• The Korean Journal of Physiology
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• v.29 no.2
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• pp.269-277
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• 1995

Membrane vesicles were prepared by differential centrifugation from epithelial cells of porcine trachea. Total activity of microsomal ATPases was measured spectrophotometrically by a coupled enzyme assay. The steady-state activity of the enzyme was $329{\pm}10$ nmol/min mg protein. Thapsigargin, a specific antagonist of intracellular $Ca^{2+}-ATPase$, inhibited about 50% of the activity, leaving $178{\pm}18\;nmol/min .mg$ protein (n=6), indicating that the $Ca^{2+}-ATPase$ is one of the major microsomal ATPases. The microsomes used in this study appeared to be tight-sealed vesicles since they showed saturation in $^{45}Ca^{2+}$ uptake experiments. Inositol 1,4,5-trisphosphate $InsP_{3}, 4\;{\mu}M$, an agonist of $InsP_{3}$-sensitive $Ca^{2+}$ release channel ($InsP_{3}$, receptor), and Ca-ionophore A23187 $(10\;{\mu}M)$ induced $^{45}Ca^{2+}$ releases of 20% and 50% of stored $^{45}Ca^{2+}$, respectively. The addition of $(10\;{\mu}M\;InsP_{3}$ also increased the microsomal ATPase activity from $282{\pm}8$ nmol/min mg protein to $334{\pm}21$ nmol/min . mg protein in the intact vesicles. Similar increase in the activity was observed by making microsomes leaky (uncoupling) using the Ca-ionophore A23187. ;$InsP_{3}-induced$ effects were blocked by either thapsigargin or heparin suggesting that: 1) the $InsP_{3}-induced$ increase in ATPase activity is mediated by microsomal $Ca^{2+}-ATPase$, and 2) dissipation of $Ca^{2+}$ gradient across the microsomal membrane is responsible for the $InsP_{3}-induced$ effect. In order to test the dependence of the $Ca^{2+}-ATPase$ activity on the activity of $InsP_{3}-induced$ the activity of ATPases was monitored in various concentrations of free $Ca^{2+}$ using $EGTA-Ca^{2+}$ buffers. The $Ca^{2+}$-dependent biphasic change is the well-known character of $InsP_{3} receptor but not of microsomal $Ca^{2+}-ATPase$ in non-excitable cells; however, the activity of microsomal ATPase appeared biphasic and a maxim진 activity of $397{\pm}36nmol/min\;.mg$ protein was obtained in the solution containing 100 nM free $Ca^{2+}$. Below or above this concentration, the activity of ATPases was lower. These results strongly support a positive correlation of microsomal $Ca^{2+}-ATPase$ to the $InsP_{3}$ receptors in epithelial microsomes.

• Journal of Korean Dental Science
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• v.10 no.2
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• pp.45-52
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• 2017

Calcium has versatile roles in diverse physiological functions. Among these functions, intracellular $Ca^{2+}$ plays a key role during the secretion of salivary glands. In this review, we introduce the diverse cellular components involved in the saliva secretion and related dynamic intracellular $Ca^{2+}$ signals. Calcium acts as a critical second messenger for channel activation, protein translocation, and volume regulation, which are essential events for achieving the salivary secretion. In the secretory process, $Ca^{2+}$ activates $K^+$ and $Cl^-$ channels to transport water and electrolyte constituting whole saliva. We also focus on the $Ca^{2+}$ signals from intracellular stores with discussion about detailed molecular mechanism underlying the generation of characteristic $Ca^{2+}$ patterns. In particular, inositol triphosphate signal is a main trigger for inducing $Ca^{2+}$ signals required for the salivary gland functions. The biphasic response of inositol triphosphate receptor and $Ca^{2+}$ pumps generate a self-limiting pattern of $Ca^{2+}$ efflux, resulting in $Ca^{2+}$ oscillations. The regenerative $Ca^{2+}$ oscillations have been detected in salivary gland cells, but the exact mechanism and function of the signals need to be elucidated. In future, we expect that further investigations will be performed toward better understanding of the spatiotemporal role of $Ca^{2+}$ signals in regulating salivary secretion.

• Biomolecules & Therapeutics
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• v.22 no.3
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• pp.223-231
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• 2014

In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its $IC_{50}$ value was $175{\mu}g/ml$. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated $[CA^{2+}]_i$ mobilization and thromboxane $A_2$ ($TXA_2$) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated $[CA^{2+}]_i$ level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor ($IP_3R$) phosphorylation. These results suggest that the inhibition of $[CA^{2+}]_i$ mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of $IP_3R$. CE-WIB801C suppressed $TXA_2$ production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and $TXA_2$ synthase (TXAS). These results suggest that the inhibition of $TXA_2$ production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent $CA^{2+}$-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

• Korean Journal of Poultry Science
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• v.42 no.1
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• pp.27-32
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• 2015

In this study, we used two breeds of chicken to identify genomic regions corresponding to necrotic enteritis (NE) resistance. We scanned the genomes of a resistant and susceptible line of Fayoumi and White Leghorn chickens (20 birds/line) using a chicken 60 K Illumina SNP panel. A total of 235 loci with divergently fixed alleles were identified across the genome in both breeds; particularly, several clusters of multiple loci with fixed alleles were found in five narrow regions. Moreover, consensus 15-SNP haplotypes that were shared by the resistant lines of both breeds were identified on chromosomes 3, 7 and 9. Genes responsible for NE resistance were identified in chicken lines selected for resistance and susceptibility. Annotation of the regions spanning clustered divergently fixed regions revealed a set of interesting candidate genes such as phosphoinositide-3-kinase, regulatory subunit 5, p101 (PIK3R5) and inositol 1,4,5-trisphosphate receptor 1 (ITPR1), which participate in immune response. Consensus haplotypes were found in regions containing possibly relevant genes, such as myostatin and myosin, which play important roles in muscle development. Thus, genome scans of divergent selection in multiple chicken lines and breeds can be used to identify genomic regions associated with NE resistance.

• Biomedical Science Letters
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• v.29 no.4
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• pp.242-248
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• 2023

Platelets are activated at the sites of vascular injury by a number of molecules, including adenosine diphosphate, collagen and thrombin. The full platelet aggregation is absolutely essential for the normal hemostasis. Glycyrrhiza glabra is a well-known medicinal herb that grows in various parts of the world and is known to have various effects such as antioxidant, anti-inflammatory, anti-atherogenic, anti-osteoporotic and skin-whitening. However, the platelet inhibitory effect of Glycyrrhiza glabra extract (GGE) has not been identified. In this study, we investigated if GGE inhibited platelet aggregation. We observed that GGE inhibited collagen-induced platelet aggregation, Ca²⁺ mobilization, and thromboxane A2 generation. In addition, GGE suppressed phosphorylation of phosphatidylinositol-3 kinase (PI3K), Akt and elevated phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R), vasodilator stimulated phosphoprotein (VASP). Taken together, GGE showed strong antiplatelet effects and may be used to block platelet-mediated cardiovascular diseases.

• Applied Biological Chemistry
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• v.39 no.3
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• pp.189-194
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• 1996

The effects of scorpion (Leiurus quinquestriatus hebraeus, Lqh) venom were evaluated on the activities of microsomal $Ca^{2+}-ATPase$ and $Ca^{2+}$ release channel prepared from the epithelial cells of pig airway. Whole venom of Lqh $(120\;{\mu}g/ml)$ increased the activity of microsomal $Ca^{2+}-ATPase$ about 32% in the tight-sealed microsomes and about 28% in the Triton X-100-treated or $Ca^{2+}$ ionophore A23187-treated leaky microsomes. Thapsigargin, a specific antagonist of $Ca^{2+}-ATPase$, inhibited 42% of total ATPase activity and also completely blocked the effects of Lqh venom, suggesting that Lqh venom directly activiates the microsomal $Ca^{2+}-ATPase$. In order to determine if Lqh venom increases the microsomal uptake of $^{45}Ca^{2+}$, Lqh venom was added in the uptake medium. The Lqh venom increased microsomal $^{45}Ca^{2+}$ uptake up to ${\sim}20%$ and the increase was only observed when heparin, an antagonist of $InsP_3$ receptor channel, was added in the uptake medium. Lqh venom in the absence of heparin unexpectedly decreased the rate and the amount of $^{45}Ca^{2+}$ uptake. These results were explained by simultaneous increases in $^{45}Ca^{2+}$ release as well as $^{45}Ca^{2+}$ uptake by Lqh venom. Lqh venom itself increased the release of $^{45}Ca^{2+}$ as much as $^{45}Ca^{2+}$ release by $4\;{\mu}m\;InsP_3$, implying that Lqh venom also activates $InsP_3$ receptor, microsomal $Ca^{2+}$ release channel. Based on these results, we suggest that the Lqh venom consists of at least two components; one activates the $InsP_3$ receptor and the other avates the $Ca^{2+}-ATPase$. Currently we a investigating the chemical and electrophysiological properties of the active components of Lqh venom.

• International Journal of Oral Biology
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• v.34 no.2
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• pp.73-79
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• 2009

Cytosolic $Ca^{2+}$ is an important regulator of tumor cell proliferation and metastasis. Recently, the strategy of blocking receptors and channels specific to certain cancer cell types has emerged as a potentially viable future treatment. Oral squamous cell carcinoma is an aggressive form of cancer with a high metastasis rate but the receptor-mechanisms involved in $Ca^{2+}$ signaling in these tumors have not yet been elucidated. In our present study, we report that bradykinin induces $Ca^{2+}$ signaling and its modulation in the human oral squamous carcinoma cell line, HSC-3. Bradykinin was found to increase the cytosolic $Ca^{2+}$ levels in a concentration-dependent manner. This increase was inhibited by pretreatment with the phospholipase C-${\beta}$ inhibitor, U73122, and also by 2-aminoethoxydiphenyl borate, an inhibitor of the inositol 1,4,5-trisphosphate receptor. Pretreatment with extracellular ATP also inhibited the peak bradykinin-induced $Ca^{2+}$ rise. In contrast, the ATP-induced rise in cytosolic $Ca^{2+}$ was not affected by pretreatment with bradykinin. Pretreatment of the cells with either forskolin or phorbol 12-myristate 13-acetate (activators of adenylyl cyclase and protein kinase C, respectively) prior to bradykinin application accelerated the recovery of cytosolic $Ca^{2+}$ to baseline levels. These data suggest that bradykinin receptors are functional in $Ca^{2+}$ signaling in HSC-3 cells and may therefore represent a future target in treatment strategies for human oral squamous cell carcinoma.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.83-91
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• 1996

The present study was designed to determine the effect of hydrocortisone on CA secretion evoked by histamine from the isolated perfused rat adrenal glands. Histamine (150 ug) given into an adrenal vein produced significantly CA secretion from the rat adrenal medulla. This histamine-evoked CA secretion was enhanced markedly by the pretreatment with the natural glucocorticoid hydrocortisone (30 uM) or the synthetic glucocorticoid dexamethasone 30 (uM) for 20 min, respectively. Hydrocortisone-induced potentiation of CA secretion evoked by histamine was inhibited by preloading with heparin (3.56 U/ml), an $IP_3$ receptor antagonist while more enhanced by forskolin (0.2 uM), a potent stimulator of adenylate cyclase. From the experiment result taken together, it is thought that hydrocortisone (glucocorticoids) can enhance the releasing effect of CA evoked by histamine from the isolated perfused rat adrenal medulla, which seems to be associated to accumulation of inositol phosphate as well as cyclic AMP in the rat adrenomedullary chromaffin cells.

• Molecules and Cells
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• v.20 no.3
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• pp.305-314
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• 2005

Investigation of chemically synthesized inositol 1,4,5-trisphosphate [$Ins(1,4,5)P_3$] analogs has led to the isolation of a novel binding protein with a molecular size of 130 kDa, characterized as a molecule with similar domain organization to phospholipase C-${\delta}1$ (PLC-${\delta}1$) but lacking the enzymatic activity. An isoform of the molecule was subsequently identified, and these molecules have been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind $Ins(1,4,5)P_3$ via the pleckstrin homology domain, the involvement of PRIP-1 in $Ins(1,4,5)P_3$-mediated $Ca^{2+}$ signaling was examined using COS-1 cells overexpressing PRIP-1 and cultured neurons prepared from PRIP-1 knock-out mice. Yeast two hybrid screening of a brain cDNA library using a unique N-terminus as bait identified GABARAP ($GABA_A$ receptor associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible involvement of PRIP in $GABA_A$ receptor signaling. For this purpose PRIP knock-out mice were analyzed for $GABA_A$ receptor function in relation to the action of benzodiazepines from the electrophysiological and behavioral aspects. During the course of these experiments we found that PRIP also binds to the b-subunit of $GABA_A$ receptors and PP2A (protein phosphtase 2A). Here, we summarize how PRIP is involved in $Ins(1,4,5)P_3$-mediated $Ca^{2+}$ signaling and $GABA_A$ receptor signaling based on the characteristics of binding molecules.

• Journal of Life Science
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• v.30 no.9
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• pp.826-833
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• 2020

Phospholipase C gamma (PLCγ) has critical roles in receptor tyrosine kinase- and non-receptor tyrosine kinase-mediated cellular signaling relating to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] to produce inositol 1,4,5 trisphosphate (IP₃) and diacylglycerol (DAG), which promote protein kinase C (PKC) and Ca²⁺ signaling to their downstream cellular targets. PLCγ has two isozymes called PLCγ1 and PLCγ2, which control cell growth and differentiation. In addition to catalytically active X- and Y-domains, both isotypes contain two Src homology 2 (SH2) domains and an SH3 domain for protein-protein interaction when the cells are activated by ligand stimulation. PLCγ also contains two pleckstrin homology (PH) domains for membrane-associated phosphoinositide binding and protein-protein interactions. While PLCγ1 is widely expressed and appears to regulate intracellular signaling in many tissues, PLCγ2 expression is restricted to cells of hematopoietic systems and seems to play a role in the regulation of immune response. A distinct mechanism for PLCγ activation is linked to an increase in phosphorylation of specific tyrosine residue, Y783. Recent studies have demonstrated that PLCγ mutations are closely related to cancer, immune disease, and brain disorders. Our review focused on the physiological roles of PLCγ by means of its structure and enzyme activity and the pathological functions of PLCγ via mutational analysis obtained from various human diseases and PLCγ knockout mice.