Plasma membrane Ca^(2+)-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

Role of platelet plasma membrane Ca^(2+)-ATPase in health and disease

Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

The investigation for the relationship among serum leptin, erythrocyte membrane Ca^(2+)-ATPase activity and hypertensive disorder complicating pregnancy

Objective： To study the significance of Leptin and the activity of erythrocyte membrane Ca^2＋-ATPase（EMCA） in the development of hypertensive disorder complicating pregnancy. Methods： Radioimmunoassay was used to test the level of serum Leptin, and the activity of EMCA was determined chemically in 38 pregnant women with hypertensive disorder complicating pregnancy and 36 normotensive pregnant women. Results： The level of serum Leptin in hypertensive disorder complicating pregnancy（gestational hypertension： 13.76 ± 3.46 ng/ml; preeclampsia： 15.76 ± 5.47 ng/ml; eclampsia： 18.32 ± 6.38 ng/ml）was significantly higher than that in normotensive pregnant women （11.33 ± 2.93 ng/ml） ,respectively. The average EMCA activity of patients with hypertensive disorder complicating pregnancy （gestational hypertension： 1.65 ± 0.24 μmol·pi/mg.h ; preeclampsia： 1.37 ± 0.19 μ mol·pi/mg·h;eclampsia：1.12 ± 0.14 μmol·pi/mg·h） was significantly lower than that of normotensive pregnant women（1.83 ± 0.38 μ mol·pi/mg·h）,respectively. There was a negative correlation between the level of serum Leptin and the activity of RMCA in hypertensive disorder complicating pregnancy （r = -0.63）. Conclusion： Inhibition of EMCA activity of erythrocyte in hypertensive disorder complicating pregnancy may increase cytoplasmic free calcium, which contributes to the development of hypertensive disorder complicating pregnancy. The negative correlation between the level of serum Leptin and the activity of EMCA, also suggested that serum Leptin and the activity of EMCA may play a role in the development of hypertensive disorder complicating pregnancy.

Effects of Calcium on the GABA_A-Coupled CI^-/HCO₃^--ATPase from Plasma Membrane of Rat Brain

The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca2+ on the multifunctional complex starts at concentration of 50·M and at concentration of 0.1 mM, it reduces the “basal” one and increases the CI-, HCO3-, -stimulated Mg2+-ATPase activities. GABA (0.1 - 100μM) activates the “basal” Mg2+-ATPase activity in the ab-sence of calcium. The effect of GABA on the enzyme in the presence of 0.01 ·M Ca2+ does not change. At the same time, 1 mM Ca2+eliminates the GABA effect on the “basal” Mg2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5 - 20 μM) in the absence of Ca2+ ions elimi-nates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the in-cubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca2+ (0.25 mM), the effect of o-vanadate on the “basal” and CI-, HCO3-, -ATPase activities does not appear. It is shown for the first time that high concentrations of Ca2+prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca2+-ATPase(PMCA)is an ATPdriven pump that is critical for the maintenance of low resting[Ca2+]i in all eukaryotic cells.Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism,has the capacity to cause ATP depletion and thus inhibit PMCA activity.This has potentially fatal consequences,particularly for non-excitable cells in which the PMCA is the major Ca2+efflux pathway.This is because inhibition of the PMCA inevitably leads to cytosolic Ca2+ overload and the consequent cell death.However,the relationship between metabolic stress,ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted.There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion.In particular,there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function.Moreover, membrane phospholipids,mitochondrial membrane potential,caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA.The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca2+ overload and cytotoxicity.

Effects of Xianzhen Tablet（仙贞片）on Na^+-K^+-ATPase and Ca^(2+)-Mg^(2+)-ATPase in Erythrocytic Membranes and Viscosity of Whole Blood in Non-Insulin-Dependent Diabetes Patients with Qi-Yin Deficiency, Kidney Deficiency and Blood Stasis

Objective: To assess the effects of Xianzhen tablet (XZT) on Na+-K+ -ATPase and Ca2+ Mg2+-ATPase on erythrocytic membranes, viscosity of whole blood, plasma glucose and clinical manifestations.Methods: Seventy-two cases of non-insulin-dependent diabetes mellitus (NIDDM) patients with deficiency of both Qi and Yin, deficiency of the Kidney and blood stasis were selected, and the effects of treatment on Na+ K + -ATPase, Ca2+-Mg2+ -ATPase, whole blood viscosity, blood sugar and clinical Symptoms were observed.Results: In XZT group (test group), activities of Na+ -K+ -ATPase and Ca2+ -Mg2+ -ATPase rose significantly(P< 0. 01, P< 0. 05) after treatment. Viscosity of whole blood and clinical manifestations also improved obviously. The total effective rate in lowering plasma glucose was 77. 8 % with fasting blood glucose (FBG) and 69.4 % with 2 hours postprandial plasma blood glucose (2°PBG). In the control group, viscosity of whole blood andclinical manifestations had no significant improvement. Its total effective rate in lowering plasma glucose was41. 7% with FBG and 38. 9% with 2°PBG. Conclusions: XZT played a certain role in increasing activities ofNa+ -K + -ATPase and Ca2+-Mg2+ -ATPase, decreasing viscosity of whole blood and plasma glucose and improving clinical manifestations. Therefore, XZT was experimentally manifested as an effective drug in treating NIDDM patients with Qi-Yin deficiency, renal deficiency and blood stasis.

Identification of H^+/Ca^(2+) Antiporter in Barley Root Plasma Membrane

On the basis of two types of calcium transport system detected in the barley root plasma membrane,the mechanisms of the calcium transport have been further studied.Ionophore CCCP has been found to inhibit Mg^(2+) -dependent calcium transport by 20%.In contrast,Mg^(2+) -independent calcium trans- port is insensitive to CCCP.The Mg^(2+) -dependent calcium transport following the collapse of H^+ gradient across the plasma membrane could be driven by the H^+ gradient either set up by ATP or imposed artificially. Any relation between Mg^(2+) -independent calcium transport and H^+ gradient has not been observed.These results indicate that Mg^(2+) -dependent calcium transport is accompanied by the decrease of H^+ gradient,and Mg^(2+) -independent calcium transport has nothing to do with the H^+ gradient.It is therefore suggested that the calcium transport across the barley root plasma membrane is driven by ATPase that is independent of Mg^(2+),and H^+/Ca^(2+) antiporter that is dependent on Mg^(2+).

Molecular Cloning and Distribution of a Plasma Membrane Calcium ATPase Homolog from the Pearl Oyster Pinctada fucata

Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2＋ homeostasis and the mechanism of mineralization in pearl oyster.

GABA_A-Coupled Cl-/ HCO₃<sup style="margin-left:-10px">-</sup>-ATPase from Plasma Membrane of the Rat Brain: Role of HCO₃<sup style="margin-left:-10px">-</sup>in the Enzyme Activation

This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - ATPase complex of the neuronal membrane and this enzyme is a Cl--pump that is coupled to GABAA receptors. The greatest (44%) activating effect on the enzyme is found with HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -ATPase activity is not sensitive to these ligands. Autoradiographic analysis of the spectrum of the partially purified enzyme phosphorylated with [γ-32P]ATP allowed us to distinguish three major 32P-labeled protein whose molecular weight are about 57, 53, and 48 kDa. In the presence of 5 mM Cl-/25mM HCO3- and 100 μM picrotoxin, the intensity of the phosphorylation of bands significantly decreased, thereby confirming the assumption about coupled of binding sites for anions and GABAA-ergic ligands. It was suggested scheme of Cl--transport through the plasma membrane by utilizing neuronal Cl-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.