mutant, suggesting that the pump is tonically stimulated by a PTK. Tonic stimulation was reduced in hippocampal neurons expressing short hairpin (sh)RNA directed to mRNA for Yes. shRNA-mediated knockdown of PMCA isoform 1 (PMCA1) removed tonic stimulation of Ca(2+) clearance, indicating that the kinase stimulates PMCA1. IL-1β accelerated Ca(2+) clearance in a manner blocked by an IL-1β receptor antagonist or by an inhibitor of neutral sphingomyelinase, the enzyme that produces ceramide. Thus IL-1β activates an SFK to stimulate the plasma membrane Ca(2+) pump, decreasing the duration of Ca(2+) transients in hippocampal neurons.

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.