• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.349-355
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• 2003

We previously shown that LES contraction depends on $M_3$ receptors linked to PTX insensitive $G_q$ protein and activation of PLC. This results in production of $IP_3$, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates $M_2$ receptors linked to PTX sensitive $G_{i3}$ protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by $G_q$ or $G{\beta}$ protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, $PLC-{\beta}1$, $PLC-{\beta}3$, and $PLC-{\gamma}1$, but not $PLC-{\beta}2$, $PLC-{\beta}4$, $PLC-{\gamma}2$, $PLC-{\delta}1$, and $PLC-{\delta}2$ from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but $PLC-{\gamma}1$ antibody incubation did not have an inhibitory effect. The inhibition by $PLC-{\beta}1$ or $PLC-{\beta}3$ antibody on Ach-induced contraction was antibody concentration dependent. The combination with $PLC-{\beta}_1$ and $PLC-{\beta}_3$ antibody completely abolished the contraction, suggesting that $PLC-{\beta}1$ and $PLC-{\beta}3$ have a synergism to inhibit the contraction in LES. $PLC-{\beta}1$, -${\beta}3$ or -${\gamma}1$ antibody did not reduce the contraction of LES cells in response to DAG ($10^{-6}$ M), suggesting that this isozyme of PLC may not activate PKC. When $G_{q/11}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}3$, but not of PLC ${\beta}_1$ was additive (Fig. 6). In contrast, when $G_{\beta}$ antibody was incubated, the inhibitory effect of the incubation of PLC ${\beta}_1$, but not of PLC ${\beta}_3$ was additive. This data suggest that $G_{q/11}$/11 or $G{\beta}$ may activate cooperatively different PLC isozyme, $PLC{\beta}_1$ or $PLC{\beta}_3$ respectively.

• 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.

• Fisheries and Aquatic Sciences
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• v.19 no.4
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• pp.18.1-18.10
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• 2016

In this study, to clarify the function of $PoPLC-{\beta}1$, in response to stress challenge, we examined the $PoPLC-{\beta}1$ expression pattern in response to external stress (pathogen-associated molecular pathogen challenge and environmental challenge including temperature and salinity). $PoPLC-{\beta}1$ expression analysis of tissue from olive flounder showed that the messenger RNA (mRNA) was predominantly expressed in the brain, heart, eye, liver, spleen, and stomach. We also tested the mRNA expression of the $PoPLC-{\beta}1$ in the spleen and kidney of olive flounder by RT-PCR and real-time PCR following stimulation with lipopolysaccharide (LPS), concanavalin A (ConA), or polyinosinic:polycytidylic acid (PolyI:C) and compared with the inflammatory cytokines IL-1b and IL-6 in the stimulated flounder tissues. Each of the spleen and kidney and mRNA transcripts of $PoPLC-{\beta}1$ were increased 30- and 10-fold than normal tissue at 1-6 h post injection (HPI) with PolyI:C when the expression of $PoPLC-{\beta}1$ transcript was similar to LPS and ConA. We also tested the expression of $PoPLC-{\beta}1$ in response to temperature and salinity stress. The expression of $PoPLC-{\beta}1$ also was affected by temperature and salinity stress. Our results provide clear evidence that the olive flounder $PLC-{\beta}1$ signal pathways may play a critical role in immune function at the cellular level and in inflammation reactions. In addition, $PLC-{\beta}1$ appears to act as an oxidative-stress suppressor to prevent cell damage in fish.

• Journal of Yeungnam Medical Science
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• v.9 no.2
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• pp.288-301
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• 1992

The objective of the present study was to identify the characteristics of phospholipase C (PLC) isozymes purified from bovine brain and to investigate their interrelationship with G protein. The purified PLC isozymes ${\beta}$, ${\gamma}$ and ${\delta}$ were obtained and the characteristics of PLC activity on various concentrations of free $Ca^{2+}$ were observed. The activity of PLC was increased with increasing $Ca^{2+}$ concentration and the activity PLC ${\delta}$ was increased higher in the presence of phosphatidyl choline(PC) than in the abscence of PC. For vesicle formation as the structure of cell membrane, cholic acid and deoxycholic acid as detergent on phosphatidylinositol bisphosphate($PIP_2$) substrate containing PC were used, and then the activity of PLC isozymes were increased with increasing concentration of cholate, from 0.2% to 1% and were increased slightly in deoxycholate. In the $PIP_2$ containing phospholipid and glycolipid as brain extract, the activity of PLC isozymes were checked in 0.2%-1% cholic acid. The activities of PLC isoyzmes were continuously increased up to 1% cholic acid. The quantitation of PLC isozymes from several bovine organs by radioimmunoassay was made. Brain was the most sufficient organ in terms of amount of PLC ${\beta}$and ${\delta}$. A large amount of PLC ${\delta}$ was existed in adrenal gland. The binding capacity of GTPrS and G protein was observed and other observations of the binding effect of GTPrS-G protein and PLC monoclonal Ab-Protein A from tissue homogenate with PLC were made. From the observation the binding capacity was revealed the range of 0.11%-1.49%. The effects of each type of G protein on the percent activity of purified PLC isozymes were observed. From the observation, activities of isozymes were increased in $Go{\alpha}$ & Gmix, and the activities of PLC ${\beta}$ and ${\delta}$ were increased in $G{\beta}{\gamma}$ and $Gi{\alpha}$. Activities of PLC ${\beta}$ and ${\gamma}$ were decreased in $Gt{\alpha}$ but PLC ${\delta}$ increased.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.4
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• pp.385-392
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• 1997

The phospholipase C (PLC)-mediated intracellular signal transduction pathway is considered to be involved in the regulation of blood pressure. However, little information is available concerning the distributional and functional significance of PLC in the genetic hypertensive rats. As the first step of knowing the role of PLC on hypertension, we investigated the distribution of 6 PLC isozymes $(PLC-{\beta}1,\;-{\beta}3,\;-{\beta}4,\;-{\gamma}1,\;-{\gamma}2\;and\;-{\delta}1)$ in the heart and brain, which are concerned with hypertension, in the normotensive Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) using the western blotting and immunocytochemistry. The immunoreactivities of PLC isozymes in brain were detected, but there were no distributional and quantitative differences between the WKY and SHR. In the heart, but the immunoreactivities to $PLC-{\beta}1$ and $-{\gamma}2$ in the SHR were higher than those in WKY. In immunocytochemistry to confirm these western blotting data, $PLC-{\beta}1$ and $-{\gamma}2$ were localized in cardiac myocytes and the intensities of immunoreactivity in SHR were stronger than that in WKY. These results suggest that $PLC-{\beta}1$ and $-{\gamma}2$ would have possibility to concern with the establishment of spontaneous hypertension.

• Journal of Fisheries and Marine Sciences Education
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• v.28 no.5
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• pp.1266-1272
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• 2016

Phospholipase C is a key enzyme of signaling pathways hydrolyzed phosphatidylinositol 4,5-bisphosphate to generate 2 second messengers. Among the PLC, $PLC-{\beta}$ subfamily consisted of 4 isoforms, $PLC-{\beta}$ 1~4. Here, we studied the tissue specific expression of $PLC-{\beta}3$ in olive flounder (Paralichthys olivaceus) following external stimulation like lipopolysaccharide (LPS), concanavalin A (ConA) and environmental stress compared with the inflammatory cytokines IL-1b. $PoPLC-{\beta}3$ gene transcripts has the effect in stimulated tissue compared to control. These results provide what we sure to be a important role for $PLC-{\beta}3$ activity in tissue and verify $PLC-{\beta}3$ as potential immune enzyme for signal transduction.

• Biomedical Science Letters
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• v.12 no.4
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• pp.419-425
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• 2006

Phospholipase $C-{\gamma}1\;(PLC-{\gamma}1)$ is an important signaling molecule for cell proliferation and differentiation. $PLC-{\gamma}1$ contains two pleckstrin homology (PH) domains, which are responsible for protein-protein interaction and protein-lipid interaction. $PLC-{\gamma}1$ also has two Src homology (SH)2 domains and a SH3 domain, which are responsible for protein- protein interaction. To identity proteins that specifically binds to PH domain of $PLC-{\gamma}1$, we prepared and incubated the glutathione S-transferase(GST)-fused PH domains of $PLC-{\gamma}1$ with COS7 cell lysate. We found that 90 kDa protein specifically binds to PH domain of $PLC-{\gamma}1$. By matrix-assisted laser desorption ionization time of flight-mass spectrometry, the 90 kDa protein revealed to be heat shock protein (Hsp) $90{\beta}$. Hsp $90{\beta}$ is a molecular chaperone that stabilizes and facilitates the folding of proteins that are involved in cell signaling, including receptors for steroids hormones and a variety of protein kinases. To know whether Hsp $90{\beta}$ affects on $PLC-{\gamma}1$ activity, we performed $PIP_2$ hydrolyzing activity of $PLC-{\gamma}1$ in the presence of purified Hsp $90{\beta}$ in vitro. Our results show that the Hsp $90{\beta}$ dose-dependently inhibits the enzymatic activity of $PLC-{\gamma}1$ and further suggest that Hsp $90{\beta}$ regulates cell growth and differentiation via regulation of $PLC-{\gamma}1$ activity.

• 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.197-197
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• 1998

Chronic electroconvulsive shock(ECS) was shown to Increase phosphatidylinositol-4,5-bisphosphate(PIP$_2$) breakdown and the activity of PLC with the accumulation of inositol-1,4,5-triphosphate(IP3). The purpose of the present study was to determine the effect of ECS on the expression of phospholipase C(PLC) isotypes in rat brain. Two groups of animals were prepared: sham and ECS treated groups. Rats in ECS treated groups received maximal ECS(70mA, 0.5second, 60㎐) by constant current stimulator through ear-clip to induce tonic extension seizures for 12 consecutive days. The expression of PLC isotypes in rat brain was determined by immunohistochemical procedure using sagital section of rat brain. The immunoreactivity of PLC${\beta}$1 was observed in corpus striatum, hippocampus, thalamus and that of PLC${\gamma}$1 in corpus striatum, hippocampus, thalamus, frontal cortex, parietooccipital cortex, limbic forebrain, pons, medulla, superior colliculus, inferior colliculus, rest of midbrain. The amount of PLC was analyzed by Western blot using antibodies against PLC${\beta}$1 and PLC${\gamma}$1. Chronic ECS reduced the immunoreactivity of PLC${\beta}$1 in corpus striatum, hippocampus, thalamus but had little effect on PLC${\gamma}$1. To quantify this change, quantitative Western blot using antibodies against PLC${\beta}$1 and PLC${\gamma}$1 was conducted. The immunoreactivity of PLC${\beta}$1 in ECS treated rat whole brain was decreased by 40 % in cytosolic fraction and 26 % in membrane fraction. This different effect of ECS on PLC isotypes may results from the difference of their activation mechanisms and the different effects of ECS on them. The results from the present study suggest that chronic ECS primalily affects neurotransmitter receptors related IP$_3$ signaling in rat brain.

• BMB Reports
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• v.44 no.9
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• pp.566-571
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• 2011

Although the phospholipase C (PLC)${\beta}$-1 isoform is associated with spontaneous seizure and distinctively expressed in the telencephalon, the distribution of PLC${\beta}$-1 expression in the epileptic gerbil hippocampus remains controversial. Therefore, we determined whether PLC${\beta}$-1 is associated with spontaneous seizure in an animal model of genetic epilepsy. In the present study, PLC${\beta}$-1 immunoreactivity was down-regulated in seizure-sensitive (SS) gerbils more than in seizure-resistant (SR) gerbils. The expression of PLC${\beta}$-1 within calretinin (CR)-positive neurons was rarely detected within the dentate hilar region of SS gerbils. PLC${\beta}$-1 immunoreactivity in the hippocampus was significantly elevated as compared to that in pre-seizure SS gerbil 3 h post-ictal. These findings suggest that alterations in PLC${\beta}$-1 immunoreactivity in the SS gerbil hippocampus may be closely related to the epileptic state of the gerbil brain and transiently elevated PLC${\beta}$-1 protein levels following seizure episodes. Such alterations may be compensatory responses in the SS gerbil hippocampus.