• The Korean Journal of Physiology and Pharmacology
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• 제7권6호
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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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• 제19권3호
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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 Yeungnam Medical Science
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• 제9권2호
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• pp.288-301
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• 1992

소의 중추신경계의 신경전달인자에 의한 세포막에서의 정보전달 과정에 관여하는 PLC 활성화에 G-단백질의 관여 여부를 관찰하기 위하여 소의 뇌조직의 PLC ${\beta}$, ${\gamma}$ 및 ${\delta}$를 얻어 각 isozyme의 특성을 관찰하였다. 기질용액에 phosphatidyl choline(PC)을 첨가시 PLC 각 isozyme 마다 정도의 차이는 있으나 증가 양상을 보였으며 PLC ${\delta}$는 $100{\mu}M$ $Ca^{2+}$ 농도에서 높은 활성도 증가를 보였다. 세포막 소포체를 형성하기 위하여 $PIP_2$기질과 PC에 detergent로 cholate와 deoxycholate 농도에 따른 PLC 효과 관찰에서 cholate 농도 0.2%에서 1%까지 증가할 때 효소 활성도의 지속적인 상승이 관찰되었고, deoxycholate는 농도가 0.2%에서 높았다가 0.4%에서 낮아졌고 1%까지 증가함에 따라 PLC 효소 활성도는 약간 증가하였다. 기질액에 뇌추출액을 첨가하여 cholic acid 농도에 따른 PLC의 효과를 관찰한 결과 cholic acid 농도 0.2%에서 보다 1%에서 각 isozyme 모두에서 PLC활성도가 증가하였다. 소의 여러 장기에서 PLC isozyme의 분포정도를 방사면역측정방법으로 관찰하였을 때 뇌조직에 가장 많이 분포하고 있으며 특히 PLC ${\beta}$, ${\gamma}$가 많았고, PLC ${\delta}$는 부신에서 가장 많이 분포하였다. 다음으로 PLC ${\beta}$는 부신과 위, PLC${\gamma}$는 부신과 폐순이었다. PLC 효소가 활성화될 때 G-단백질의 관여 여부에 관하여 cholate 0.2%와 0.1%에서 G-단백질과 GTPrS 및 PLC의 결합정도의 관찰은 조직분쇄시료를 소의 뇌 및 부신조직을 이용하여 $^{35}S$-GTPrS 첨가시와 단세포군 항체를 이용한 경우 모두에서 1.49% 이하의 낮은 결합 정도를 관찰하였다. 그래서 정제된 PLC isozyme과 G-단백질 $Go{\alpha}$, $G{\beta}{\gamma}$, Gmix, $Gi{\alpha}$ 및 $Gt{\alpha}$ 각각에 대한 효과 관찰에 서 $Go{\alpha}$와 $G{\beta}{\gamma}$는 PLC ${\beta}$와 ${\delta}$의 활성도를 증가시켰고, PLC ${\gamma}$는 별 영향이 없었으며 Gmix에서는 세효소 모두 증가시켰다. $Gi{\alpha}$는 PLC ${\beta}$와 ${\gamma}$에서만 증가하였다. $Gt{\alpha}$는 PLC ${\beta}$ 및 ${\gamma}$에서 억제하였고 PLC ${\delta}$에서는 증가 양상을 보였다. 그러므로 PLC 활성화에 G-단백질의 관여가 인지되며 PLC isozyme과 G-단백질의 종류에 따라 대개의 경우 증가하는 경향이나 일부는 억제 내지는 별 영향이 없는 것으로 나타났다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 제4회 추계심포지움
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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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• 제1권4호
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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.

• 수산해양교육연구
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• 제28권5호
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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.

• 대한의생명과학회지
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• 제12권4호
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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.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1993년도 제2회 신약개발 연구발표회 초록집
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• pp.63-63
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• 1993

Phospoinositide-specific phospholipase C (PLC)는 세포막의 phosphoinositide를 분해하여 inositol phosphates와 diacylglycerol을 전달하는데 핵심적인 효소이다. PLC는 분자량과 1차구조의 비교에 의하여 type (PLC-$\beta$, ${\gamma}$, $\delta$)로 구분되며, 각 type마다 2-4종의 subtype이 존재하고 PLC isozyme들에 대한 현재가지의 각종 신호 전달 및 조절에 대한 연구를 종합하면: (1) PLC-$\beta$ type은 G-protein과 연결되어 신호를 전달받고, (2) PLC-${\gamma}$ type은growth factor receptor tyrosine kinase에 의하여 인산화 되어 활성화됨으로, 세포의 성장 신호를 전달하며. (3) PLC-$\delta$ type에 대한 신호 전달이나 조절은 밝혀지지 않고 있다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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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.

• 생명과학회지
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• 제28권12호
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• pp.1516-1522
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• 2018

죽상동맥경화는 대동맥의 만성염증에 의해 주로 발병되는 폐쇄동맥질환이다. 혈관평활근세포의 증식 및 이동은 죽상동맥경화 발병의 주된 병리적 반응이다. 본 연구에서는 죽상동맥경화 발병기전을 유도하는 표적 염증반응 물질의 탐색 및 이들에 의한 신호전달 기전을 연구하였다. 혈관평활근세포의 증식 및 이동은 prostaglandin $F_{2{\alpha}}$ ($PGF_{2{\alpha}}$)에 의해 의미 있게 증가하였으나 tumor necrosis factor ${\alpha}$ ($TNF{\alpha}$)에 의해서는 증가하지 않았다. Prostacyclin $I_2$ ($PGI_2$)는 혈관평활근세포의 증식은 촉진시켰으나 이동은 오히려 억제하였다. prostaglandin $D_2$ ($PGD_2$) 및 prostaglandin $E_2$ ($PGE_2$)는 혈관평활근세포의 증식을 촉진시켰으나 이동에는 영향을 미치지 않았다. $PGF_{2{\alpha}}$는 용량 의존적으로 혈관평활근세포의 증식 및 이동을 촉진시켰고 EC50는 약 $0.1{\mu}M$로 관찰되었다. 혈관평활근세포에서 phospholipase $C-{\beta}3$ ($PLC-{\beta}3$) 아형의 발현은 매우 높았으나 $PLC-{\beta}1$, $PLC-{\beta}2$, 및 $PLC-{\beta}4$의 발현은 관찰되지 않았다. U73122 처리를 통해 PLC의 활성을 억제하면 $PGF_{2{\alpha}}$에 의한 혈관평활근세포의 이동이 억제되었다. 또한 $PLC-{\beta}3$의 발현을 억제하면 $PGF_{2{\alpha}}$에 의한 혈관평활근세포의 증식 및 이동이 억제되었다. 이러한 결과들을 바탕으로 $PGF_{2{\alpha}}$ 는 혈관평활근세포의 증식 및 이동에 중요한 역할을 수행하고, 여기에는 $PLC-{\beta}3$가 필수적인 역할을 담당하고 있음을 제안한다.