• Development and Reproduction
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• v.24 no.4
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• pp.297-306
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• 2020

Repetitive changes in the intracellular calcium concentration ([Ca²⁺]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca²⁺]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca²⁺ ion elevation during [Ca²⁺]i oscillations are Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca²⁺ ion influx through Ca²⁺ channel on the plasma membrane. Ca²⁺ channels have been characterized into voltage-dependent Ca²⁺ channels (VDCCs), ligand-gated Ca²⁺ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca²⁺]i oscillation was observed in a Ca²⁺ contained medium with sperm factor or adenophostin A injection but disappeared in Ca²⁺ free medium. Ca²⁺ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca²⁺]i oscillation profiles in SrCl₂ treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca²⁺ influx is essential for [Ca²⁺]i oscillation during mammalian fertilization. This Ca²⁺ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

• Molecules and Cells
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• v.43 no.6
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• pp.572-580
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• 2020

Transient receptor potential ankyrin 1 from rattlesnakes (rsTRPA1) and boas (bTRPA1) was previously proposed to underlie thermo-sensitive infrared sensing based on transcript enrichment in infrared-sensing neurons and hyper-thermosensitivity expressed in Xenopus oocytes. It is unknown how these TRPA1s show thermosensitivities that overwhelm other thermoreceptors, and why rsTRPA1 is more thermosensitive than bTRPA1. Here, we show that snake TRPA1s differentially require Ca²⁺ for hyper-thermosensitivity and that predisposition to cytosolic Ca²⁺ potentiation correlates with superior thermosensitivity. Extracellularly applied Ca²⁺ upshifted the temperature coefficients (Q10s) of both TRPA1s, for which rsTRPA1, but not bTRPA1, requires cytosolic Ca²⁺. Intracellular Ca²⁺ chelation and substitutive mutations of the conserved cytosolic Ca²⁺-binding domain lowered rsTRPA1 thermosensitivity comparable to that of bTRPA1. Thapsigargin-evoked Ca²⁺ or calmodulin little affected rsTRPA1 activity or thermosensitivity, implying the importance of precise spatiotemporal action of Ca²⁺. Remarkably, a single rattlesnake-mimicking substitution in the conserved but presumably dormant cytosolic Ca²⁺-binding domain of bTRPA1 substantially enhanced thermosensitivity through cytosolic Ca²⁺ like rsTRPA1, indicating the capability of this single site in the determination of both cytosolic Ca²⁺ dependence and thermosensitivity. Collectively, these data suggest that Ca²⁺ is essential for the hyper-thermosensitivity of these TRPA1s, and cytosolic potentiation by permeating Ca²⁺ may contribute to the natural variation of infrared senses between rattlesnakes and boas.

• Molecules and Cells
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• v.43 no.1
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• pp.66-75
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• 2020

Saturated fatty acids contribute to β-cell dysfunction in the onset of type 2 diabetes mellitus. Cellular responses to lipotoxicity include oxidative stress, endoplasmic reticulum (ER) stress, and blockage of autophagy. Palmitate induces ER Ca²⁺ depletion followed by notable store-operated Ca²⁺ entry. Subsequent elevation of cytosolic Ca²⁺ can activate undesirable signaling pathways culminating in cell death. Mitochondrial Ca²⁺ uniporter (MCU) is the major route for Ca²⁺ uptake into the matrix and couples metabolism with insulin secretion. However, it has been unclear whether mitochondrial Ca²⁺ uptake plays a protective role or contributes to lipotoxicity. Here, we observed palmitate upregulated MCU protein expression in a mouse clonal β-cell, MIN6, under normal glucose, but not high glucose medium. Palmitate elevated baseline cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and reduced depolarization-triggered Ca²⁺ influx likely due to the inactivation of voltage-gated Ca²⁺ channels (VGCCs). Targeted reduction of MCU expression using RNA interference abolished mitochondrial superoxide production but exacerbated palmitate-induced [Ca²⁺]_i overload. Consequently, MCU knockdown aggravated blockage of autophagic degradation. In contrast, co-treatment with verapamil, a VGCC inhibitor, prevented palmitate-induced basal [Ca²⁺]_i elevation and defective [Ca²⁺]_i transients. Extracellular Ca²⁺ chelation as well as VGCC inhibitors effectively rescued autophagy defects and cytotoxicity. These observations suggest enhanced mitochondrial Ca²⁺ uptake via MCU upregulation is a mechanism by which pancreatic β-cells are able to alleviate cytosolic Ca²⁺ overload and its detrimental consequences.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.4
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• pp.335-344
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• 2024

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca²⁺ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca²⁺ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC₅₀ of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca²⁺ transients by DPI. The L-type Ca²⁺ current (I_Ca) was decreased concentration-dependently by DPI (IC₅₀ of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca²⁺ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca²⁺ and Na⁺. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca²⁺ channel and RyR, thereby attenuating Ca²⁺-induced Ca²⁺ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

• Korean Chemical Engineering Research
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• v.60 no.2
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• pp.175-183
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• 2022

With the expansion of the automobile market, the demand for Nd as an essential rare earth material for automobile motors is rapidly increasing. Research on the calcio-thermic reduction process between Nd₂O₃ and calcium-based alloys has been extensively studied in order to manufacture Nd. In this study, Ca-Cu, as a reducing for Nd₂O₃, was prepared by electrolysis in CaCl₂ molten salt. Cu wire and graphite were employed as a working electrode and a counter electrode for electrolysis reaction, respectively. The reference electrode was manufactured by putting Ag wire in a mixture of AgCl and CaCl₂ at a ratio of 1:99 mol%. The cyclic voltammetry results showed that the deposition of Ca²⁺ on the surface of working electrode was observed from a potential of -1.8 V, and the reduction potential of Ca²⁺ decreased as the reaction temperature increased. The diffusion coefficient of Ca²⁺ calculated by the chronoamperometry experiment was found to be 5.4(±6.8)×10^-6 cm²/s. In addition, Ca-Cu liquid alloy was prepared by applying a constant potential to Cu electrodes. The element ratio of Ca-Cu alloy formed by applying a potential of -2.0 V was found to Ca:Cu=1:4.

• International Journal of Oral Biology
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• v.47 no.3
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• pp.33-40
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• 2022

Store-operated Ca²⁺ entry (SOCE) represents one of the major Ca²⁺ entry routes in non-excitable cells. It is involved in a variety of fundamental biological processes and the maintenance of Ca²⁺ homeostasis. The Ca²⁺ release-activated Ca²⁺ (CRAC) channel consists of stromal interaction molecule and Orai; however, the role and action of Homer proteins as an adaptor protein to SOCE-mediated Ca²⁺ signaling through the activation of CRAC channels in non-excitable cells still remain unknown. In the present study, we investigated the role of Homer2 in the process of Ca²⁺ signaling induced by the interaction between CRACs and Homer2 proteins in non-excitable cells. The response to Ca²⁺ entry by thapsigargin-mediated Ca²⁺ store depletion remarkably decreased in pancreatic acinar cells of Homer2^-/- mice, as compared to wild-type cells. It also showed critical differences in regulated patterns by the specific blockers of SOCE in pancreatic acinar cells of Homer2^-/- mice. The response to Ca²⁺ entry by the depletion in Ca²⁺ store markedly increased in the cellular overexpression of Orai1 and STIM1 as compared to the overexpression of Homer2 in cells; however, this response was remarkably inhibited by the overexpression of Orai1, STIM1, and Homer2. These results suggest that Homer2 has a critical role in the regulatory action of SOCE activity and the interactions between CRAC channels.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.3
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• pp.219-225
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• 2022

Glucagon like peptide-1 (GLP-1) released from enteroendocine L-cells in the intestine has incretin effects due to its ability to amplify glucose-dependent insulin secretion. Promotion of an endogenous release of GLP-1 is one of therapeutic targets for type 2 diabetes mellitus. Although the secretion of GLP-1 in response to nutrient or neural stimuli can be triggered by cytosolic Ca²⁺ elevation, the stimulus-secretion pathway is not completely understood yet. Therefore, the aim of this study was to investigate the role of reverse Na⁺/Ca²⁺ exchanger (rNCX) in Ca²⁺ entry induced by muscarinic stimulation in NCI-H716 cells, a human enteroendocrine GLP-1 secreting cell line. Intracellular Ca²⁺ was repetitively oscillated by the perfusion of carbamylcholine (CCh), a muscarinic agonist. The oscillation of cytosolic Ca²⁺ was ceased by substituting extracellular Na⁺ with Li⁺ or NMG⁺. KB-R7943, a specific rNCX blocker, completely diminished CCh-induced cytosolic Ca²⁺ oscillation. Type 1 Na⁺/Ca²⁺ exchanger (NCX₁) proteins were expressed in NCI-H716 cells. These results suggest that rNCX might play a crucial role in Ca²⁺ entry induced by cholinergic stimulation in NCI-H716 cells, a GLP-1 secreting cell line.

• International Journal of Oral Biology
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• v.46 no.3
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• pp.134-139
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• 2021

Under physiological conditions, calcium (Ca²⁺) regulates essential functions of polarized secretory cells by the stimulation of specific Ca²⁺ signaling mechanisms, such as increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the store-operated Ca²⁺ entry (SOCE) and the receptor-operated Ca²⁺ entry (ROCE). Homer proteins are scaffold proteins that interact with G protein-coupled receptors, inositol 1,4,5-triphosphate (IP₃) receptors, Orai1-stromal interaction molecule 1, and transient receptor potential canonical (TRPC) channels. However, their role in the Ca²⁺ signaling in exocrine cells remains unknown. In this study, we investigated the role of Homer2 in the Ca²⁺ signaling and regulatory channels to mediate SOCE and ROCE in pancreatic acinar cells. Deletion of Homer2 (Homer2^-/-) markedly increased the expression of TRPC3, TRPC6, and Orai1 in pancreatic acinar cells, whereas these expressions showed no difference in whole brains of wild-type and Homer2^-/- mice. Furthermore, the response of Ca²⁺ entry by carbachol also showed significant changes to the patterns regulated by specific blockers of SOCE and ROCE in pancreatic acinar cells of Homer2^-/- mice. Thus, these results suggest that Homer2 plays a critical role in the regulatory action of the [Ca²⁺]_i via SOCE and ROCE in mouse pancreatic acinar cells.

• Journal of Interdisciplinary Genomics
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• v.5 no.2
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• pp.35-41
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• 2023

Background: Ca²⁺ signaling plays a vital role in neuronal signaling and altered Ca²⁺ homeostasis in Parkinson's disease (PD). Overexpression of αSYN significantly promote the Ca²⁺-Calmodulin (CaM) activity and subsequent nuclear translocation of nuclear factor of activated T cells (NFAT) transcription factor in dopaminergic neurons of midbrain. However, the exact role of Ca²⁺-CaM and NFAT in PD pathology is yet to be elucidated. Methods: We designed the CaM-NFAT-oligodeoxynucleotide (ODN), a synthetic short DNA containing complementary sequence for NFAT transcription factor and CaM mRNA. Then, the effect of CaM-NFAT-ODN on 1-methyl-4-phenylpyridinium (MPP⁺)-mediated neurotoxicity was investigated in mimic PD model in vitro. Results: First, the expression of αSYN and CaM was strongly increased in substantia nigra (SN) of PD and the expression of tyrosine hydroxylase (TH) was strongly increased in control SN. Additionally, the expression of apoptosis marker proteins was strongly increased in SN of PD. Transfection of CaM-NFAT-ODN repressed CaM and pNFAT, the target genes of this ODN in rat embryo primary mesencephalic neurons. It also reduced ERK phosphorylation, a downstream target of these genes. These results demonstrated that CaM-NFAT-ODN operated successfully in rat embryo primary mesencephalic neurons. Transfection of CaM-NFAT-ODN repressed TH reduction, αSYN accumulation, and apoptosis by MPP⁺-induced neurotoxicity response through Ca²⁺ signaling and mitogen-activated protein kinases (MAPK) signaling. Conclusion: Synthetic CaM-NFAT-ODN has substantial therapeutic feasibility for the treatment of neurodegenerative diseases.

• Clean Technology
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• v.25 no.4
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• pp.311-315
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• 2019

The cation extraction and impurity separation were studied in order to investigate the recyclability of a slag produced from the steel refinery industry. Two types of slag (Slag-A, B) were collected and characterized in this study. The initial characterization by X-ray diffraction (XRD) and X-ray fluorescence (XRF) confirmed the existence of various kinds of ions in the slag such as Ca²⁺ (30 ~ 40%), Fe³⁺ (20 ~ 30%), Si⁴⁺ (15%), Al³⁺ (10%), Mn²⁺ (7%), and Mg²⁺ (3 ~ 5%). Inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis on the extracted slag using 2 M HCl as a solvent indicated that a higher concentration of Ca²⁺ was extracted as the S/L ratio was increased. The Ca²⁺ extraction concentration were found to be 8,940 mg L^-1 (Slag-A) and 10,690 (Slag-B) mg L^-1 when the S/L ratio for Ca²⁺ extraction was 0.1. However, the extract was strongly acidic ( < pH 1) at 0.1 S/L. Also the other ions (impurities) were extracted simultaneously in addition to Ca²⁺. To increase the purity of Ca²⁺ in order to transform the slag to a high value resource, a pH-swing was conducted. The impurities tended to precipitate at higher rate as the pH was increased. Notably, the Ca²⁺ rapidly precipitated above a certain pH and at a pH of 10.5, while the selectivity of Ca²⁺ was over 99%. It is expected that the aqueous solution in which high contents of Ca²⁺ was selectively dissolved in this study would be suitable for the carbonation process for reducing CO₂ and for the production of calcium carbonate.