• The Korean Journal of Physiology and Pharmacology
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• v.8 no.1
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• pp.57-63
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• 2004

Fluoxetine, a widely used anti-depressant compound, has several additional effects, including blockade of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells by using fura-2-based digital calcium imaging and assay for $[^3H]-inositol$ phosphates (IPs). Treatment with ATP $(100\;{\mu}M)$ for 2 min induced $[Ca^{2+}]_i$ increases. The ATP-induced $[Ca^{2+}]_i$ increases were significantly decreased by removal of extracellular $Ca^{2+}$ and treatment with the inhibitor of endoplasmic reticulum $Ca^{2+}$ ATPase thapsigargin $(1\;{\mu}M)$. Treatment with fluoxetine for 5 min blocked the ATP-induced $[Ca^{2+}]_i$ increase concentration-dependently. Treatment with fluoxetine $(30\;{\mu}M)$ for 5 min blocked the ATP-induced $[Ca^{2+}]_i$ increase following removal of extracellular $Ca^{2+}$ and depletion of intracellular $Ca^{2+}$ stores. While treatment with the L-type $Ca^{2+}$ channel antagonist nimodipine for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ increases significantly, treatment with fluoxetine alone blocked the ATP-induced responses. Treatment with fluoxetine also inhibited the 50 mM $K^+-induced$ $[Ca^{2+}]_i$ increases completely. However, treatment with fluoxetine did not inhibit the ATP-induced $[^3H]-IPs$ formation. Collectively, we conclude that fluoxetine inhibits ATP-indueed $[Ca^{2+}]_i$ increases in PC12 cells by inhibiting both an influx of extracellular $Ca^{2+}$ and a release of $Ca^{2+}$ from intracellular stores without affecting IPs formation.

• Journal of Life Science
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• v.31 no.9
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• pp.796-805
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• 2021

Hepatic endoplasmic reticulum (ER) stress contributes to the development of steatosis and insulin resistance. The components of unfolded protein response (UPR) regulate lipid metabolism. Recent studies have reported an association between ER stress and aberrant cellular lipid control; moreover, research has confirmed the involvement of sterol regulatory element-binding proteins (SREBPs)-the central regulators of lipid metabolism-in the process. However, the exact role of SREBPs in controlling lipid metabolism during ER stress and its contribution to fatty liver disease remain unknown. Here, we show that SREBP-1c deficiency protects against ER stress-induced hepatic steatosis in mice by regulating UPR, inflammation, and fatty acid oxidation. SREBP-1c directly regulated inositol-requiring kinase 1α (IRE1α) expression and mediated ER stress-induced tumor necrosis factor-α activation, leading to a reduction in expression of peroxisome proliferator-activated receptor γ coactivator 1-α and subsequent impairment of fatty acid oxidation. However, the genetic ablation of SREBP-1c prevented these events, alleviating hepatic inflammation and steatosis. Although the mechanism by which SREBP-1c deficiency prevents ER stress-induced inflammatory signaling remains to be elucidated, alteration of the IRE1α signal in SREBP-1c-depleted Kupffer cells might be involved in the signaling. Overall, the results suggest that SREBP-1c plays a crucial role in the regulation of UPR and inflammation in ER stress-induced hepatic steatosis.

• Molecules and Cells
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• v.19 no.3
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• pp.375-381
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• 2005

Phospholipase C-${\beta}$ (PLC-${\beta}$) hydrolyses phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate in response to activation of various G protein-coupled receptors (GPCRs). Using glial cells from knock-out mice lacking either PLC-${\beta}1$ [PLC-${\beta}1$ (-/-)] or PLC-${\beta}3$ [PLC-${\beta}3$ (-/-)], we examined which isotype of PLC-${\beta}$ participated in the cellular signaling events triggered by thrombin. Generation of inositol phosphates (IPs) was enhanced by thrombin in PLC-${\beta}1$ (-/-) cells, but was negligible in PLC-${\beta}3$ (-/-) cells. Expression of PLC-${\beta}3$ in PLC-${\beta}3$ (-/-) cells resulted in an increase in pertussis toxin (PTx)-sensitive IPs in response to thrombin as well as to PAR1-specific peptide, while expression of PLC-${\beta}1$ in PLC-${\beta}1$ (-/-) cells did not have any effect on IP generation. The thrombin-induced $[Ca^{2+}]_i$ increase was delayed and attenuated in PLC-${\beta}3$ (-/-) cells, but normal in PLC-${\beta}1$ (-/-) cells. Pertussis toxin evoked a delayed $[Ca^{2+}]_i$ increase in PLC-${\beta}3$ (-/-) cells as well as in PLC-${\beta}1$ (-/-) cells. These results suggest that activation of PLC-${\beta}3$ by pertussis toxin-sensitive G proteins is responsible for the transient $[Ca^{2+}]_i$ increase in response to thrombin, whereas the delayed $[Ca^{2+}]_i$ increase may be due to activation of some other PLC, such as PLC-${\beta}4$, acting via PTx-insensitive G proteins.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.354-364
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• 2015

Background: Intracellular $Ca^{2+}$($[Ca^{2+}]_i$) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of $[Ca^{2+}]_i$mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the $Ca^{2+}$-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). Methods: We investigated the $Ca^{2+}$-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I ($IP_3RI$) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. Results: The inhibition of $[Ca^{2+}]_i$ mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-BrcAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)- dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) ($Thr^{197}$) by KRG-TS. The phosphorylation of $IP_3RI$ ($Ser^{1756}$) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-BrcGMPS. These results suggest that the inhibitory effect of $[Ca^{2+}]_i$ mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. Conclusion: These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits $[Ca^{2+}]_i$ mobilization in thrombin-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

• Journal of Life Science
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• v.18 no.9
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• pp.1219-1224
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• 2008

The embryonic zebrafish (Danio rerio) is rapidly becoming an important model organism for studies of early events in vertebrate development. Neural crest-derived pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores, and/or iridophores. Cell-signaling mechanisms related to the development of pigmentation and pigment pattern formation remain obscure. In this study, zebrafish embryos were treated with various signaling-related molecules - LiCl (an inositol-phosphatase inhibitor), forskolin (a protein kinase-A activator), a combination of LiCl/forskolin, and LiCl/heparin (an IP3 inhibitor) in order to identify the mechanisms involved in pigmentation. LiCl treatment resulted in ultrastructural and morphological alterations of melanophores. To identify the possible proteins responsible for this ultrastructural and morphological change, phosphorylation patterns in vitro and in vivo were analyzed. LiCl and LiCl/forskolin treatment elicited dramatic increases in the phosphorylation of a 55-kDa protein which was inhibited by heparin treatment. LiCl treatment also induced phosphorylation of a 55-kDa protein in melanophores purified from adult zebrafish. Collectively these results suggest that a LiCl-induced 55-kDa phosphoprotein plays a role in melanophore morphology and ultrastructure and ultimately effects gross pigmentation.

• 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.11a
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• pp.75-84
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• 1996

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate (IP$_3$), mobilization of intracellular Ca$\^$2+/, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation. A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, seven, out often known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC-${\gamma}$2 > PLC-${\beta}$2 > PLC-${\beta}$3 > PLC-${\beta}$l > PLC-${\gamma}$ > PLC-$\delta$1 > PLC-${\beta}$4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-${\beta}$2, whereas PLC-${\beta}$4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-${\beta}$2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.89-94
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• 2014

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces $Ca^{2+}$ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in $Ca^{2+}$ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated $Ca^{2+}$-activated $Cl^-$ channels (CaCCs) and increased intracellular calcium concentrations ($[Ca^{2+}]_i$) in primary cultured human conjunctival cells. DA-6034 also increased $[Ca^{2+}]_i$ in mouse salivary gland cells and human corneal epithelial cells. $[Ca^{2+}]_i$ increase of DA-6034 was dependent on the $Ca^{2+}$ entry from extracellular and $Ca^{2+}$ release from internal $Ca^{2+}$ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate ($IP_3$) pathway and lysosomal $Ca^{2+}$ stores. These results suggest that DA-6034 induces $Ca^{2+}$ signaling via extracellular $Ca^{2+}$ entry and RyRs-sensitive $Ca^{2+}$ release from internal $Ca^{2+}$ stores in epithelial cells.

• Archives of Pharmacal Research
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• v.25 no.6
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• pp.747-758
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• 2002

The skin displays a highly active metabolism of polyunsaturated fatty acids (PUFA). Dietary deficiency of linoleic acid (LA), an 18-carbon (n-6) PUFA, results in characteristic scaly skin disorder and excessive epidermal water loss. Although arachidonic acid (AA), a 20-carbon (n6) PUFA, is metabolized via cyclooxygenase pathway into predominantly prostaglandin $E_2(PGE_2)$ and $PGF_{2{\alpha}}$, the metabolism of AA via the 15-lipoxygenase (15-LOX) pathway, which is very active in skin epidermis and catalyzes the transformation of M into predominantly 15S-hydroxyeicosatetraenoic acid (15S-HETE). Additionally, the 15-LOX also metabolizes the 18-carbon LA into 13S-hydroxyoctadecadienoic acid (13S-HODE), respectively. Interestingly, 15-LOX catalyzes the transformation of $dihomo-{\gamma}-linolenic$ acid (DGLA), derived from dietary gamma-linolenic acid, to 15S-hydroxyeicosatrienoic acid (15S-HETrE). These monohydroxy fatty acids are incorporated into the membrane inositol phospholipids which undergo hydrolytic cleavage to yield substituted-diacylglycerols such as 13S-HODE-DAG from 13S-HODE and 15S-HETrE-DAG from 15S-HETrE. These substituted-monohydroxy fatty acids seemingly exert anti-inflammatory/antiproliferative effects via the modulation of selective protein kinase C as well as on the upstream/down-stream nuclear MAP-kinase/AP-1/apoptotic signaling events.

• 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.