Serum from patients with hypertension promotes endothelial dysfunction to induce trophoblast invasion through the miR­27b­3p/ATPase plasma membrane Ca2+ transporting 1 axis.

Pregnancy­induced hypertension is often accompanied by preeclampsia. The present study investigated whether microRNA (miR)­27b­3p affected the occurrence of preeclampsia by regulating the function of endothelial cells. Expressions levels of miR­27b­3p and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) were determined using reverse­transcription quantitative PCR. miR­27b­3p targeting ATP2B1 was predicted using bioinformatics and further confirmed by dual­luciferase reporter assays. Cell Counting Kit­8, Transwell and Matrigel tube formation assays were performed to detect the effects of miR­27b­3p on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), respectively. Moreover, HTR8/SVneos cells were co­cultured with HUVECs to detect the invasion of trophoblast cells, and the expression levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)­2 and MMP­9 of HUVECs and HTR8/SVneos were detected by western blotting. Expression levels of miR­27b­3p were upregulated in the serum of patients with hypertension and preeclampsia, which could target and regulate the expression of ATP2B1. The expression levels of miR­27b­3p were increased and those of ATP2B1 were reduced in HUVECs from hypertensive serums. Moreover, miR­27b­3p mimics reduced the expression level of ATP2B1, and miR­27b­3p inhibitor reversed the effect of hypertensive serum on ATP2B1 expression. Furthermore, patients with hypertension showed increased endothelial dysfunction, reduced trophoblastic invasion and the expressions of VEGF, MMP­2 and MMP­9, and miR­27b­3p mimics and silencing of ATP2B1 produced similar results to HUVECs. The miR­27b­3p inhibitor reversed the effect of hypertensive serum, and silencing of ATP2B1 inhibited the improvement of miR­27b­3p inhibitor to HUVECs and HTR­8/SVneo cells in proliferation, migration and tube formation. The current findings suggested that miR­27b­3p promoted proliferation, migration and tube formation of HUVECs and enhanced invasion of trophoblast cells, via regulation of ATP2B1. Thus, miR­27b­3p could be considered as a molecular risk factor in the pathogenesis and development of preeclampsia.

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

Peroxyl-induced oxidative stress in aging erythrocytes of rat.

This study aims at determining the possible changes in intracellular calcium (Ca (i) (2+) ), plasma membrane calcium ATPase (PMCA) activity and phosphatidylserine (PS) along with glutathione (GSH) level in response to an oxidant challenge in vitro. Erythrocytes were isolated on Percoll and incubated with 2, 2'azobis (2-aminopropane) hydrochloride (AAPH) as well as with vitamin C preceding AAPH incubation. Membrane integrity in terms of hemolysis was negatively related to acetylcholine esterase (AChE) activity with the extent of reduction under OS being higher in the old erythrocyte than in the young. A divergent pattern was seen towards lower PMCA and higher (Ca (i) (2+) ) in the young and old cells. However, the PMCA activity in the stressed young cell was high when pre-treated with vitamin C. PS externalization in the young under OS is perhaps analogous to normal aging, with vitamin C preventing premature death. These findings suggest that young erythrocytes may benefit from vitamin C in therapies addressed towards the mechanisms underlying the reduced effects of OS.

Changes in erythrocyte glutathione and plasma membrane calcium pump in preterm newborns treated antenatally with MgSO4.

BACKGROUND: Magnesium is required for the proper activity of many metabolic pathways in every cell type. Mg deficiency gives rise to preterm birth and low body weight that are associated with pathological circumstances, including disturbed ions homeostasis and insufficient antioxidant protection. Antenatal MgSO(4) treatment has been reported to exhibit a protective effect on the developing fetus; however, the molecular mechanism of this protection remains not fully understood. OBJECTIVES: Since Mg ions very rapid cross the placenta, our study tested the hypothesis whether prenatal exposure to MgSO(4) may modify the expression and activity of erythrocyte plasma membrane calcium pump (PMCA), and total erythrocyte glutathione (GSH) concentration in preterm newborns. METHODS: Immunocharacteristics of erythrocyte PMCA in control and MgSO(4)-treated newborns were done using general and isoform-specific antibodies. The hydrolytic activity of PMCA was determined with and without calmodulin. Total GSH erythrocyte levels were analyzed by a quantitative assay using Ellman reagent. All assays were done after delivery and 24 h later. RESULTS: Significant differences were present in PMCA amount, isoform composition, basal activity and sensitivity for stimulation by calmodulin in both groups of neonates. The total GSH content altered during examined time, but only in the control group. CONCLUSIONS: Prenatal treatment with MgSO(4) may exert a significant influence on calcium homeostasis and nonenzymatic antioxidant reserve in the erythrocytes of preterm newborns.

Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability.

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.

Phosphorylation of the plasma membrane calcium pump at high ATP concentration. On the mechanism of ATP hydrolysis.

The plasma membrane calcium ATPase (PMCA) reacts with ATP to form acid-stable phosphorylated intermediates (EP) that can be measured using (gamma-32P)ATP. However, the steady-state level of EP at [ATP] higher than 100 microM has not yet been studied due to methodological problems. Using a microscale method and a purified preparation of PMCA from human red blood cells, we measured the steady-state concentration of EP as a function of [ATP] up to 2 mM at different concentrations of Mg2+, both at 4 and 25 degrees C. We have measured the Ca2+-ATPase activity (v) under the same conditions as those used for phosphorylation experiments. While the curves of ATPase activity vs [ATP] were well described by the Michaelis-Menten equation, the corresponding curves of EP required more complex fitting equations, exhibiting at least a high- and a low-affinity component. Mg2+ increases the apparent affinity for ATP of this latter component, but it shows no significant effect on its high-affinity one or on the Ca2+-ATPase activity. We calculated the turnover of EP (k(pEP)) as the ratio v/EP. At 1 mM Mg2+, k(pEP) increases hyperbolically with [ATP], while at 8 microM Mg2+, it exhibits a behavior that cannot be explained by the currently accepted mechanism for ATP hydrolysis. These results, together with measurements of the rate of dephosphorylation at 4 degrees C, suggest that ATP is acting in additional steps involving the interconversion of phosphorylated intermediates during the hydrolysis of the nucleotide.

Ca2+-Mg2+-dependent ATP-ase activity and calcium homeostasis in children with chronic kidney disease.

Extracellular calcium concentrations in humans are thousands times higher than within cells. Maintenance of such gradient requires specific regulation including intracellular stores, Ca binding proteins and transmembrane protein systems. The aim of the study was to estimate PMCA (plasma membrane Ca-transporting adenosine triphosphatase; ATPase 3.6.1.38) activity and calcium homeostasis in erythrocytes of children with chronic kidney disease (CKD). Twenty-one children wth CKD stages 1-3 (group I) and 18 healthy children (group II) were examined. Group I was divided into two subgroups: Ia (8 patients with normal intact parathyroid hormone, iPTH, serum levels) and Ib (13 patients with increased iPTH). iPTH, urea, creatinine, inorganic phosphorus, cytosolic Ca2+ in red blood cells (R-Ca), and PMCA were determined. Significantly elevated R-Ca levels were observed in children from subgroup Ib in comparison with group II and subgroup Ia. The lowest activity of PMCA was found in subgroup Ia and Ib in comparison with group II. There was a negative correlation between PMCA and R-Ca in group Ia and Ib (r=-0.8, r=-0.9, respectively). In children with CKD treated conservatively, activity of PMCA in erythrocytes is disturbed. An increase in R-Ca and decrease in PMCA activity are also observed.

[Calpain-calpastatin system in erythrocytes of children with chronic renal failure]. / Uklad kalpaina-kalpastatyna w erytrocytach dzieci z przewlekla niewydolnoscia nerek.

INTRODUCTION: Disturbances of erythrocyte calcium homeostasis in the course of chronic renal failure (CRF) may result from impaired function of a membrane calcium pump (PMCA). Plasma membrane Ca+2-transport ATP-ase is responsible for maintaining cytoplasmic level of intracellular Ca+2. PMCA activity is regulated by high Ca levels and calmodulin, in case of Ca+2 deficiency--by intracellular cysteine protease--calpain, and its natural inhibitor--calpastatin. The aim of the study was to assess the relationship between the activity of PMCA and calpain-calpastatin system in erythrocytes of children with CRF treated conservatively. MATERIAL AND METHODS: The study was performed on 21 CRF children treated conservatively (gr. I) aged 6-18 years. The control group consisted of 18 healthy age matched children. The parameters evaluated in erythrocytes were as follows: calpain-calpastatin system (Cp-Cs) activity, PMCA activity, level of intracellular Ca (Cai+2) and calmodulin (Ca) activity. RESULTS: Our investigation revealed: calmodulin concentrations decrement in CRF children (2.08 +/- 0.51 mg/L) vs. controls (4.54 +/- 1.09 nmol/min/mg) (p<0.01), calpain activity in CRF children (60.56 +/- 12.43 U/mg) higher than in controls (45.34 +/- 5.65 U/mg) (p<0.01) and calpastatin activity increment in gr. I (40.03 +/- 10.24 U/mg) vs. control group (20.123.05 U/mg) (p<0.01). Decreased activity of a system containing an enzyme and its inhibitor (calpain-calpastatin) was also observed: 1 : 0.66 ratio in gr. I vs 1:0.4 ratio in controls. CONCLUSIONS: There is a decrease inPMCA activity and an increase of intracellular calcium in erythrocytes of children with mild CRF. The concomitant increment of erythrocyte calpastatin is the cause of disturbances in calpain-calpastatin system, thus unabling normalization of PMCA pump activity.