Reduced Calcium Uptake Research Articles

A case of nephrocalcinosis in a 7‐month‐old with congenital hypothyroidism: Insights from targeted exome sequencing

AbstractNephrocalcinosis is a complex disease with a multitude of triggering factors. An association with congenital hypothyroidism has been described in the literature, but the mechanisms leading to its development remain unclear. A 7‐month‐old infant presented with muscular hypotonia and signs of malnutrition was diagnosed with congenital hypothyroidism, nephrocalcinosis of unclear origin, and multiple kidney stones. Urine analysis revealed the presence of calcium oxalate crystals and slightly elevated oxaluria without hypercalciuria. Targeted exome sequencing found no variants in the four causative genes of primary oxaluria, namely AGXT, CBX5, GRHPR, and HOGA1. The clinical outcome was favorable with thyroid hormone and potassium citrate treatment. Ultrasound follow‐up showed progressive improvement of nephrocalcinosis. The low level of urinary oxalate and the prevalence of dihydrate calcium oxalate crystals spoke against primary or secondary oxaluria. Recent evidence from mitochondrial research shows that thyroid hormone T3 enhances mitochondrial calcium levels by stimulating calcium uptake by the mitochondrial calcium uniporter. A reduced calcium uptake and metabolism in mitochondria might represent an explanation for cytoplasmic calcium accumulation in tubular cells, subsequently precipitating nephrocalcinosis in the absence of thyroid hormone. Even if the pathophysiological mechanisms are not yet fully understood, recent evidence is supportive of a causal relationship between hypothyroidism and nephrocalcinosis, a sometimes overlooked association.

Establishing the role of the mitochondrial voltage-dependent anion channel (VDAC) in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the causative agent of Chagas Disease, a neglected tropical disease that affects millions globally. As it transitions from insect vector to human host, T. cruzi faces homeostatic challenges, including oxidative stress, fluctuations in osmolarity, and nutrient availability. Ion channels regulate the parasite's ability to respond to environmental stressors. Our group has previously identified a mechanosensitive channel located in the contractile vacuole that, when knocked out, leads to reduced infectivity, abnormal morphology, and impaired motility. Transcriptomic analysis showed that Voltage-Dependent Anion Channel (VDAC) was significantly downregulated in the knockouts, suggesting a functional link between the two proteins. VDAC is a highly expressed outer mitochondrial membrane protein that, in other eukaryotes, regulates cell death, mitochondrial dynamics and metabolism. In protozoans, VDAC has been shown to share similar functions, with additional roles in tRNA and protein import. However, the function of VDAC in T. cruzi remains uncharacterized. The genome of T. cruzi contains five copies of VDAC sharing 99% homology. Four of them are organized in tandem, suggesting a gene expansion event as their most probable origin. To elucidate the function of this channel in the parasites, we targeted a conserved region using CRISPR/Cas9 strategies to generate a VDAC-deficient cell line. The mutant strain exhibited abnormal morphology and significant growth defects relative to the wild type, suggesting that VDAC is critical for parasite replication. Fluorometric analysis indicates that VDAC downregulation results in mitochondrial depolarization and reduced calcium uptake supporting its role in maintenance of mitochondrial functions.

Abstract 13138: Dissection of Cardiomyocyte versus Fibroblast Effects of Interleukin-11 in Heart Failure

Introduction: Interleukin-11 (IL-11) is a cytokine produced in response to tissue damage and cellular stress and is secreted from and acts on both parenchymal and stromal cells across tissues. The relative contributions of IL-11 to cardiomyocyte dysfunction and fibroblast activation in heart failure pathologies are ill-defined. Hypothesis: IL-11 causes both cardiomyocyte dysfunction and fibroblast activation that leads to distinct pathologies common to heart failure syndromes. Methods: Cardiomyocytes were isolated from adult C57BL/6 mice and treated with recombinant mouse IL-11 (rmIL-11) or control for 2 hours. Cardiomyocytes were then incubated with a calcium indicator (Fluo-4AM) and stimulated at 1Hz to allow recordings of contractility and calcium transients. In vivo studies were performed using C57BL/6 mice which were injected with rmIL-11 2 hours prior to echocardiography under isoflurane anaesthesia. HFpEF was induced in C57BL/6 mice with diet containing 60% fat and drinking water containing the nitric oxide inhibitor L-NAME. IL-11 was conditionally overexpressed in cardiomyocytes of C57BL/6 mice using a Myh6 dependent cre-loxp system. Ventricular function was measured by echocardiography and molecular analysis was performed 6 weeks following induction of overexpression. Results: Acute treatment for 2 hours with rmIL-11 impaired contractility at both a cellular level in isolated cardiomyocytes and of the whole heart in vivo, as measured by echocardiography. The impairment in systolic function was associated with reduced calcium uptake in cardiomyocytes, an effect which was rapidly reversible. In a mouse model of HFpEF, IL-11 was elevated in the ventricle of treated mice as compared to controls with an associated increase in IL-11 dependent profibrotic gene expression. IL-11 expression in cardiomyocytes results in extensive fibrotic changes in the myocardium, notably peri-vascularly, and significantly reduces systolic function, as compared to control animals. Conclusions: IL-11 is increased in a mouse model of HFpEF, is negatively inotropic in vitro and in vivo and induces fibrosis when secreted from cardiomyocytes. Hence, IL-11 contributes to heart failure pathologies across cellular compartments in the failing heart.

Pharmacological intervention of the FGF-PTH axis as a potential therapeutic for craniofacial ciliopathies.

ABSTRACTCiliopathies represent a disease class characterized by a broad range of phenotypes including polycystic kidneys and skeletal anomalies. Ciliopathic skeletal phenotypes are among the most common and most difficult to treat due to a poor understanding of the pathological mechanisms leading to disease. Using an avian model (talpid2) for a human ciliopathy with both kidney and skeletal anomalies (orofaciodigital syndrome 14), we identified disruptions in the FGF23–PTH axis that resulted in reduced calcium uptake in the developing mandible and subsequent micrognathia. Although pharmacological intervention with the U.S. Food and Drug Administration (FDA)-approved pan-FGFR inhibitor AZD4547 alone rescued expression of the FGF target SPRY2, it did not significantly rescue micrognathia. In contrast, treatment with a cocktail of AZD4547 and teriparatide acetate, a PTH agonist and FDA-approved treatment for osteoporosis, resulted in molecular, cellular and phenotypic rescue of ciliopathic micrognathia in talpid2 mutants. Together, these data provide novel insight into pathological molecular mechanisms associated with ciliopathic skeletal phenotypes and a potential therapeutic strategy for a pleiotropic disease class with limited to no treatment options.

TMBIM5 loss of function alters mitochondrial matrix ion homeostasis and causes a skeletal myopathy.

Ion fluxes across the inner mitochondrial membrane control mitochondrial volume, energy production, and apoptosis. TMBIM5, a highly conserved protein with homology to putative pH-dependent ion channels, is involved in the maintenance of mitochondrial cristae architecture, ATP production, and apoptosis. Here, we demonstrate that overexpressed TMBIM5 can mediate mitochondrial calcium uptake. Under steady-state conditions, loss of TMBIM5 results in increased potassium and reduced proton levels in the mitochondrial matrix caused by attenuated exchange of these ions. To identify the in vivo consequences of TMBIM5 dysfunction, we generated mice carrying a mutation in the channel pore. These mutant mice display increased embryonic or perinatal lethality and a skeletal myopathy which strongly correlates with tissue-specific disruption of cristae architecture, early opening of the mitochondrial permeability transition pore, reduced calcium uptake capability, and mitochondrial swelling. Our results demonstrate that TMBIM5 is an essential and important part of the mitochondrial ion transport system machinery with particular importance for embryonic development and muscle function.

Cardiovascular benefits of Eruca sativa mill. Defatted seed meal extract: Potential role of hydrogen sulfide.

Eruca sativa Mill. is an edible plant belonging to the Brassicaceae botanical family with a long story as a medicinal material, mainly linked to the presence of glucoerucin. One of the main products of this glucosinolate is erucin, a biologicallly active isothiocyanate recently recognized as a hydrogen sulfide (H2S) donor. In this work, an Eruca sativa extract has been obtained from a defatted seed meal (DSM), achieving a powder rich in thiofunctionalized glucosinolates, glucoerucin, and glucoraphanin, accounting for 95% and 5% of the total glucosinolate content (17% on a dry weight basis), associated with 13 identified phenolic acids and flavonoids accounting for 2.5%. In a cell‐free model, Eruca sativa DSM extract slowly released H2S. Moreover, this extract promoted significant hypotensive effects in hypertensive rats, and evoked dose‐dependent cardioprotection in in vivo model of acute myocardial infarct, obtained through a reversible coronary occlusion. This latter effect was sensitive to blockers of mitochondrial KATP and Kv7.4 potassium channels, suggesting a potential role of these mitochondrial channels in the protective effects of Eruca sativa DSM extract. Accordingly, Eruca sativa DSM extract reduced calcium uptake and apoptotic cell death in isolated cardiac mitochondria. Taken together, these results demonstrate that Eruca sativa DSM extract is endowed with an interesting nutraceutical profile on the cardiovascular system due to, at least in part, its H2S releasing properties. These results pave the way for future investigations on active metabolites.

Hypoxic hagfish gills binge calcium

Slimy, tentacle-faced hagfish look like aliens and, accordingly, possess several PhD theses’ worth of unusual traits. Their body fluids have a similar number of dissolved salts to seawater, although because hagfish control the levels of some ions like calcium in their blood, the mix of salts is slightly different. Hagfish are also famously tolerant of low oxygen conditions (hypoxia), which comes in handy when they burrow inside rotting whales to dine. In some fishes, hypoxia disrupts how they manage the delicate balance between their internal salts and the salts in their surroundings as they cut back on the energy-intensive process of ion movement to save energy. But what about hagfish with their unusual balance of salts? And what happens when oxygen is scarce? Chris Glover of Athabasca University, Canada, and Greg Goss of the University of Alberta, Canada, decided to find out.Glover and Goss hypothesized that their slimy beasts would do the same as other fish when oxygen was in short supply: decreasing calcium uptake and accumulation. They tested this idea by placing hagfish in air-tight jars and tracking the movement of mildly radioactive calcium into their tissues as they used up the available oxygen.Unlike the hagfish in open jars, the hagfish in sealed jars moved calcium quickly into most of their tissues, including skin, liver, muscle, heart, plasma and brain. Low oxygen levels also bumped up the rates of calcium build-up in their gut and especially their gills; the guts of the fish in the low oxygen conditions had nearly 4 times more calcium than the guts of the fish in oxygen-rich conditions after adjusting for calcium contained in the blood flowing through the tissue. Since hagfish have no bones to leech calcium from, these results meant that, contrary to expectations, hagfish responded to hypoxia by importing more calcium into their bodies.When an animal does something you don't expect, the next questions are usually: how and why? Tackling the first question, Glover and Goss focused on how calcium moved across hagfish skin and gut tissue when it was isolated from the rest of the body with the goal of understanding the role each tissue played in the hypoxic fish's calcium binge.They discovered that low oxygen in the environment reduced calcium uptake by the skin, but only when the experiment was performed in unnaturally low calcium conditions, so this was unlikely to be a major mechanism of calcium accumulation in living animals. However, the results in the gut tissue were more puzzling. Despite surging calcium levels in the gut under hypoxia, there were no differences in the calcium uptake rates between the guts of the hagfish that had been kept in normal levels of oxygen and those that were oxygen starved. The authors mused that perhaps the sealed-in hagfish had gulped down seawater as a reaction to the stress, accidently flushing the tissue with calcium in the process. With the skin and gut ruled out as major importers of calcium into the body, the researchers determined that the gills were the source of the calcium detected in the closed-jar hagfish, which squares with the impressive rate of calcium accumulation observed in this tissue.Hagfish are peculiar creatures, so it's perhaps unsurprising that they respond so differently from most other earthbound fishes. As for why hypoxic hagfish pull in so much calcium through their gills, that remains a mystery. Calcium could possibly aid in coping with low oxygen conditions by constricting blood vessels or protecting brains cells. Alternatively, the calcium accumulation might not be a strategy at all and could simply be a result of hagfish moving more water – and consequently calcium – through their gills; but that's a study for another time.

Effects of G-Rh2 on mast cell-mediated anaphylaxis via AKT-Nrf2/NF-κB and MAPK-Nrf2/NF-κB pathways

BackgroundThe effect of ginsenoside Rh2 (G-Rh2) on mast cell-mediated anaphylaxis remains unclear. Herein, we investigated the effects of G-Rh2 on OVA-induced asthmatic mice and on mast cell-mediated anaphylaxis. MethodsAsthma model was established for evaluating airway changes and ear allergy. RPMCs and RBL-2H3 were used for in vitro experiments. Calcium uptake, histamine release and degranulation were detected. ELISA and Western blot measured cytokine and protein levels, respectively. ResultsG-Rh2 inhibited OVA-induced airway remodeling, the production of TNF-α, IL-4, IL-8, IL-1β and the degranulation of mast cells of asthmatic mice. G-Rh2 inhibited the activation of Syk and Lyn in lung tissue of OVA-induced asthmatic mice. G-Rh2 inhibited serum IgE production in OVA induced asthmatic mice. Furthermore, G-Rh2 reduced the ear allergy in IgE-sensitized mice. G-Rh2 decreased the ear thickness. In vitro experiments G-Rh2 significantly reduced calcium uptake and inhibited histamine release and degranulation in RPMCs. In addition, G-Rh2 reduced the production of IL-1β, TNF-α, IL-8, and IL-4 in IgE-sensitized RBL-2H3 cells. Interestingly, G-Rh2 was involved in the FcεRI pathway activation of mast cells and the transduction of the Lyn/Syk signaling pathway. G-Rh2 inhibited PI3K activity in a dose-dependent manner. By blocking the antigen-induced phosphorylation of Lyn, Syk, LAT, PLCγ2, PI3K ERK1/2 and Raf-1 expression, G-Rh2 inhibited the NF-κB, AKT-Nrf2, and p38MAPK-Nrf2 pathways. However, G-Rh2 up-regulated Keap-1 expression. Meanwhile, G-Rh2 reduced the levels of p-AKT, p38MAPK and Nrf2 in RBL-2H3 sensitized IgE cells and inhibited NF-κB signaling pathway activation by activating the AKT-Nrf2 and p38MAPK-Nrf2 pathways. ConclusionG-Rh2 inhibits mast cell-induced allergic inflammation, which might be mediated by the AKT-Nrf2/NF-κB and p38MAPK-Nrf2/NF-κB signaling pathways.

Impaired Mitochondrial ATP Production Downregulates Wnt Signaling via ER Stress Induction

Wnt signaling affects fundamental development pathways and, if aberrantly activated, promotes the development of cancers. Wnt signaling is modulated by different factors, but whether the mitochondrial energetic state affects Wnt signaling is unknown. Here, we show that sublethal concentrations of different compounds that decrease mitochondrial ATP production specifically downregulate Wnt/β-catenin signaling invitro in colon cancer cells and invivo in zebrafish reporter lines. Accordingly, fibroblasts from a GRACILE syndrome patient and a generated zebrafish model lead to reduced Wnt signaling. We identify a mitochondria-Wnt signaling axis whereby a decrease in mitochondrial ATP reduces calcium uptake into the endoplasmic reticulum (ER), leading to endoplasmic reticulum stress and to impaired Wnt signaling. In turn, the recovery of the ATP level or the inhibition of endoplasmic reticulum stress restores Wnt activity. These findings reveal a mechanism that links mitochondrial energetic metabolism to the control of the Wnt pathway that may be beneficial against several pathologies.

Increased FoxO3a expression prevents osteoblast differentiation and matrix calcification

Forkhead Box O transcription factors play important roles in bone metabolism by defending against oxidative stress and apoptosis. FoxO3a is of special interest as it is the predominant isoform expressed in bone. In osteoblasts, the administration of 1,25 dihydroxyvitamin D3 (1,25D3) increases FoxO3a expression, and alters calcium handling. We therefore queried whether FoxO3a participates in vitamin D-mediated regulation of calcium transport pathways or matrix calcification, independent of reactive oxygen species (ROS) formation. To examine this possibility, we differentiated MC3T3-E1 cells into mature osteoblast-like cells over 7 days. This coincided with an increased ability to mineralize extracellular matrix. FoxO3a expression increased throughout differentiation. 1,25D3 enhanced both FoxO3a mRNA and protein expression. Immunofluorescence microscopy found increased FoxO3a nuclear localization with differentiation and after treatment with 1,25D3. Live cell ratiometric imaging with Fura-2AM identified significant L-type calcium channel mediated calcium uptake that was enhanced by 1,25D3. We observed expression of both Cav1.2 and Cav1.3, although expression decreased throughout differentiation and was not altered by 1,25D3 treatment. FoxO3a overexpression reduced calcium uptake and calcium deposition. FoxO3a overexpression also prevented alterations in calcium channel expression and the cell differentiation associated decrease in expression of Runx2 and increased expression of osteocalcin, findings consistent with a failure for the cells to differentiate. Based on both our expression and functional data, we suggest that high levels of FoxO3a prevent osteoblast differentiation and matrix calcification.

The vitamin D positive feedback hypothesis of inflammatory bowel diseases

While it appears that there are a variety of factors that exacerbate IBD, it is frustrating that symptoms can persist and worsen even when environmental insults are removed. We suggest that there may be a positive feedback loop which perpetuates the inflammatory response in IBD patients. The loop is triggered by vitamin D deficiency which reduces calcium uptake. Lowered vitamin D and calcium interfere with anti-inflammatory pathways. Inflammation of the mucosa inhibits absorption of calcium and thus perpetuates the reduced anti-inflammatory response. A number of predictions follow from this hypothesis and are supported by geographic and lifestyle patterns in IBD incidence and prevalence.

Effect of acute peritonitis on rocuronium-induced intraperitoneal pressure reduction and the uptake function of the sarcoplasmic reticulum.

Previous studies have reported the incomplete relaxation effect of neuromuscular blockers on skeletal muscles in acute peritonitis (AP) and other inflammatory processes; however, the underlying mechanisms responsible for this effect have not yet been satisfactorily identified. The impaired removal of cytosolic Ca2+ through sarcoendoplasmic Ca2+-ATPase (SERCA) and defects in sarcoplasmic reticulum (SR) Ca2+ uptake are the major contributing factors to diastolic dysfunction. Previous studies on the effects of neuromuscular blockers have primarily focused on neuromuscular transmission. Because of the reduced calcium uptake in the SR itself, even when neuromuscular transmission is fully blocked, the muscle is not able to relax effectively. In the present study, the impact of AP on rocuronium-induced intraperitoneal pressure reduction and rectus abdominal muscle relaxation, and SERCA uptake function was investigated. AP was induced via gastric perforation and changes in the intraperitoneal pressure before and after the administration of rocuronium were recorded. Muscle contractile properties, uptake and release functions and SERCA activity in the rectus abdominal muscles of AP model rats were measured. The half-relaxation time in the AP group was significantly prolonged compared with that in the control group (P<0.01). The peak rate of SR Ca2+ uptake for whole muscle homogenates was significantly reduced (P<0.05) in AP model rats without reduction of the rate of Ca2+ release evoked through AgNO3. In conclusion, gastric perforation-induced AP attenuates the intraperitoneal pressure-reducing effect of rocuronium, and AP induces diastolic dysfunction of the rectus abdominal muscle. The SR Ca2+-ATPase uptake rate was also reduced by AP.

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

G.P.120: The Sodium/Hydrogen Exchanger (NHE-1): A promising novel target for DMD

Abnormal intracellular calcium load is one of the most consistent findings in Duchenne muscular dystrophy (DMD) but previous attempts to improve the dystrophic pathology with calcium antagonists have been unsuccessful. However, there is an intriguing possibility of restoring calcium homeostasis indirectly by exploiting the relationship between the Sodium/Hydrogen Exchanger (NHE-1) and the Sodium/Calcium Exchanger (NCX). NHE-1 has been reported to be abnormally overactive in DMD models, leading to an increased influx of sodium which in turn switches the NCX into reserve mode leading to calcium influx. Using selective NHE-1 inhibitors the influx of sodium can be reduced thereby allowing the NCX to revert back to normal mode, where calcium is extruded, thus reducing the cellular calcium load. In collaboration with our industrial partner Peacock Pharma, we have demonstrated the potential of NHE-1 inhibition in the mdx mouse model for DMD by chronically treating animals with a novel NHE-1 inhibitor. The pilot study showed significantly reduced calcium uptake in treated mice (<i>n</i>=6) using manganese enhanced MRI (MEMRI). Manganese acts as a contrast agent in T1 weighted MRI and is thought to be taken up by cardiac and skeletal muscle by the same receptors as calcium. There was also an increase in functional grip strength, although not statistically significant the results are nevertheless encouraging and indicate the potential of this novel approach in DMD. This is currently being investigated more thoroughly using a greater number of animals (<i>n</i>=12) treated chronically via drug in chow over 6-months and assessed using MEMRI and grip test. In addition, in vitro calcium-flux assays (e.g. Fluo-4) are used to determine the potency and investigate the mechanism of action of this drug. The objective of our study is to provide the requisite pre-clinical data that would enable these drugs to progress towards clinical trials.

Tail-anchored membrane protein SLMAP is a novel regulator of cardiac function at the sarcoplasmic reticulum

Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dt(max)) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca(2+)-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function.

Evaluation of serum nitrite, nitrate and malonyldialdehyde levels in preeclampsia

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca++ uptake by inside–out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49±3% in the preeclamptic women as compared to the gestational controls (8.06±0.11 nmol Ca++/mg protein min, gestational controls; 4.08±0.1 nmol Ca++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05±0.96 nmol Pi/mg protein min, gestational controls; 8.85±0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.

Anti-allergic effects of scoparone on mast cell-mediated allergy model

Scoparone is known to have a wide range of pharmacological properties in vitro. However, the roles of scoparone in immediate-type allergic reactions have not yet been investigated. In this study, we demonstrated that scoparone attenuated IgE-mediated allergic response in mast cells. Oral administration of scoparone inhibited passive cutaneous anaphylaxis in rats. Presence of scoparone dose-dependently decreased histamine release from rat peritoneal mast cells (RPMC) stimulated by anti-dinitrophenyl IgE. Moreover, scoparone reduced the expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-6 in RPMC. Pretreatment with scoparone inhibited the calcium uptake and p38 mitogen-activated protein kinase (MAPK) activity. Furthermore, scoparone blocked translocation of nuclear factor-kappa B (NF-kappaB) p65 subunit by suppressing IkappaBalpha phosphorylation in RPMC. Reduced calcium uptake as well as the suppressed activity of p38 MAPK and NF-kappaB might be involved in the inhibitory effect of scoparone on the secretory response. Our findings suggest that scoparone may serve as an effective therapeutic agent for allergic diseases.

Regulation of store‐operated calcium entries and mitochondrial uptake by minidystrophin expression in cultured myotubes

Defective expression of dystrophin in muscle cells is the primary feature of Duchenne muscular dystrophy (DMD), which is accompanied by fiber necrosis and intracellular calcium mishandling. These features led to the hypothesis that dystrophin could control calcium movements. Calcium mishandling in human DMD myotubes is dependent on contraction and/or calcium release activity, suggesting the involvement of channels being activated during these processes. Forced expression of minidystrophin at the plasma membrane of dystrophin-deficient Sol8 myotubes reactivates appropriate sarcolemmal expression of dystrophin-associated proteins and results in normal calcium homeostasis. In active dystrophic myotubes, store-operated calcium channels could be responsible for a sustained calcium influx in muscle cells. We show here that depletion of calcium stores (sarcoplasmic reticulum) by repetitive activation of calcium release and blockade of SERCA leads to a calcium influx. In myotubes expressing recombinant minidystrophin, these store-dependent influxes were reduced to a level similar to that observed in myotubes expressing native dystrophin. High store-dependent calcium influxes in dystrophin-deficient myotubes were associated with sustained cytosolic calcium transients and high intramitochondrial entries, while lower store-dependent calcium influx in myotubes expressing minidystrophin resulted in shorter calcium transients and reduced calcium uptake into mitochondria. We propose that minidystrophin negatively regulates sarcolemmal store-dependent calcium channels, which reduces store-dependent calcium influx, as well as its mitochondrial uptake. Forced expression of minidystrophin in dystrophic cells might restore the regulation of sarcolemmal store-dependent channels, which could protect against calcium mishandling.

Regulation of N-Methyl-D-aspartate Receptors by Calpain in Cortical Neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.

Lipid peroxidation and active calcium transport in inside–out vesicles of red blood cells from preeclamptic women

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca ++ uptake by inside–out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49±3% in the preeclamptic women as compared to the gestational controls (8.06±0.11 nmol Ca ++/mg protein min, gestational controls; 4.08±0.1 nmol Ca ++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05±0.96 nmol Pi/mg protein min, gestational controls; 8.85±0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.