• Biomedical Science Letters
• /
• v.13 no.4
• /
• pp.333-339
• /
• 2007

Recently, much attention has been paid to human retinoblastoma since it provide a good model system for studying mechanisms underlying cell growth, differentiation, proliferation, and apoptosis, and for developing cancer therapy. However, until now it is unclear whether purinergic receptors are involved in the calcium mobilization in the retinoblastoma cells. In this regard, we measured possible purinergic signaling in WERI-Rb-1 cells using $Ca^{2+}$ imaging technique and RT-PCR method. ATP-induced $[Ca^{2+}]_i$ transients was maintained to about $90.7{\pm}1.0%$ of the control (n=48) even in the absence of extracellular calcium. The ATP-induced intracellular calcium response was only attained to $10.4{\pm}1.8%$ (n=55) of peak amplitude of the control after preincubation of 1 ${\mu}MU-73122$, a PLC inhibitor, but it was not affected by 1 ${\mu}MU-73343$, a inactive form of U-73122. And also ATP-induced $[Ca^{2+}]_i$ rise was almost attenuated by 20 ${\mu}M$ 2-APB, a putative $IP_3$ receptor inhibitor. Two subtypes of $IP_3$ receptor $(IP_{3-1}R,\;IP_{3-2}R)$ were identified by a RT-PCR method. These findings suggest that purinergic stimuli can cause calcium mobilization via $PLC-IP_3$ pathway after the activation of P2Y receptors in the retinoblastoma cells, which may play important roles in cell proliferation, differentiation, growth, and cell death.

• YAKHAK HOEJI
• /
• v.54 no.1
• /
• pp.8-12
• /
• 2010

Inositol 1,4,5-trisphosphate ($IP_3$) receptor ($IP_3R$)-mediated signaling pathway is involved in many cellular processes including fertilization, apoptosis and neuronal function. Although cardiac myocytes express the $IP_3R$, its pathophysiological role has not been clearly understood because of limited selectivity of currently available pharmacological blockers. In the present study we constructed shRNA-expressing adenovirus to knock-down the type 2 $IP_3R$ ($IP_3R2$), a major subtype in cardiac ventricular myocytes, and demonstrated that the virus successfully eliminated the expression and localization of the $IP_3R2$. These results may provide a reliable tool for probing pathophysiological roles of the $IP_3R2$ in isolated intact cardiac myocytes.

• Journal of Animal Science and Technology
• /
• v.51 no.4
• /
• pp.289-294
• /
• 2009

Inositol 1,4,5-triphosphate receptor type 1 (IP3R1) is a $Ca^{2+}$ release channel that responds to the second messenger IP3 and that modulates diverse cellular functions such as contraction/excitation, secretion, gene expression and cellular growth. We discovered single nucleotide polymorphisms (SNPs) within IP3R1 gene and analyzed associations between gene polymorphisms and carcass traits in Korean cattle (Hanwoo) in order to develop novel DNA markers at genomic level. Three SNPs were detected at the position of g.1428617A>G, g.1418843C>T and g.1414377C>T with 24 unrelated Hanwoo samples by direct sequencing of the PCR products. We found that genotype of g.1414377C>T SNP was associated with live weight (P<0.05) and carcass weight (P<0.01) using the general linear model of SAS package. These results suggest that polymorphism of IP3R1 gene was associated with weight-related traits in Hanwoo.

• The Korean Journal of Physiology and Pharmacology
• /
• v.16 no.1
• /
• pp.31-36
• /
• 2012

The receptor activator of NF-${\kappa}B$ ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-${\kappa}B$ and other signal transduction pathways essential for osteoclastogenesis, such as $Ca^{2+}$ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate ($IP_3$) and $IP_3$-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of $IP_3$ and evaluated $IP_3$-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of $Ca^{2+}$ signaling proteins such as $IP_3$ receptors ($IP_3Rs$), plasma membrane $Ca^{2+}$ ATPase, and sarco/endoplasmic reticulum $Ca^{2+}$ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of $IP_3$ was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) ${\delta}$, a probe specifically detecting intracellular $IP_3$ levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)[ and of $IP_3Rs$ with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of $IP_3Rs$) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular $IP_3$ levels and the $IP_3$-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

• Molecules and Cells
• /
• v.21 no.3
• /
• pp.315-329
• /
• 2006

Two, well characterized cationic channels, the ryanodine receptor (RyR) and the canonical transient receptor potential cation channel (TRPC) are briefly reviewed with a particular attention on recent developments related to the interplay between the two channel families.

• Development and Reproduction
• /
• v.23 no.1
• /
• pp.1-9
• /
• 2019

The ability of oocytes to undergo normal fertilization and embryo development is acquired during oocyte maturation which is transition from the germinal vesicle stage (GV), germinal vesicle breakdown (GVBD) to metaphase of meiosis II (MII). Part of this process includes redistribution of inositol 1, 4, 5-triphosphate receptor (IP3R), a predominant $Ca^{2+}$ channel on the endoplasmic reticulum membrane. Type 1 IP3R (IP3R1) is expressed in mouse oocytes dominantly. At GV stage, IP3R1 are arranged as a network throughout the cytoplasm with minute accumulation around the nucleus. At MII stage, IP3R1 diffuses to the entire cytoplasm in a more reticular manner, and obvious clusters of IP3R1 are observed at the cortex of the egg. This structural reorganization provides acquisition of $[Ca^{2+}]_i$ oscillatory activity during fertilization. In this review, general properties of IP3R1 in somatic cells and mammalian oocyte are introduced.

• Proceedings of the Korean Biophysical Society Conference
• /
• 1999.06a
• /
• pp.55-55
• /
• 1999

Imperatoxin A (IpTx$_{a}$), a 3.7 kDa peptide from the African scorpion Pandinus imperator, has been known as an agonist of skeletal ryanodine receptor (RyR). In order to study the structure and function of the toxins on RyR, the IpTx$_{a}$ cDNA was PCR-amplified using 3 pairs of primers and the toxin was expressed in E. coli expression system.(omitted)ted)

• Molecules and Cells
• /
• v.42 no.6
• /
• pp.470-479
• /
• 2019

Interstitial cells of Cajal (ICCs) are pacemaker cells that exhibit periodic spontaneous depolarization in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of ghrelin and motilin on the pacemaker potentials of ICCs isolated from the mouse small intestine. Using the whole-cell patch-clamp configuration, we demonstrated that ghrelin depolarized pacemaker potentials of cultured ICCs in a dose-dependent manner. The ghrelin receptor antagonist [D-Lys] GHRP-6 completely inhibited this ghrelin-induced depolarization. Intracellular guanosine 5'-diphosphate-${\beta}$-S and pre-treatment with $Ca^{2+}$-free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate ($IP_3$), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the $IP_3$ receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C, the Rho kinase inhibitor Y-27632, and the PKC inhibitors staurosporine, Go6976, and rottlerin. All inhibitors except rottlerin blocked the ghrelin-induced pacemaker potential depolarization of ICCs. In addition, motilin depolarized the pacemaker potentials of ICCs in a similar dose-dependent manner as ghrelin, and this was also completely inhibited by [D-Lys] GHRP-6. These results suggest that ghrelin induced the pacemaker potential depolarization through the ghrelin receptor in a G protein-, $IP_3$-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular $Ca^{2+}$ regulation. In addition, motilin was able to depolarize the pacemaker potentials of ICCs through the ghrelin receptor. Therefore, ghrelin and its receptor may modulate GI motility by acting on ICCs in the murine small intestine.

• Molecules and Cells
• /
• v.42 no.3
• /
• pp.252-261
• /
• 2019

The omega-3 fatty acid docosahexaenoic acid (DHA) is known to induce apoptosis and cell cycle arrest via the induction of reactive oxygen species (ROS) production and endoplasmic reticulum (ER) stress in many types of cancers. However, the roles of DHA in drug-resistant cancer cells have not been elucidated. In this study, we investigated the effects of DHA in cisplatin-resistant gastric cancer SNU-601/cis2 cells. DHA was found to induce ROS-dependent apoptosis in these cells. The inositol 1,4,5-triphosphate receptor ($IP_3R$) blocker 2-aminoethyl diphenylboninate (2-APB) reduced DHA-induced ROS production, consequently reducing apoptosis. We also found that G-protein-coupled receptor 120 (GPR120), a receptor of long-chain fatty acids, is expressed in SNU-601/cis2 cells, and the knockdown of GPR120 using specific shRNAs alleviated DHA-mediated ROS production and apoptosis. GPR120 knockdown reduced the expression of ER stress response genes, similar to the case for the pre-treatment of the cells with N-acetyl-L-cysteine (NAC), an ROS scavenger, or 2-APB. Indeed, the knockdown of C/EBP homologous protein (CHOP), a transcription factor that functions under ER stress conditions, markedly reduced DHA-mediated apoptosis, indicating that CHOP plays an essential role in the anti-cancer activity of DHA. These results suggest that GPR120 mediates DHA-induced apoptosis by regulating $IP_3R$, ROS, and ER stress levels in cisplatin-resistant cancer cells, and that GPR120 is an effective chemotherapeutic target for cisplatin resistance.

• Molecules and Cells
• /
• v.22 no.3
• /
• pp.328-335
• /
• 2006

Imperatoxin A ($IpTx_a$), a 3.7 kDa peptide from the African scorpion Pandinus imperator, is an agonist of the skeletal muscle ryanodine receptor (RyR1). In order to study the structure of the toxin and its effect on RyR1, $IpTx_a$ cDNA was PCR-amplified using 3 pairs of primers, and the toxin was expressed in E. coli. The toxin was further purified by chromatography, and various point mutants in which basic amino acids were substituted by alanine were prepared by site-directed mutagenesis. Studies of single channel properties by the planar lipid bilayer method showed that the recombinant $IpTx_a$ was identical to the synthetic $IpTx_a$ with respect to high-performance liquid chromatography mobility, amino acid composition and specific effects on RyR1. Mutations of certain basic amino acids ($Lys^{19}$, $Arg^{23}$, and $Arg^{33}$) dramatically reduced the capacity of the peptide to activate RyRs. A subconductance state predominated when $Lys^8$ was substituted with alanine. These results suggest that some basic amino acid residues in $IpTx_a$ are important for activation of RyR1, and that $Lys^8$ plays an important role in regulating the gating mode of RyR1.