PMCA Activity Research Articles

Role of Piezo1 in Terminal Density Reversal of Red Blood Cells.

Density reversal of senescent red blood cells has been known for a long time, yet the identity of the candidate ion transporter(s) causing the senescent cells to swell is still elusive. While performing fractionation of RBCs from healthy individuals in Percoll density gradient and characterization of the separated fractions, we identified a subpopulation of cells in low-density fraction (1.02% ± 0.47) showing signs of senescence such as loss of membrane surface area associated with a reduction in band 3 protein abundance, and Phosphatidylserine (PS) exposure to the outer membrane. In addition, we found that these cells are overloaded with Na+ and Ca2+. Using a combination of blockers and activators of ion pumps and channels, we revealed reduced activity of Plasma membrane Ca2+ ATPase and an increase in Ca2+ and Na+ leaks through ion channels in senescent-like cells. Our data revealed that Ca2+ overload in these cells is a result of reduced PMCA activity and facilitated Ca2+ uptake via a hyperactive Piezo1 channel. However, we could not exclude the contribution of other Ca2+-permeable ion channels in this scenario. In addition, we found, as a universal mechanism, that an increase in intracellular Ca2+ reduced the initially high selectivity of Piezo1 channel for Ca2+ and allowed higher Na+ uptake, Na+ accumulation, and swelling.

Tubulin Regulates Plasma Membrane Ca2+-ATPase Activity in a Lipid Environment-dependent Manner.

Ca2+ plays a crucial role in cell signaling, cytosolic Ca2+ can change up to 10,000-fold in concentration due to the action of Ca2+-ATPases, including PMCA, SERCA and SCR. The regulation and balance of these enzymes are essential to maintain cytosolic Ca2+ homeostasis. Our laboratory has discovered a novel PMCA regulatory system, involving acetylated tubulin alone or in combination with membrane lipids. This regulation controls cytosolic Ca2+ levels and influences cellular properties such as erythrocyte rheology. This review summarizes the findings on the regulatory mechanism of PMCA activity by acetylated tubulin in combination with lipids. The combination of tubulin cytoskeleton and membrane lipids suggests a novel regulatory system for PMCA, which consequently affects cytosolic Ca2+ content, depending on cytoskeletal and plasma membrane dynamics. Understanding the interaction between acetylated tubulin, lipids and PMCA activity provides new insights into Ca2+ signaling and cell function. Further research may shed light on potential therapeutic targets for diseases related to Ca2+ dysregulation. This discovery contributes to a broader understanding of cellular processes and offers opportunities to develop innovative approaches to treat Ca2+-related disorders. By elucidating the complex regulatory mechanisms of Ca2+ homeostasis, we advance our understanding of cell biology and its implications for human health.

Alterations of the Ca2+ clearing mechanisms by type 2 diabetes in aortic smooth muscle cells of Zucker diabetic fatty rat.

Type 2 Diabetes Mellitus (T2DM) is a rapidly rising disease with cardiovascular complications constituting the most common cause of death among diabetic patients. Chronic hyperglycemia can induce vascular dysfunction through damage of the components of the vascular wall, such as vascular smooth muscle cells (VSMCs), which regulate vascular tone and contribute to vascular repair and remodeling. These functions are dependent on intracellular Ca2+ changes. The mechanisms by which T2DM affects Ca2+ handling in VSMCs still remain poorly understood. Therefore, the objective of this study was to determine whether and how T2DM affects Ca2+ homeostasis in VSMCs. We evaluated intracellular Ca2+ signaling in VSMCs from Zucker Diabetic Fatty rats using Ca2+ imaging with Fura-2/AM. Our results indicate that T2DM decreases Ca2+ release from the sarcoplasmic reticulum (SR) and increases the activity of store-operated channels (SOCs). Moreover, we were able to identify an enhancement of the activity of the main Ca2+ extrusion mechanisms (SERCA, PMCA and NCX) during the early stage of the decay of the ATP-induced Ca2+ transient. In addition, we found an increase in Ca2+ entry through the reverse mode of NCX and a decrease in SERCA and PMCA activity during the late stage of the signal decay. These effects were appreciated as a shortening of ATP-induced Ca2+ transient during the early stage of the decay, as well as an increase in the amplitude of the following plateau. Enhanced cytosolic Ca2+ activity in VSMCs could contribute to vascular dysfunction associated with T2DM.

Evaluation of the neuroprotective activity of gamma‐benzylidenes digoxin derivatives against brain ischemia

Brain ischemia is one of the diseases with the highest mortality rate today. Among the victims who do not die, most will have some functional disability as a sequel. Currently, tissue plasminogen activator (rt‐PA) is the only pharmacological treatment for ischemia and there are no treatments with neuroprotective effects for this disease. Recently, cardiotonic steroids (CTS) have been shown to promote neuroprotection against ischemia. However, CTS are drugs that have a narrow therapeutic index and their utilization in clinic are restrict. BD‐15 is a new a non‐toxic benzylidine digoxin derivative that was demonstrated to direct increase of the α3‐Na,K‐ATPase activity, and to prevent chemical ischemia in N2a cells model, but it was never tested in a ischemia model in animal. We report here the results of the use of BD‐15 against cognitive damage and neurotoxic actions caused during brain ischemia. Wistar rats were divided in 4 groups (6 animals per group): Control, Control + BD 100 µg/kg treatment, Ischemia and Ischemia + BD‐15 100 µg/kg treatment. Transient brain ischemia was induced with bilateral occlusion of both common carotid arteries for 30 minutes, and after that the animal were followed by three days of reperfusion, with or without BD‐15 treatment (one dose per day IP, 100 µg/kg) and the hippocampus was collected to perform biochemical analysis such as: membrane lipid profile and peroxidation and membrane enzyme activities (PMCA, SERCA, and acetylcholinesterase); histological analysis for infarct volume quantification using TTC dye, and behavioral parameters assessed by the open field behavior test (the parameters evaluated were the number of times the animal changed quadrant, raised its body and presented self‐cleaning behavior and the number of visits to the central quadrants) were also evaluated. In histological analysis, the ischemia provoked a brain tissue damage after 3 days reperfusion, and BD‐15 treatment was able to impair this damage. Ischemia provoked an decrease in the total Na,K‐ATPase activity and an increase of the expression levels of the pump and BD‐15 prevent that changes caused by ischemia. The same profile was found for total PMCA activity and PMCA4 expression levels. BD‐15 was also able to prevent the decrease of activity of SERCA, Mg‐ATPase and acetylcholinesterase caused by ischemia. The membrane lipid peroxidation (thiobarbituric acid reactive substance), was increased in the ischemic group, and again BD‐15 was able to prevent this effect. Regarding the lipid profile, we observed an increase in the content of phospholipids and cholesterol in the ischemic group, and an decrease in the groups treated with BD‐15. The behavioral parameters demonstrated that BD‐15 prevents the motor damage caused by ischemia. In conclusion, BD‐15 is a non‐toxic CTS that demonstrate the prevention of brain damages provoked by ischemia, and appear to promote neuroprotection in animals exposed to global ischemia.

Impaired regulation of PMCA activity by defective CFTR expression promotes epithelial cell damage in alcoholic pancreatitis and hepatitis.

Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl- channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis.

Effect of Mg-Gluconate on the Osmotic Fragility of Red Blood Cells, Lipid Peroxidation, and Ca2+-ATPase (PMCA) Activity of Placental Homogenates and Red Blood Cell Ghosts From Salt-Loaded Pregnant Rats.

Preeclampsia (PE) is a pregnancy-specific syndrome with multisystem involvement which leads to fetal, neonatal, and maternal morbidity and mortality. A model of salt-loaded pregnant rats has been previously studied, sharing several pathological characteristics of preeclamptic women. In this study, it was compared the effects of the treatment with an oral magnesium salt, magnesium gluconate (Mg-gluconate), on the osmotic fragility of red blood cells, lipid peroxidation, and PMCA activity of placental homogenates and red blood cell ghosts in salt-loaded pregnant rats. Mg-gluconate has a higher antioxidant capacity than MgSO4 due to the presence of several hydroxyl groups in the two anions of this salt. Salt-loaded pregnant rats received 1.8% NaCl solution ad libitum as a beverage during the last week of pregnancy. On day 22nd of pregnancy, the rats were euthanized and red blood cells and placenta were obtained. Salt-loaded pregnant rats showed an increased level of lipid peroxidation and a lowered PMCA activity in placental and red blood cell ghosts, as well as an increased osmotic fragility of their red blood cells. The treatment of the salt-loaded pregnant rats with Mg-gluconate avoids the rise in the level of lipid peroxidation and the concomitant lowering of the PMCA activity of their red blood cell membranes, reaching values similar to those from control pregnant rats. Also, this treatment prevents the increase of the osmotic fragility of their red blood cells, keeping values similar to those from control pregnant rats. Mg-gluconate seems to be an important candidate for the replacement of the MgSO4 treatment of preeclamptic women.

Tubular (T-) System SOCE-PMCA Balance Is Regulated by RyR Leak in Resting Skeletal Muscle

Skeletal muscle possesses a unique, rapidly activating store-operated Ca2+ entry (SOCE) mechanism. SOCE is activated when the activity of the ryanodine receptors increase, following action potential-induced Ca2+release or even increased RyR Ca2+ leak (Koenig et al 2018, Comm Biol; Cully et al 2018, PNAS). The role of SOCE in muscle remains poorly defined, but a role in calcium content regulation is likely, which means it must work in concert with fibre Ca2+ extrusion mechanisms for balance. Kinetics of Ca2+ movements in imaging experiments performed with high spatiotemporal resolution have shown that the t-system PMCA exists in the same microdomain as RyRs and the SOCE protein components; and all components are equally distributed throughout the muscle fibre (Cully et al 2012, AJP). We studied RyR mutant mice (RyR KI; Lopez et al 2018, BJA) and calsequestrin 1 null (CSQKO) mice (Paolini et al 2007, J.Physiol) to examine the regulatory relationship of RyR Ca2+ leak with SOCE and PMCA activity using mechanically skinned fibres. We determined that RyR Ca2+ leak increased in the order WT<RyR KI HET<RyR KI HOM<CSQKO. This leak caused a steady state reduction in SR calcium content, which we quantified in absolute terms. We found PMCA activity to be increased in same order as increased RyR Ca2+ leak, where CSQKO muscle showed the most rapid rate of Ca2+ extrusion when [Ca2+]t-sys was initially 0.1 mM and [Ca2+]cyto was varied between 28-1342 nM. The increase in Ca2+ extrusion was offset by increasing SOCE so that physiological, mM [Ca2+]t-sys levels were maintained. Our data conform with Cell Boundary Theorem (Rios, 2010, J.Physiol Sci), providing a platform to explain the increased [Ca2+]cyto in resting intact fibres (Lopez et al 2018) in the presence of increased RyR Ca2+ leak.

Immunomodulator mediated changes in plasma membrane calcium ATPase in controlling visceral leishmaniasis

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCβ, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.

Cutting off the fuel supply to calcium pumps in pancreatic cancer cells: role of pyruvate kinase-M2 (PKM2)

BackgroundPancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca2+-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived ATP supply to the PMCA as a potential therapeutic locus.MethodsPDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca2+ overload and in situ Ca2+ clearance. PKM2 knockdown was achieved using siRNA.ResultsThe PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death.ConclusionsCutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.

New bufadienolides extracted from Rhinella marina inhibit Na,K-ATPase and induce apoptosis by activating caspases 3 and 9 in human breast and ovarian cancer cells

Bufadienolide compounds have been used for growth inhibition and apoptosis induction in tumor cells. Those families of cardiotonic steroids can bind the Na,K-ATPase, causing its inhibition. The use of bufadienolides is widely described in the literature as an anticancer function. The aim of this study was to evaluate the effects of bufadienolides and alkaloid isolated from venom samples from R. marina on tumor cells. We performed cytotoxicity assay in MDA-MB-231 and TOV-21G cells and evaluated the activity of Caspases (3 and 9), Na, K-ATPase, PMCA and SERCA. Four compounds were extrated from the venom of R. marina. The compound 1 showed higher cytotoxicity in MDA-MB-231cells. Compound 1 also showed activation of Caspase 3 and 9. This compound caused an inhibition of the activity and expression of Na, K-ATPase, and also showed activation of both caspase-9 and caspase-3 in MDA-MB-231 cells. We also observed that Compound 1 had a direct effect on some ATPases, such as Na, K-ATPase, PMCA and SERCA. Compound 1 was able to inhibit the activity of the purified Na, K-ATPase enzyme from the concentration of 5 µM. It also caused inhibition of PMCA at all concentrations tested (1 nM-30 µM). However, the compound 1 led to an increase of the activity of purified SERCA between the concentrations of 7.5–30 µM. Thus, we present a Na, K-ATPase and PMCA inhibitor, which may lead to the activation of caspases 3 and 9, causing the cells to enter into apoptosis. Our study suggests that compound 1 may be an interesting molecule as an anticancer agent.

Erythrocyte ion content and dehydration modulate maximal Gardos channel activity in KCNN4 V282M/+ hereditary xerocytosis red cells.

Hereditary xerocytosis (HX) is caused by missense mutations in either the mechanosensitive cation channel PIEZO1 or the Ca2+-activated K+ channel KCNN4. All HX-associated KCNN4 mutants studied to date have revealed increased current magnitude and red cell dehydration. Baseline KCNN4 activity was increased in HX red cells heterozygous for KCNN4 mutant V282M. However, HX red cells maximally stimulated by Ca2+ ionophore A23187 or by PMCA Ca2+-ATPase inhibitor orthovanadate displayed paradoxically reduced KCNN4 activity. This reduced Ca2+-stimulated mutant KCNN4 activity in HX red cells was associated with unchanged sensitivity to KCNN4 inhibitor senicapoc and KCNN4 activator Ca2+, with slightly elevated Ca2+ uptake and reduced PMCA activity, and with decreased KCNN4 activation by calpain inhibitor PD150606. The altered intracellular monovalent cation content of HX red cells prompted experimental nystatin manipulation of red cell Na and K contents. Nystatin-mediated reduction of intracellular K+ with corresponding increase in intracellular Na+ in wild-type cells to mimic conditions of HX greatly suppressed vanadate-stimulated and A23187-stimulated KCNN4 activity in those wild-type cells. However, conferral of wild-type cation contents on HX red cells failed to restore wild-type-stimulated KCNN4 activity to those HX cells. The phenotype of reduced, maximally stimulated KCNN4 activity was shared by HX erythrocytes expressing heterozygous PIEZO1 mutants R2488Q and V598M, but not by HX erythrocytes expressing heterozygous KCNN4 mutant R352H or PIEZO1 mutant R2456H. Our data suggest that chronic KCNN4-driven red cell dehydration and intracellular cation imbalance can lead to reduced KCNN4 activity in HX and wild-type red cells.

Cortical cytoskeleton dynamics regulates plasma membrane calcium ATPase isoform-2 (PMCA2) activity

We have previously shown that purified actin can directly bind to human plasma membrane Ca2+ ATPase 4b (hPMCA4b) and exert a dual modulation on its Ca2+-ATPase activity: F-actin inhibits PMCA while short actin oligomers may contribute to PMCA activation. These studies had to be performed with purified proteins given the nature of the biophysical and biochemical approaches used. To assess whether a functional interaction between the PMCAs and the cortical cytoskeleton is of physiological relevance, we characterized this phenomenon in the context of a living cell by monitoring in real-time the changes in the cytosolic calcium levels ([Ca2+]CYT). In this study, we tested the influence of drugs that change the actin and microtubule polymerization state on the activity and membrane expression of the PMCA transiently expressed in human embryonic kidney (HEK293) cells, which allowed us to observe and quantify these relationships in a live cell, for the first time. We found that disrupting the actin cytoskeleton with cytochalasin D significantly increased PMCA-mediated Ca2+ extrusion (~50–100%) whereas pre-treatment with the F-actin stabilizing agent jasplakinolide caused its full inhibition. When the microtubule network was disrupted by pretreatment of the cells with colchicine, we observed a significant decrease in PMCA activity (~40–60% inhibition) in agreement with the previously reported role of acetylated tubulin on the calcium pump. In none of these cases was there a difference in the level of expression of the pump at the cell surface, thus suggesting that the specific activity of the pump was the regulated parameter. Our results indicate that PMCA activity is profoundly affected by the polymerization state of the cortical cytoskeleton in living cells.

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca(2+)-ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein-undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l(-1), respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) activity, Ca(2+)/CaM activated EGM Ca(2+) ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca(2+)/CaM to activate EGM Ca(2+)-ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition.

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca(2+)-ATPase (PMCA), which is critical for maintaining a low intracellular Ca(2+) concentration ([Ca(2+)]i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca(2+)]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca(2+)]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca(2+)]i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.

P-Methoxycinnamic acid, an active phenylpropanoid induces mitochondrial mediated apoptosis in HCT-116 human colon adenocarcinoma cell line.

Among the eight phytochemicals (dihydrocarveol, sinapic acid, vanillic acid, ethylgallate, myrtenol, transcarveol, p-methoxycinnamic acid, and isoferulic acid) we tested, p-methoxycinnamic acid (p-MCA) [10 μM] showed the most potent in vitro growth inhibition on human colon adenocarcinoma (HCT-116 cells). Antiproliferative activity of p-MCA at 24h was associated with DNA damage, morphological changes and the results were comparable with doxorubicin. p-MCA induced phosphatidylserine translocation, increased the levels of reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), protein carbonyl content (PCC) and decreased enzymic antioxidant status (SOD, CAT, GPx) in HCT-116. p-MCA treatment increased the percentage of apoptotic cells, decreased the mitochondrial membrane potential and triggered cytochrome C release to cytosol. The induction of apoptosis by p-MCA was accompanied by an increase in caspase 3 and caspase 9 activities, increased expression of Bax and decreased expression of Bcl-2. Thus p-MCA induces mitochondria mediated intrinsic pathway of apoptosis in HCT-116 and has potential for treatment and prevention of colon cancer.

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities.

Inhibition of PMCA activity by tau as a function of aging and Alzheimer's neuropathology

Ca2+-ATPases are plasma membrane and intracellular membrane transporters that use the energy of ATP hydrolysis to pump cytosolic Ca2+ out of the cell (PMCA) or into internal stores. These pumps are the main high-affinity Ca2+ systems involved in the maintenance of intracellular free Ca2+ at the properly low level in eukaryotic cells. The failure of neurons to keep optimal intracellular Ca2+ concentrations is a common feature of neurodegeneration by aging and aging-linked neuropathologies, such as Alzheimer's disease (AD). This disease is characterized by the accumulation of β-amyloid senile plaques and neurofibrillary tangles of tau, a protein that plays a key role in axonal transport. Here we show a novel inhibition of PMCA activity by tau which is concentration-dependent. The extent of inhibition significantly decreases with aging in mice and control human brain membranes, but inhibition profiles were similar in AD-affected brain membrane preparations, independently of age. No significant changes in PMCA expression and localization with aging or neuropathology were found. These results point out a link between Ca2+-transporters, aging and neurodegeneration mediated by tau protein.

ATPases, ion exchangers and human sperm motility.

Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na(+)/Ca(2) (+)-exchanger (NCX) and the Na(+)/H(+)-exchanger (NHE). On the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with two Ki (7.9 × 10(-9) and 9.8 × 10(-5) M), which is consistent with the presence of two isoforms of α subunit of the NKA in the sperm plasma membranes (α1 and α4), being α4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca(2) (+). We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H(+) and Ca(2) (+), and therefore inhibition of sperm motility.

Preeclampsia and preeclampsia superimposed on chronic hypertension in Buenos Aires

Preeclampsia and preeclampsia superimposed on chronic hypertension in Buenos Aires

Asenapine Effects On Peroxidation and Calcium Movements in HL-1 Cells

Introduction Bipolar patients are at higher risk for cardiovascular morbidity and mortality than their counterparts in the general population. In a recent in vitro study, Asenapine, a new antipsychotic for the treatment of mania/mixed mania, was found to keep physiological endothelial function by activation of eNOS-related NO release and to protect endothelial cells against peroxidation by interference with mitochondria, apoptosis and cell survival. Objective To examine the cardiac protective effects elicited by Asenapine against peroxidation and on the Ca2+ movements. Methods In HL-1 that had undergone oxidative stress by 20 min hydrogen peroxide the effects of 30 min pre-treatment with Asenapine on survival and proliferation will be examined. In Fura-2AM loaded HL-1 we will next analyze the effects of Asenapine on Ca2+ movements and the related involvement of cAMP/PKA and PLC pathways, CaMKII, L and T type Ca2+ channels and 5HT1A receptors. The role of ‘capacitative” Ca2+ entry, plasma-membrane Ca2+ pump inhibitor (PMCA) and Na+/Ca2+ exchanger will be analyzed. Changes of membrane potential caused by interference with K+ channels will be examined, as well. Results We expect to find a proliferative and anti-peroxidative effect of Asenapine in HL-1 cells. Asenapine could also affect Ca2+ movements through cAMP/PKA and PLC-dependent signalling and the involvement of 5HT1A receptors. The effects of Asenapine could also be related to changes of plasma membrane by interference with K+ channels and the modulation of PMCA activity and Na+/Ca2+ exchanger. Conclusions We expect to further confirm the protective effect of Asenapine against peroxidative injuries. Implications will be discussed