Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca²⁺-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca²⁺ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca²⁺ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca²⁺ entry were investigated in Fura-2 Ca²⁺-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca²⁺ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S₁₁₇₂A mutant displayed enhanced Ca²⁺ entry. The TRPM3-mediated Ca²⁺ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.     

Chlorogenic Acid Decreases Glutamate Release from Rat Cortical Nerve Terminals by P/Q-Type Ca2+ Channel Suppression: A Possible Neuroprotective Mechanism

The glutamatergic neurotransmitter system has received substantial attention in research on the pathophysiology and treatment of neurological disorders. The study investigated the effect of the polyphenolic compound chlorogenic acid (CGA) on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). CGA inhibited 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes. This inhibition was prevented in the absence of extracellular Ca²⁺ and was associated with the inhibition of 4-AP-induced elevation of Ca²⁺ but was not attributed to changes in synaptosomal membrane potential. In line with evidence observed through molecular docking, CGA did not inhibit glutamate release in the presence of P/Q-type Ca²⁺ channel inhibitors; therefore, CGA-induced inhibition of glutamate release may be mediated by P/Q-type Ca²⁺ channels. CGA-induced inhibition of glutamate release was also diminished by the calmodulin and Ca²⁺/calmodilin-dependent kinase II (CaMKII) inhibitors, and CGA reduced the phosphorylation of CaMKII and its substrate, synapsin I. Furthermore, pretreatment with intraperitoneal CGA injection attenuated the glutamate increment and neuronal damage in the rat cortex that were induced by kainic acid administration. These results indicate that CGA inhibits glutamate release from cortical synaptosomes by suppressing P/Q-type Ca²⁺ channels and CaMKII/synapsin I pathways, thereby preventing excitotoxic damage to cortical neurons.     

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition,Driving Impaired Ca2+ Handling and Arrhythmogenesis

Phospholamban (PLN), a key modulator of Ca²⁺-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca²⁺-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a’s Ca²⁺-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca²⁺-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients.     

Skeletal Ryanodine Receptors Are Involved in Impaired Myogenic Differentiation in Duchenne Muscular Dystrophy Patients

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting following repeated muscle damage and inadequate regeneration. Impaired myogenesis and differentiation play a major role in DMD as well as intracellular calcium (Ca²⁺) mishandling. Ca²⁺ release from the sarcoplasmic reticulum is mostly mediated by the type 1 ryanodine receptor (RYR1) that is required for skeletal muscle differentiation in animals. The study objective was to determine whether altered RYR1-mediated Ca²⁺ release contributes to myogenic differentiation impairment in DMD patients. The comparison of primary cultured myoblasts from six boys with DMD and five healthy controls highlighted delayed myoblast differentiation in DMD. Silencing RYR1 expression using specific si-RNA in a healthy control induced a similar delayed differentiation. In DMD myotubes, resting intracellular Ca²⁺ concentration was increased, but RYR1-mediated Ca²⁺ release was not changed compared with control myotubes. Incubation with the RYR-calstabin interaction stabilizer S107 decreased resting Ca²⁺ concentration in DMD myotubes to control values and improved calstabin1 binding to the RYR1 complex. S107 also improved myogenic differentiation in DMD. Furthermore, intracellular Ca²⁺ concentration was correlated with endomysial fibrosis, which is the only myopathologic parameter associated with poor motor outcome in patients with DMD. This suggested a potential relationship between RYR1 dysfunction and motor impairment. Our study highlights RYR1-mediated Ca²⁺ leakage in human DMD myotubes and its key role in myogenic differentiation impairment. RYR1 stabilization may be an interesting adjunctive therapeutic strategy in DMD.     

Different pools of esterified arachidonic acid in rabbit kidney medulla: Relationship to Ca2+-stimulated prostaglandin biosynthesis

We investigated the effect of Ca²⁺ ions on renal medulla metabolism of endogenous esterified arachidonic acid in contrast to that of radioactive arachidonate incorporated into medullary lipids. Some striking differences between the release of unlabeled prostaglandin E₂ and of¹⁴C-labeled prostaglandin E₂ and arachidonic acid were seen in incubations in absence or presence of Ca²⁺ ions. These differences indicated that exogenous [¹⁴C] arachidonate incubated with medulla slices is incorporated into both Ca²⁺-sensitive and Ca²⁺-insensitive lipid pools of esterified arachidonate and furthermore, the Ca²⁺-sensitive pool is itself heterogeneous and consists of at least 2 functionally different lipid pools of esterified arachidonate. The first Ca²⁺-sensitive pool is characterized by a higher arachidonate turnover rate and incorporates more rapidly added radioactive arachidonate. The acylhydrolase activity which releases arachidonate from this pool is not efficiently coupled to prostaglandin endoperoxide synthase. In contrast, the second Ca²⁺-sensitive lipid pool has a slower arachidonate turnover rate and, consequently, a slower incorporation of added¹⁴C-acid. The acylhydrolase activity associated with this pool is more tightly coupled to prostaglandin endoperoxide synthase, so that a higher portion of released arachidonate is converted to prostaglandin E₂. Studies on arachidonic acid metabolic transformations using exogenously radioactive free acid added to tissues should therefore be inerpreted with caution because the results obtained may not reflect accurately the metabolic fate of endogenous, lipid-esterified arachidonate which is released and metabolized under physiological conditions in vivo.     

Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus

Calcium (Ca²⁺) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca²⁺ toolkit and its fine subcellular compartmentalization. Study of the role of Ca²⁺ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca²⁺]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca²⁺] changes. To this aim, as nucleoplasm Ca²⁺ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca²⁺ probe. In particular, we modified the previously described nuclear Ca²⁺ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca²⁺ measurements. The affinity for Ca²⁺ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca²⁺ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca²⁺ signal dynamics in different subcellular compartments in the very same cells are also presented.     

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.     

IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP₃ receptors /IP₃R) via its coiled-coil domain and modulates Ca²⁺ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca²⁺ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP₃ receptors and its impact on intracellular Ca²⁺ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP₃R and altered Ca²⁺ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca²⁺, suggesting that IRAG2 leads to activation of IP₃R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca²⁺ signaling, which regulates exocrine pancreatic function.     

Comprehensive Simulation of Ca2+ Transients in the Continuum of Mouse Skeletal Muscle Fiber Types

Mag-Fluo-4 has revealed differences in the kinetics of the Ca²⁺ transients of mammalian fiber types (I, IIA, IIX, and IIB). We simulated the changes in [Ca²⁺] through the sarcomere of these four fiber types, considering classical (troponin –Tn–, parvalbumin –Pv–, adenosine triphosphate –ATP–, sarcoplasmic reticulum Ca²⁺ pump –SERCA–, and dye) and new (mitochondria –MITO–, Na⁺/Ca²⁺ exchanger –NCX–, and store-operated calcium entry –SOCE–) Ca²⁺ binding sites, during single and tetanic stimulation. We found that during a single twitch, the sarcoplasmic peak [Ca²⁺] for fibers type IIB and IIX was around 16 µM, and for fibers type I and IIA reached 10–13 µM. The release rate in fibers type I, IIA, IIX, and IIB was 64.8, 153.6, 238.8, and 244.5 µM ms⁻¹, respectively. Both the pattern of change and the peak concentrations of the Ca²⁺-bound species in the sarcoplasm (Tn, PV, ATP, and dye), the sarcolemma (NCX, SOCE), and the SR (SERCA) showed the order IIB ≥ IIX > IIA > I. The capacity of the NCX was 2.5, 1.3, 0.9, and 0.8% of the capacity of SERCA, for fibers type I, IIA, IIX, and IIB, respectively. MITO peak [Ca²⁺] ranged from 0.93 to 0.23 µM, in fibers type I and IIB, respectively, while intermediate values were obtained in fibers IIA and IIX. The latter numbers doubled during tetanic stimulation. In conclusion, we presented a comprehensive mathematical model of the excitation–contraction coupling that integrated most classical and novel Ca²⁺ handling mechanisms, overcoming the limitations of the fast- vs. slow-fibers dichotomy and the use of slow dyes.     

Metabolism of phosphatidylinositol in plasma membranes and synaptosomes of rat cerebral cortex: A comparison between endogenous Vs exogenous substrate pools

The metabolism of phosphatidylinositols (PI) labeled with [¹⁴C]arachidonic acid within plasma membranes or synaptosomes was compared to the metabolism of PI prelabeled with [¹⁴C]arachidonic acid and added exogenously to the same membranes. Incubation of membranes containing the endogenously-labeled PI pool in the presence of Ca²⁺ resulted in the release of labeled arachidonic acid, as well as a small amount of labeled diacylglycerol. Labeled arachidonic acid was effectively reutilized and returned to the membrane phospholipids in the presence of adenosine triphosphate (ATP), CoA, and lysoPI. Although Ca²⁺ promoted the release of labeled diacylglycerol from prelabeled plasma membranes, this amount was only 17% of the maximal release, i.e., release in the presence of deoxycholate and Ca²⁺. This latter condition is known to fully activate the PI-phospholipase C, and incubation of prelabeled plasma membranes resulted in a six-fold increase in labeled diacylglycerols. On the other hand, when exogenously labeled PI were incubated with plasma membranes in the presence of Ca²⁺, the labeled diacylglycerols released were 59% of that compared to the fully activated condition. The phospholipase C action was calcium-dependent, regardless of whether exogenous or endogenous substrates were used in the incubation. In contrast to plasma membranes, intact synaptosomes had limited ability to metabolize exogenous PI even in the presence of Ca²⁺, although the activity of phospholipase C was similar to that in the plasma membranes when assayed in the presence of deoxycholate and Ca²⁺. These results suggest that discrete pools of PI are present in plasma membranes, and that the pool associated with the acyltransferase is apparently not readily accessible to hydrolysis by phospholipase C.     

Electrophysiological Properties of Endogenous Single Ca2+ Activated Cl− Channels Induced by Local Ca2+ Entry in HEK293

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca²⁺ entry (SOCE). Ca²⁺ release through inositol 1,4,5-trisphosphate receptor (IP₃R) and subsequent Ca²⁺ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca²⁺, which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca²⁺ entry through TRPC1 channels activated endogenous Ca²⁺-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca²⁺ concentration and/or transmembrane potential, conversely lowering of intracellular Ca²⁺ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca²⁺ concentration. Experiments with Ca²⁺ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca²⁺-activated chloride channels are involved in Ca²⁺ entry microdomains.     

Enmein Decreases Synaptic Glutamate Release and Protects against Kainic Acid-Induced Brain Injury in Rats

This study investigated the effects of enmein, an active constituent of Isodon japonicus Hara, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) and evaluated its neuroprotective potential in a rat model of kainic acid (KA)-induced glutamate excitotoxicity. Enmein inhibited depolarization-induced glutamate release, FM1-43 release, and Ca²⁺ elevation in cortical nerve terminals but had no effect on the membrane potential. Removing extracellular Ca²⁺ and blocking vesicular glutamate transporters, N- and P/Q-type Ca²⁺ channels, or protein kinase C (PKC) prevented the inhibition of glutamate release by enmein. Enmein also decreased the phosphorylation of PKC, PKC-α, and myristoylated alanine-rich C kinase substrates in synaptosomes. In the KA rat model, intraperitoneal administration of enmein 30 min before intraperitoneal injection of KA reduced neuronal cell death, glial cell activation, and glutamate elevation in the hippocampus. Furthermore, in the hippocampi of KA rats, enmein increased the expression of synaptic markers (synaptophysin and postsynaptic density protein 95) and excitatory amino acid transporters 2 and 3, which are responsible for glutamate clearance, whereas enmein decreased the expression of glial fibrillary acidic protein (GFAP) and CD11b. These results indicate that enmein not only inhibited glutamate release from cortical synaptosomes by suppressing Ca²⁺ influx and PKC but also increased KA-induced hippocampal neuronal death by suppressing gliosis and decreasing glutamate levels by increasing glutamate uptake.     

Efficient and tunable Mn2+ sensitized luminescence via energy transfer of a novel red phosphor Ca19Mn2(PO4)14: Eu2+ for white LED

The exploration of efficient and high-purity red phosphors is an urgent need in LED development. Due to the compact and compositional-tunable structure of whitlockite compound, manganese-based Ca₁₉Mn₂(PO₄)₁₄ is chosen as phosphor host for Eu²⁺ sensitization. Rietveld refinement, steady-state spectra, decay lifetime analysis and temperature-dependent emission spectra were investigated and clearly discussed. Under 360 nm excitation, Ca₁₉Mn₂(PO₄)₁₄: Eu²⁺ shows a strong Mn²⁺ sensitized emission at 655 nm with FWHM of 82 nm, benefiting from the short-distance-induced high-efficient Eu² -Mn²⁺ energy transfer. Emission engineering of Ca₁₉Mn₂(PO₄)₁₄: Eu²⁺ is achieved by Sr²⁺ co-doping, leading to both tunable peak wavelength (ranging from 650 to 610 nm) and improved intensity (130% of original value). Moreover, Ca₁₉Mn₂(PO₄)₁₄: Eu²⁺ exhibits a promising thermal stability where only 40% of emission intensity is lost at 200 °C. Finally, we explored the working performance of the fabricated RGB phosphor-converted white LED. The present work indicates that Ca₁₉Mn₂(PO₄)₁₄: Eu²⁺ phosphor is of great potential as a promising and efficient red phosphor in phosphor-converted white LED.     

The Effect of Isosaponarin Derived from Wasabi Leaves on Glutamate Release in Rat Synaptosomes and Its Underlying Mechanism

Excessive glutamate release is known to be involved in the pathogenesis of neurological diseases, and suppression of glutamate release from nerve terminals is considered to be a treatment strategy. In this study, we investigated whether isosaponarin, a flavone glycoside isolated from wasabi leaves, could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by an online enzyme-coupled fluorimetric assay. Isosaponarin produced a concentration-dependent inhibition of 4-AP-evoked glutamate release with a half-maximum inhibition of release value of 22 μM. The inhibition caused by isosaponarin was prevented by eliminating extracellular Ca²⁺ or by using bafilomycin A1, an inhibitor of synaptic vesicle exocytosis. Isosaponarin decreased intrasynaptosomal rises in Ca²⁺ levels that were induced by 4-AP, without affecting the synaptosomal membrane potential. The isosaponarin-induced inhibition of glutamate release was significantly prevented in synaptosomes that were pretreated with a combination of the calcium channel blockers ω-conotoxin GVIA (N-type) and ω-agatoxin IVA (P/Q-types). The protein kinase C (PKC) pan-inhibitor GF109203X and the Ca²⁺-dependent PKC inhibitor Go6976 abolished the inhibition of glutamate release by isosaponarin, while the Ca²⁺-independent PKC inhibitor rottlerin did not show any effect. The results from immunoblotting assays also showed that isosaponarin lowered PKC, PKCα, synaptosomal-associated protein of 25 kDa (SNAP-25), and myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation induced by 4-AP. In addition, FM1-43-labeled synaptic vesicles in synaptosomes showed that treatment with isosaponarin resulted in an attenuation of the 4-AP-induced decrease in fluorescence intensity that is consistent with glutamate release. Transmission electron microscopy of synaptosomes also provided evidence that isosaponarin altered the number of synaptic vesicles. These results indicate that isosaponarin suppresses the Ca²⁺-dependent PKC/SNAP-25 and MARCKS pathways in synaptosomes, causing a decrease in the number of available synaptic vesicles, which inhibits vesicular glutamate release from synaptosomes.     

Enhancement by ganglioside GT1b of annexin I phosphorylation in bovine mammary gland in the presence of phosphatidylserine and Ca2+

Ganglioside GT1b and, to a lesser extent, GD3, enhanced phosphorylation of a 36 kDa protein (the substrate of protein kinase C) in the particulate fraction from bovine mammary gland. Sialic acids, asialogangliosides, and GM3 were without effect, and GD1a conversely inhibited phosphorylation of the 36 kDa protein. The enhanced phosphorylation by GT1b required the simultaneous presence of phosphatidylserine (PS) and Ca²⁺. The 36 kDa protein reacted with anti-annexin I in Western blot analysis. Addition of purified annexin I to the reaction mixture containing the particulate fraction increased the extent of phosphorylated 36 kDa protein, and the phosphorylation was further enhanced by GT1b. The enhanced phosphorylation of annexin I by GT1b was also dependent on PS and Ca²⁺. When annexin I was phosphorylated by purified protein kinase C, GT1b inhibited the annexin I phosphorylation. Addition of epidermal growth factor or insulin to the particulate fraction had little effect on the enhancement. These results suggest that an enzyme or enzymes other than protein kinase C, epidermal growth factor receptor kinase, or insulin receptor kinase is responsible for the GT1b- and GD3-enhanced phosphorylation of annexin I in the presence of PS and Ca²⁺.     

Calcium ion modulates protein release from chitosan‐hyaluronic acid poly electrolyte gel

Polyelectrolyte complex (PEC) of chitosan (CH) and hyaluronic acid (HA) are widely used for skin, cartilage, and bone tissue engineering. However, no reports are seen on their response at high ionic media, like increased Ca²⁺ where they are likely to be exposed in the form of bone constructs and the influence of these ions on modulating the release of incorporated entities such as drugs and growth factors. Here, we prepared freeze dried scaffolds of PEC of CH and HA (CH‐HA) and characterized them by FTIR, TGA, SEM, and ESEM. FITC conjugated BSA, designated as FA, was incorporated into the PEC to study the release properties in response to Ca²⁺. The swellability of CH‐HA and the extent of drug release from the matrix, FA loaded CH‐HA was studied in deionised water and aqueous Na⁺ and Ca²⁺ solutions. Swelling and drug release were high for the matrix in aqueous Ca²⁺ whereas it was remarkably low in water and Na⁺. Drug released was found to increase with concentrations of Ca²⁺ (0.02–1.0M) indicating that CH‐HA is a promising matrix for Ca²⁺ responsive delivery of agents to accelerate healing of bone cracks, which is known to release high amount of Ca²⁺. POLYM. ENG. SCI., 55:2089–2097, 2015. © 2015 Society of Plastics Engineers     

PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca2+ Imbalance

PSMD14, a subunit of the 19S regulatory particles of the 26S proteasome, was recently identified as a potential prognostic marker and therapeutic target in diverse human cancers. Here, we show that the silencing and pharmacological blockade of PSMD14 in MDA-MB 435S breast cancer cells induce paraptosis, a non-apoptotic cell death mode characterized by extensive vacuolation derived from the endoplasmic reticulum (ER) and mitochondria. The PSMD14 inhibitor, capzimin (CZM), inhibits proteasome activity but differs from the 20S proteasome subunit-inhibiting bortezomib (Bz) in that it does not induce aggresome formation or Nrf1 upregulation, which underlie Bz resistance in cancer cells. In addition to proteasome inhibition, the release of Ca²⁺ from the ER into the cytosol critically contributes to CZM-induced paraptosis. Induction of paraptosis by targeting PSMD14 may provide an attractive therapeutic strategy against cancer cells resistant to proteasome inhibitors or pro-apoptotic drugs.     

The dissolution behavior in alkaline solutions of a borosilicate glass with and without P2O5

There are a variety of applications for glasses in alkaline environments, including glass fibers and glass‐coated steel to reinforce concrete structures. To understand how a simple glass reacts in such environments, the dissolution behavior of a 25Na₂O–25B₂O₃–50SiO₂ (mol%) glass, doped with and without 3 mol% P₂O₅, in pH 12 KOH and pH 12 KOH saturated with Ca²⁺ ions was studied. Ca²⁺ ions in the solution significantly reduce the glass dissolution rate by forming a passivating calcium silicate hydrate (C–S–H) gel layer on the glass surface. When these corroded glasses were then exposed to Ca‐free KOH, the C–S–H layer redissolves into the undersaturated solution and the glass dissolution rate increases. For phosphate‐doped borosilicate glass, PO₄^3? units released from the dissolving glass react with Ca²⁺ ions in saturated solutions to form crystalline hydroxylapatite on the glass surface, but this layer does not protect the glass from corrosion as well as the C–S–H does. The nature of the C–S–H layer was characterized by Raman spectroscopy, which reveals a gel layer constituted mainly of silicate anions.     

The Mechanisms Underlying the Protective Action of Selenium Nanoparticles against Ischemia/Reoxygenation Are Mediated by the Activation of the Ca2+ Signaling System of Astrocytes and Reactive Astrogliosis

In recent years, much attention has been paid to the study of the therapeutic effect of the microelement selenium, its compounds, especially selenium nanoparticles, with a large number of works devoted to their anticancer effects. Studies proving the neuroprotective properties of selenium nanoparticles in various neurodegenerative diseases began to appear only in the last 5 years. Nevertheless, the mechanisms of the neuroprotective action of selenium nanoparticles under conditions of ischemia and reoxygenation remain unexplored, especially for intracellular Ca²⁺ signaling and neuroglial interactions. This work is devoted to the study of the cytoprotective mechanisms of selenium nanoparticles in the neuroglial networks of the cerebral cortex under conditions of ischemia/reoxygenation. It was shown for the first time that selenium nanoparticles dose-dependently induce the generation of Ca²⁺ signals selectively in astrocytes obtained from different parts of the brain. The generation of these Ca²⁺ signals by astrocytes occurs through the release of Ca²⁺ ions from the endoplasmic reticulum through the IP3 receptor upon activation of the phosphoinositide signaling pathway. An increase in the concentration of cytosolic Ca²⁺ in astrocytes leads to the opening of connexin Cx43 hemichannels and the release of ATP and lactate into the extracellular medium, which trigger paracrine activation of the astrocytic network through purinergic receptors. Incubation of cerebral cortex cells with selenium nanoparticles suppresses ischemia-induced increase in cytosolic Ca²⁺ and necrotic cell death. Activation of A2 reactive astrocytes exclusively after ischemia/reoxygenation, a decrease in the expression level of a number of proapoptotic and proinflammatory genes, an increase in lactate release by astrocytes, and suppression of the hyperexcitation of neuronal networks formed the basis of the cytoprotective effect of selenium nanoparticles in our studies.