Increase In Ca2 Research Articles

Pathological Mechanisms Induced by TRPM2 Ion Channels Activation in Renal Ischemia-Reperfusion Injury.

Renal ischemia-reperfusion (IR) injury triggers a cascade of signaling reactions involving an increase in Ca2 + charge and reactive oxygen species (ROS) levels resulting in necrosis, inflammation, apoptosis, and subsequently acute kidney injury (AKI).Transient receptor potential (TRP) channels include an essential class of Ca2+ permeable cation channels, which are segregated into six main channels: the canonical channel (TRPC), the vanilloid-related channel (TRPV), the melastatin-related channel (TRPM), the ankyrin-related channel (TRPA), the mucolipin-related channel (TRPML) and polycystin-related channel (TRPP) or polycystic kidney disease protein (PKD2). TRP channels are involved in adjusting vascular tone, vascular permeability, cell volume, proliferation, secretion, angiogenesis and apoptosis.TRPM channels include eight isoforms (TRPM1-TRPM8) and TRPM2 is the second member of this subfamily that has been expressed in various tissues and organs such as the brain, heart, kidney and lung. Renal TRPM2 channels have an important role in renal IR damage. So that TRPM2 deficient mice are resistant to renal IR injury. TRPM2 channels are triggered by several chemicals including hydrogen peroxide, Ca2+, and cyclic adenosine diphosphate (ADP) ribose (cADPR) that are generated during AKI caused by IR injury, as well as being implicated in cell death caused by oxidative stress, inflammation, and apoptosis.

µ-Opioid Receptor Activation Reduces Glutamate Release in the PreBötzinger Complex in Organotypic Slice Cultures.

The inspiratory rhythm generator, located in the brainstem preBötzinger complex (preBötC), is dependent on glutamatergic signaling and is affected profoundly by opioids. Here, we used organotypic slice cultures of the newborn mouse brainstem of either sex in combination with genetically encoded sensors for Ca2+, glutamate, and GABA to visualize Ca2+, glutamatergic and GABAergic signaling during spontaneous rhythm and in the presence of DAMGO. During spontaneous rhythm, the glutamate sensor SF-iGluSnFR.A184S revealed punctate synapse-like fluorescent signals along dendrites and somas in the preBötC with decay times that were prolonged by the glutamate uptake blocker (TFB-TBOA). The GABA sensor iGABASnFR showed a more diffuse fluorescent signal during spontaneous rhythm. Rhythmic Ca2+- and glutamate transients had an inverse relationship between the spontaneous burst frequency and the burst amplitude of the Ca2+ and glutamate signals. A similar inverse relationship was observed when bath applied DAMGO reduced spontaneous burst frequency and increased the burst amplitude of Ca2+, glutamate, and GABA transient signals. However, a hypoxic challenge reduced both burst frequency and Ca2+ transient amplitude. Using a cocktail that blocked glutamatergic, GABAergic, and glycinergic transmission to indirectly measure the release of glutamate/GABA in response to an electrical stimulus, we found that DAMGO reduces the release of glutamate in the preBötC but has no effect on GABA release. This suggest that the opioid mediated slowing of respiratory rhythm involves presynaptic reduction of glutamate release, which would impact the ability of the network to engage in recurrent excitation, and may result in the opioid-induced slowing of inspiratory rhythm.SIGNIFICANCE STATEMENT Opioids slow down breathing rhythm by affecting neurons in the preBötzinger complex (preBötC) and other brainstem regions. Here, we used cultured slices of the preBötC to better understand this effect by optically recording Ca2+, glutamate, and GABA transients during preBötC activity. Spontaneous rhythm showed an inverse relationship between burst frequency and burst amplitude in the Ca2+ and glutamate signals. Application of the opioid DAMGO slowed the rhythm, with a concomitant increase in Ca2+, glutamate, and GABA signals. When rhythm was blocked pharmacologically, DAMGO reduced the presynaptic release of glutamate, but not GABA. These data suggest the mechanism of action of opioids involves presynaptic reduction of glutamate release, which may play an important role in the opioid-induced slowing of inspiratory rhythm.

Abstract 058: ACE2 Ubiquitination Regulates Neuronal Activity In Hypertension

ACE2 expression on GABAergic neurons could maintain the inhibitory tone over pre-sympathetic neurons in the paraventricular nucleus (PVN) and thus contribute to the regulation of blood pressure (BP). Here we investigated the role of ACE2 ubiquitination in the bed nucleus of the stria terminalis (BNST), a region containing GABAergic neurons and involved in cardiovascular responses to stress. C57BL6/J male mice were injected in the BNST with a ubiquitination-resistant human ACE2 mutant (AAV-hACE2-5R). A subset of mice was also injected with AAV-GCaMP6s-eYFP in the PVN and BNST to record intracellular Ca 2+ activity via fiber photometry. After 6 weeks, the mice were chronically infused with Ang-II (600 ng/kg/min) for 2 weeks while BP was monitored by telemetry. Ang-II-infusion resulted in a significant increase in mean BP compared to controls (152±7 vs. 123±4 mmHg, respectively; n=6, p<0.05) that was blunted in mice expressing hACE2-5R (133±5 mmHg, n=6, p<0.05). Injection of bicuculline (GABA A antagonist, 1 mmol/L in 100nL) in the PVN produced a blunted BP rise in Ang-II-infused mice compared to controls (AUC: 261±36 vs. 392±45, respectively; n=4, p<0.05) and an enhanced pressor response in the hACE2-5R+Ang-II group (AUC: 690±81, n=5, p<0.05). Acute injection of Ang-II (250 ng iv) caused an immediate increase in Ca 2+ mediated fluorescence in the PVN and a parallel reduction of neuronal activity in the BNST (ΔF/F 0 : +0.9±0.02 and -0.1±0.03; respectively). Surprisingly, hACE2-5R expression in the BNST produced a rapid and persistent inhibition of neuronal activity. Capillary Western analysis showed a 3-fold increase in NEDD4-2 (n=4, p<0.05) with a parallel reduction of ACE2 levels (-50%, n=4, p<0.05) in the BNST of Ang-II-infused mice, while hACE2-5R prevented NEDD4-2 upregulation and ACE2 downregulation (n=4, p<0.05). Our data show that increases in peripheral Ang-II levels result in an immediate regulation of neuronal activity in the PVN and BNST. Hypertension is associated with NEDD4-2 mediated ubiquitination of ACE2 blunting GABAergic input to the PVN and potentially increasing the activity of pre-sympathetic neurons. ACE2 ubiquitination by NEDD4-2 in key brain regions is a new mechanism contributing to the development of neurogenic hypertension.

Misfolded protein oligomers induce an increase of intracellular Ca2+ causing an escalation of reactive oxidative species

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.

Saponins from Camellia sinensis Seeds Stimulate GIP Secretion in Mice and STC-1 Cells via SGLT1 and TGR5.

Glucose-dependent insulinotropic polypeptide (GIP) is one of the important incretins and possesses lots of physiological activities such as stimulating insulin secretion and maintaining glucose homeostasis. The pentacyclic triterpenoid saponins are the major active ingredients in tea (Camellia sinensis) seeds. This study aimed to investigate the effect of tea seed saponins on the GIP secretion and related mechanisms. Our data showed that the total tea seed saponins (TSS, 65 mg/kg BW) and theasaponin E1 (TSE1, 2–4 µM) could increase the GIP mRNA and protein levels in mice and STC-1 cells. Phlorizin, the inhibitor of Sodium/glucose cotransporter 1 (SGLT1), reversed the TSE1-induced increase in Ca2+ and GIP mRNA level. In addition, TSE1 upregulated the protein expression of Takeda G protein-coupled receptor 5 (TGR5), and TGR5 siRNA significantly decreased GIP expression in TSE1-treated STC-1 cells. Network pharmacology analysis revealed that six proteins and five signaling pathways were associated with SGLT1, TGR5 and GIP regulated by TSE1. Taken together, tea seed saponins could stimulate GIP expression via SGLT1 and TGR5, and were promising natural active ingredients for improving metabolism and related diseases.

Segregated cation flux by TPC2 biases Ca2+ signaling through lysosomes

Two-pore channels are endo-lysosomal cation channels with malleable selectivity filters that drive endocytic ion flux and membrane traffic. Here we show that TPC2 can differentially regulate its cation permeability when co-activated by its endogenous ligands, NAADP and PI(3,5)P2. Whereas NAADP rendered the channel Ca2+-permeable and PI(3,5)P2 rendered the channel Na+-selective, a combination of the two increased Ca2+ but not Na+ flux. Mechanistically, this was due to an increase in Ca2+ permeability independent of changes in ion selectivity. Functionally, we show that cell permeable NAADP and PI(3,5)P2 mimetics synergistically activate native TPC2 channels in live cells, globalizing cytosolic Ca2+ signals and regulating lysosomal pH and motility. Our data reveal that flux of different ions through the same pore can be independently controlled and identify TPC2 as a likely coincidence detector that optimizes lysosomal Ca2+ signaling.

Effect and Mechanism of Ca2+ on the Corrosion of Hot-Rolled Strip in Turbid Ring Water Containing Cl− and SO2- 4

After the hot-rolled strip is cooled by laminar flow, Cl−, SO2- 4 and Ca2+ in turbid ring water will remain on the surface of the steel plate, which makes the steel plate corrode during storage in a humid environment. In order to clarify the effect and mechanism of residual Ca2+ on the corrosion of strip, simulations were carried out by setting different Ca2+ concentrations in solutions containing Cl− and SO2- 4 with different times of alternate immersion. Through the analyses of corrosion pit morphology, corrosion rate, electrochemical characteristics and corrosion products, it was found that the corrosion rate increased first and then decreased regardless of the Ca2+ concentration. When the Ca2+ concentration is 50~300 mg/L, the increase of Ca2+ concentration will increase the adsorption rate of the matrix and increase the corrosion pits. In addition, the corrosion rate decreases, the depth of corrosion pit becomes shallow and the polarization resistance increases due to the inhibition of the calcium salt layer, and the increase of Ca2+ concentration will enhance the inhibition of corrosion. When the Ca2+ concentration is 1000 mg/L, the denseness of the calcium salt layer is easily destroyed and the corrosion is intensified due to the large amount of adsorption of ions by the matrix.

Identification of OSCA gene family in Solanum habrochaites and its function analysis under stress

BackgroundOSCA (hyperosmolality-gated calcium-permeable channel) is a calcium permeable cation channel protein that plays an important role in regulating plant signal transduction. It is involved in sensing changes in extracellular osmotic potential and an increase in Ca2+ concentration. S. habrochaites is a good genetic material for crop improvement against cold, late blight, planthopper and other diseases. Till date, there is no report on OSCA in S. habrochaites. Thus, in this study, we performed a genome-wide screen to identify OSCA genes in S. habrochaites and characterized their responses to biotic and abiotic stresses.ResultsA total of 11 ShOSCA genes distributed on 8 chromosomes were identified. Subcellular localization analysis showed that all members of ShOSCA localized on the plasma membrane and contained multiple stress-related cis acting elements. We observed that genome-wide duplication (WGD) occurred in the genetic evolution of ShOSCA5 (Solhab04g250600) and ShOSCA11 (Solhab12g051500). In addition, repeat events play an important role in the expansion of OSCA gene family. OSCA gene family of S. habrochaites used the time lines of expression studies by qRT-PCR, do indicate OSCAs responded to biotic stress (Botrytis cinerea) and abiotic stress (drought, low temperature and abscisic acid (ABA)). Among them, the expression of ShOSCAs changed significantly under four stresses. The resistance of silencing ShOSCA3 plants to the four stresses was reduced.ConclusionThis study identified the OSCA gene family of S. habrochaites for the first time and analyzed ShOSCA3 has stronger resistance to low temperature, ABA and Botrytis cinerea stress. This study provides a theoretical basis for clarifying the biological function of OSCA, and lays a foundation for tomato crop improvement.

"Calcium bombs" as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions.

Excitotoxicity is thought to be an important factor in the onset and progression of amyotrophic lateral sclerosis (ALS). Evidence from human and animal studies also indicates that early signs of ALS include degeneration of motor nerve terminals at neuromuscular junctions (NMJs), before degeneration of motor neuron cell bodies. Here we used a model of excitotoxicity at NMJs in isolated mouse muscle, utilizing the organophosphorus (OP) compound omethoate, which inhibits acetylcholinesterase activity. Acute exposure to omethoate (100 μM) induced prolonged motor endplate contractures in response to brief tetanic nerve stimulation at 20–50 Hz. In some muscle fibers, Fluo-4 fluorescence showed association of these contractures with explosive increases in Ca2+ (“calcium bombs”) localized to motor endplates. Calcium bombs were strongly and selectively mitigated by increasing Mg2+ concentration in the bathing medium from 1 to 5 mM. Overnight culture of nerve-muscle preparations from WldS mice in omethoate or other OP insecticide components and their metabolites (dimethoate, cyclohexanone, and cyclohexanol) induced degeneration of NMJs. This degeneration was also strongly mitigated by increasing [Mg2+] from 1 to 5 mM. Thus, equivalent increases in extracellular [Mg2+] mitigated both post-synaptic calcium bombs and degeneration of NMJs. The data support a link between Ca2+ and excitotoxicity at NMJs and suggest that elevating extracellular [Mg2+] could be an effective intervention in treatment of synaptic pathology induced by excitotoxic triggers.

406 Modulation of calcium channel activity in Darier’s disease keratinocytes improves disease phenotypes

Darier’s disease (DD) is an autosomal dominant skin disorder characterized by acantholysis and dyskeratosis associated with mutations in ATP2A2 encoding for the sarco/endoplasmic reticulum Ca2+-ATPase pump type 2 (SERCA2), resulting in patients being functionally haploinsufficient for SERCA2. DD keratinocytes displayed a 1.5-fold increase in Ca2+ levels above that observed in control keratinocytes. This Ca2+ imbalance negatively influenced localization of protein constituents of the cell-cell adhesive junction, the desmosome, resulting in decreased desmosome function and loss of epidermal integrity.

Molecular and Cellular Response of the Myocardium (H9C2 Cells) Towards Hypoxia and HIF-1α Inhibition.

IntroductionOxidative phosphorylation is an essential feature of Animalian life. Multiple adaptations have developed to protect against hypoxia, including hypoxia-inducible-factors (HIFs). The major role of HIFs may be in protecting against oxidative stress, not the preservation of high-energy phosphates. The precise mechanism(s) of HIF protection is not completely understood.Materials and MethodsTo better understand the role of hypoxia-inducible-factor-1, we exposed heart/myocardium cells (H9c2) to both normoxia and hypoxia, as well as cobalt chloride (prolyl hydroxylase inhibitor), echniomycin (HIF inhibitor), A2P (anti-oxidant), and small interfering RNA to beclin-1. We measured cell viability, intracellular calcium and adenosine triphosphate, NADP/NADPH ratios, total intracellular reactive oxidative species levels, and markers of oxidative and antioxidant levels measured.ResultsHypoxia (1%) leads to increased intracellular Ca2+ levels, and this response was inhibited by A2P and echinomycin (ECM). Exposure of H9c2 cells to hypoxia also led to an increase in both mRNA and protein expression for Cav 1.2 and Cav 1.3. Exposure of H9c2 cells to hypoxia led to a decrease in intracellular ATP levels and a sharp reduction in total ROS, SOD, and CAT levels. The impact of hypoxia on ROS was reversed with HIF-1 inhibition through ECM. Exposure of H9c2 cells to hypoxia led to an increase in Hif1a, VEGF and EPO protein expression, as well as a decrease in mitochondrial DNA. Both A2P and ECM attenuated this response to varying degrees.ConclusionHypoxia leads to increased intracellular Ca2+, and inhibition of HIF-1 attenuates the increase in intracellular Ca2+ that occurs with hypoxia. HIF-1 expression leads to decreased adenosine triphosphate levels, but the role of HIF-1 on the production of reactive oxidative species remains uncertain. Anti-oxidants decrease HIF-1 expression in the setting of hypoxia and attenuate the increase in Ca2+ that occurs during hypoxia (with no effect during normoxia). Beclin-1 appears to drive autophagy in the setting of hypoxia (through ATG5) but not in normoxia. Additionally, Beclin-1 is a powerful driver of reactive oxidative species production and plays a role in ATP production. HIF-1 inhibition does not affect autophagy in the setting of hypoxia, suggesting that there are other drivers of autophagy that impact beclin-1.

Platelet-derived growth factor (PDGF)-BB regulates the airway tone via activation of MAP2K, thromboxane, actin polymerisation and Ca2+-sensitisation

BackgroundPDGFR-inhibition by the tyrosine kinase inhibitor (TKI) nintedanib attenuates the progress of idiopathic pulmonary fibrosis (IPF). However, the effects of PDGF-BB on the airway tone are almost unknown. We studied this issue and the mechanisms beyond, using isolated perfused lungs (IPL) of guinea pigs (GPs) and precision-cut lung slices (PCLS) of GPs and humans.MethodsIPL: PDGF-BB was perfused after or without pre-treatment with the TKI imatinib (perfused/nebulised) and its effects on the tidal volume (TV), the dynamic compliance (Cdyn) and the resistance were studied. PCLS (GP): The bronchoconstrictive effects of PDGF-BB and the mechanisms beyond were evaluated. PCLS (human): The bronchoconstrictive effects of PDGF-BB and the bronchorelaxant effects of imatinib were studied. All changes of the airway tone were measured by videomicroscopy and indicated as changes of the initial airway area.ResultsPCLS (GP/human): PDGF-BB lead to a contraction of airways. IPL: PDGF-BB decreased TV and Cdyn, whereas the resistance did not increase significantly. In both models, inhibition of PDGFR-(β) (imatinib/SU6668) prevented the bronchoconstrictive effect of PDGF-BB. The mechanisms beyond PDGF-BB-induced bronchoconstriction include activation of MAP2K and TP-receptors, actin polymerisation and Ca2+-sensitisation, whereas the increase of Ca2+ itself and the activation of EP1–4-receptors were not of relevance. In addition, imatinib relaxed pre-constricted human airways.ConclusionsPDGFR regulates the airway tone. In PCLS from GPs, this regulatory mechanism depends on the β-subunit. Hence, PDGFR-inhibition may not only represent a target to improve chronic airway disease such as IPF, but may also provide acute bronchodilation in asthma. Since asthma therapy uses topical application. This is even more relevant, as nebulisation of imatinib also appears to be effective.

The neuroprotective effect of Xylopia parviflora against aluminum chloride-induced neurotoxicity in rats

Neurodegenerative disease such as Alzheimer’s disease, are progressive disorders which has been linked to oxidative imbalance and associated perturbations characterised by loss of memory, cognition and cholinergic deficit. To date, cholinesterase inhibition and neuroprotection are the two major strategies in drug development. Xylopia parviflora (Annonacea family) is a spice consumed in Cameroon and has been used in traditional medicine to treat various pains. In this study, X. parviflora was evaluated on behavioural studies, ion homeostasis, cholinesterase inhibitory and antioxidant activities. Rats were exposed to aluminium chloride (75 mg/kg) during 60 days, and were treated with the extract of X. parviflora (150 and 300 mg/kg BW) and two drugs references (Donepezil and Curcumin). Behavioural parameters were assessed using the Morris-Maze test and the Open Field, followed by biochemical investigations, namely, cholinesterase enzyme activity (AChE and BChE), oxidative stress (NO, MDA, GSH level, SOD and Catalase activities) and ion homeostasis (Mg2+ and Ca2+ levels). AlCl3 administration shows a decrease in learning and memory improvement during behavioural studies, significant alteration of the central cholinergic system characterised by an increase in AChE and BChE activities to 2.72 ± 0.002 mol/min/g and 5.74 ± 0.12 mol/min/g respectively, disturbance of ion homeostasis with an increase in Ca2+ level (25.68 ± 3.78 μmol/mg protein) and a decrease in Mg2+ level (15.97 ± 2.05 μmol/mg protein) and an increase in oxidative stress compared to the positive control group. Treatment with the different doses of X. parviflora increased memory and improved locomotion, improved cholinesterase activities, ion homeostasis and stabilized brain oxidative stress levels. The study suggests that X. parviflora could potentially be used for the management of some biochemical alterations associated with Alzheimer’s disease. It could even be a good alternative to chemical drugs for neurotoxicity and memory enhancement.

Novel experimental evidence on the impact of surface carboxylic acid site density on the role of individual ions in the electrical behavior of crude oil/water

Perceiving the electrical behavior of the rock/brine and the crude oil/brine interfaces gives insight into the performance of engineered waterflooding. Compared to the rock surface, few studies have attempted to comprehend the complex behavior of the crude-oil surface electrical behavior. To reveal the impact of each ion on the surface charge of crude oil, the zeta potential of crude oil/single-salt brines (including NaCl, CaCl2, MgCl2, Na2SO4, and NaHCO3) was measured in a wide range of salinity. Then, the counterpart interfacial tension (IFT) was measured to determine the capability of each brine in bringing carboxylic acid groups from crude-oil bulk (COOH) to crude oil/brine interface (>COOH). Based on the values obtained for the zeta potential and IFT, chloride and sulfate ions show neutral affinity toward the charge of the crude-oil surface. The results showed that an increase in Ca2+ and Mg2+ results in higher values for zeta potential, which approves the adsorption of these divalent ions on the crude-oil surface. Also, the tendency of Mg2+ to be adsorbed on the >COO– sites is less than that of Ca2+ ions. The role of calcium and magnesium in crude oil/brine electrical behavior is not significantly affected by the variation of >COOH. On the contrary, the impact of Na+ on the zeta potential varies for different trends of >COOH density vs. salinity. In the case of constant IFT values for different salinities, an increase in Na+ concentration leads to higher zeta potential. The observed behavior is attributed to the shrinkage of the electrical double layer due to the higher ionic strength. Based on the results, an increase in Na+ does not lead to an increase in Na+ adsorption on the >COO–. Therefore, similar to Cl− and SO42−, Na+ is also neutral specious toward the charge of the crude-oil surface. However, as IFT decreases with salinity, an increase in Na+ concentration leads to a significant reduction of the zeta potential. The observed behavior is attributed to the transport of more carboxylic acid groups to the interface for the low IFT condition.

Selenoprotein K Is Essential for the Migration and Phagocytosis of Immature Dendritic Cells.

Selenoprotein K (SELENOK) is an endoplasmic reticulum stress (ERS)-regulated protein required for the calcium (Ca2+) flux-mediated migration of T cells and neutrophils, and the migration and phagocytosis of macrophages and microglia. However, the effect of SELENOK on the regulation of the immune function of dendritic cells (DCs), including immature DCs (imDCs) and mature DCs (mDCs), is still unclear. In this study, imDCs prepared from SELENOK knockout mice were used to evaluate the effect of SELENOK on the migration and phagocytosis of imDCs. The results showed that ERS-induced downregulation of imDCs phenotypic markers led to a reduction in Ras homolog gene family member A (RhoA)-dependent migration and enhanced Ca2+/CD205-mediated phagocytosis. SELENOK deficiency-induced upregulation of selenoprotein S (SELENOS) attenuated ERS levels in imDCs. An increase in Ca2+ levels resulted in increased migration and decreased phagocytosis with or without ERS conditions. The migration was RhoA-dependent, and Ca2+ or CD205 was associated with regulating phagocytosis in imDCs. Our study found that SELENOK is required for imDC migration and phagocytosis.

Ginsenoside Rg-1 prevents elevated cytosolic Ca2+ via store-operated Ca2+ entry in high-glucose–stimulated vascular endothelial and smooth muscle cells

BackgroundGinsenoside Rg-1 (Rg-1), a triterpenoid saponin abundantly present in Panax ginseng, is a type of naturally occurring steroid with known anti-diabetic and anti-inflammatory effects. In this study, we sought to confirm the effects and mechanisms of action of Rg-1 on store-operated Ca2+ entry (SOCE) in human vascular endothelial cell line (EA) and murine aortic vascular smooth muscle cell line (MOVAS) cells exposed to high glucose.MethodsCytosolic Ca2+ concentrations in EA and MOVAS cells were measured by monitoring fluorescence of the ratiometric Ca2+-indicator, Fura-2 AM.ResultsHigh glucose significantly increased Ca2+ influx by abnormally activating SOCE in EA and MOVAS cells. Notably, this high glucose-induced increase in SOCE was restored to normal levels in EA and MOVAS cells by Rg-1. Moreover, Rg-1 induced reductions in SOCE in cells exposed to high glucose were significantly inhibited by the plasma membrane Ca2+ ATPase (PMCA) blocker lanthanum, the Na+/K+-ATPase blocker ouabain, or the Na+/Ca2+ exchanger (NCX) blockers Ni2+ and KB-R7943. These observations suggest that the mechanism of action of Rg-1 inhibition of SOCE involves PMCA and Na+/K+-ATPase, and an increase in Ca2+ efflux via NCXs in both EA and MOVAS cells exposed to high glucose.ConclusionsThese findings indicate that Rg-1 may protect vascular endothelial and smooth muscle cells from Ca2+ increases following exposure to hyperglycemic conditions.

Ca2+ attenuates nucleation activity of leiomodin.

A transient increase in Ca2+ concentration in sarcomeres is essential for their proper function. Ca2+ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin‐binding protein C were also found to be Ca2+ sensitive. However, the effects of Ca2+ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene‐actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca2+. Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin‐binding sites binds Ca2+. We propose that Ca2+ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca2+ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function.Summary StatementCa2+ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca2+ on the activity of leiomodin—an actin nucleator and thin filament length regulator—have remained unknown. In this study, we demonstrate that Ca2+ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca2+ in the regulation of the sarcomeric protein interactions.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12

AbstractThe critical role of G protein–coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5′-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2−/− platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2−/− platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2−/− platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2−/− mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gβγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

Structure dependence of dielectric properties in Ca-doped bismuth magnesium niobate pyrochlores

The cubic pyrochlore Bi1.5MgNb1.5O7 (BMN) materials have large permittivity, good temperature stability, and extremely low dielectric loss. The structure and dielectric properties of Bi1.5Mg1−xCaxNb1.5O7 (BMCN, x = 0.0–0.5) are discussed systematically. All the samples except x = 0.5 were found that the dominant crystalline phase was cubic pyrochlore structure. The BMN (x = 0.0) cubic pyrochlore phase contained little impurity Mg4Nb2O9. Although Ca-doping inhibited the formation of the impurity phase Mg4Nb2O9, excessive Ca-doping (x ≥ 0.4) led to another second phase CaBi2Nb2O9 in BMCN structure. The substitution of Ca2+ brought about the reduction of structure disorder which were caused by abnormal cell shrinkage. It evidenced that Ca2+ ions are prone to substitute Mg2+ ions on A-site, then take on the B-site when the more Ca2+ ions doping in the pyrochlores. The permittivity of doped BMCN (x = 0.1–0.5) was smaller than that of BMN, and decreased with the increase of Ca2+ doping. The dielectric loss of doped BMCN increased due to the lattice defects caused by larger Ca2+ substitution for Mg2+, and the existence of the second phase with x = 0.4–0.5. The temperature dependence of the dielectric properties become unstable by Ca-doping. The Ca2+ substitution led to a closer association of BO6 octahedra, which had adverse impact on temperature stability of BMCN (x = 0.1–0.3). The coexistence of the second phase with positive temperature coefficient improved the dielectric temperature stability of the BMCN (x = 0.4–0.5). The dielectric loss was interpreted on the basis of the interfacial polarization at the grain-boundary region, which would become sensitive to temperature/frequency.

1454-P: A Transcriptomic Signature of Pancreatic Islet Leader Beta Cells

Pulsatile increases in Ca2+ within beta cells underlie the stimulation of insulin secretion at elevated glucose concentrations. Recent data obtained by imaging these transients in intact islets have revealed the existence of subpopulations of beta cells (“leaders”) which initiate Ca2+ waves across the syncytium and are critical for these dynamics. Whether leader cells possess unique transcriptional features is unknown. By combining Ca2+ imaging with cell labelling and isolation, we sought to address this question. Method: Islets were isolated from C57BL6/J mice expressing the Ca2+ probe GCaMP6f selectively in beta cells. After infection with an adenovirus expressing photo-activable- (PA) mCherry, Ca2+ imaging was performed at 11mM glucose at 5 Hz. Leader (or follower) cells were identified and photopainted by PA-mCherry activation at 405nm. After islet dispersal, cell sorting was used to isolate single cells. cDNA libraries were obtained using NEBNext Single cell kit and used for sequencing (Illumina HiSeq 4000) . Reads were aligned to the mmtranscriptome with Salmon v1.3 and differential expression analysis performed with DESeq2. Differentially expressed genes were subjected to Gene Ontology analysis. Results: cDNA was obtained from 13 leader cells, identified as those exhibiting the first Ca2+ signal increase during Ca2+ waves, and 9 follower cells. Of 96 differentially expressed genes, the most strongly affected included cell adhesion proteins (Cdh22; Scarf2) and the calcium sensor Mctp1. Other genes identified were involved in mitochondrial Ca2+ uptake (Mcub) , cytochrome assembly (Tmem177) and type 2 diabetes risk (Tcf7l2) . GO analysis identified 9 enriched functional clusters, including protein ubiquitination and metal ion binding. Conclusion: Leader cells possess a stably altered transcriptome, and the affected genes include several which may regulate cell adhesion. The extent to which the identified genes reflect the contribution of these cells to Ca2+ wave initiation requires further study. Disclosure P.L.Chabosseau: n/a. S.Yong: None. L.Lopez: None. I.Leclerc: None. Y.Ali: n/a. A.Martinez-sanchez: None. G.A.Rutter: Advisory Panel; Sun Pharmaceutical Industries Ltd., Research Support; Sun Pharmaceutical Industries Ltd.