scholarly journals Plasmodium falciparum Phospholipase C Hydrolyzing Sphingomyelin and Lysocholinephospholipids Is a Possible Target for Malaria Chemotherapy

2001 ◽  
Vol 195 (1) ◽  
pp. 23-34 ◽  
Author(s):  
Kentaro Hanada ◽  
Nirianne Marie Q. Palacpac ◽  
Pamela A. Magistrado ◽  
Ken Kurokawa ◽  
Ganesh Rai ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Recombinant Protein ◽  
Phospholipase C ◽  
Dependent Manner ◽  
Glutathione S Transferase ◽  
Trophozoite Stage ◽  
Severe Impairment ◽  
Active Transcription ◽  
Biochemical Analyses ◽  
Dose Dependent

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is ∼25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum–infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID50 values for SM/LCPL-PLC activities and the parasite growth at 3–5 μM and ∼7 μM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

scholarly journals Inhibition of Fungal Appressorium Formation by Pepper (Capsicum annuum) Esterase

2001 ◽  
Vol 14 (1) ◽  
pp. 80-85 ◽  
Author(s):  
Young Soon Kim ◽  
Hyun Hwa Lee ◽  
Moon Kyung Ko ◽  
Chae Eun Song ◽  
Cheol-Yong Bae ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Capsicum Annuum ◽  
Magnaporthe Grisea ◽  
Dependent Manner ◽  
Appressorium Formation ◽  
Glutathione S Transferase ◽  
Porcine Liver ◽  
Esterase Gene ◽  
Dose Dependent ◽  
Dependent Signaling Pathway

A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned. Glutathione-S-transferase-tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters. Inoculation of compatible unripe pepper fruits with C. gloeosporioides spores amended with the recombinant protein did not cause anthracnose symptoms on the fruit. The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner. An esterase from porcine liver also inhibited appressorium formation, and the recombinant protein inhibited appressorium formation in the rice blast fungus, Magnaporthe grisea. Inhibition of appressorium formation in M. grisea by the recombinant protein was reversible by treatment with cyclic AMP (cAMP) or 1,16-hexadecanediol. The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus. Taken together, the PepEST esterase activity can inhibit appressorium formation of C. gloeosporioides, which may result in protection of the unripe fruit against the fungus.

scholarly journals Tea saponin reduces the damage of Ectropis obliqua to tea crops, and exerts reduced effects on the spiders Ebrechtella tricuspidata and Evarcha albaria compared to chemical insecticides

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4534 ◽  
Author(s):  
Chi Zeng ◽  
Lingbing Wu ◽  
Yao Zhao ◽  
Yueli Yun ◽  
Yu Peng
Keyword(s):  
Biological Control ◽  
Field Trials ◽  
Control Measures ◽  
Dependent Manner ◽  
Glutathione S Transferase ◽  
Tea Plantation ◽  
Tea Saponin ◽  
Yield And Quality ◽  
Significant Damage ◽  
Dose Dependent

Background Tea is one of the most economically important crops in China. However, the tea geometrid (Ectropis obliqua), a serious leaf-feeding pest, causes significant damage to tea crops and reduces tea yield and quality. Spiders are the most dominant predatory enemies in the tea plantation ecosystem, which makes them potentially useful biological control agents of E. obliqua. These highlight the need for alternative pest control measures. Our previous studies have shown that tea saponin (TS) exerts insecticidal activity against lepidopteran pests. Here, we investigate whether TS represents a potentially new alternative insecticide with no harm to spiders. Methods We investigated laboratory bioactivities and the field control properties of TS solution against E. obliqua. (i) A leaf-dip bioassay was used to evaluate the toxicity of TS to 3rd-instar E. obliqua larvae and effects of TS on the activities of enzymes glutathione-S-transferase (GST), acetylcholinesterase (AChE), carboxylesterase (CES) and peroxidase (POD) of 3rd-instar E. obliqua larvae in the laboratory. (ii) Topical application was used to measure the toxicity of 30% TS (w/v) and two chemical insecticides (10% bifenthrin EC and 50% diafenthiuron SC) to two species of spider, Ebrechtella tricuspidata and Evarcha albaria. (iii) Field trials were used to investigate the controlling efficacy of 30% TS against E. obliqua larvae and to classify the effect of TS to spiders in the tea plantation. Results The toxicity of TS to 3rd-instar E. obliqua larvae occurred in a dose-dependent manner and the LC50 was 164.32 mg/mL. Activities of the detoxifying-related enzymes, GST and POD, increased in 3rd-instar E. obliqua larvae, whereas AChE and CES were inhibited with time by treatment with TS. Mortalities of E. tricuspidata and E. albaria after 48 h with 30% TS treatment (16.67% and 20%, respectively) were significantly lower than those with 10% bifenthrin EC (80% and 73.33%, respectively) and 50% diafenthiuron EC (43.33% and 36.67%, respectively). The highest controlling efficacy of 30% TS was 77.02% at 5 d after treatment, which showed no difference to 10% bifenthrin EC or 50% diafenthiuron SC. 30% TS was placed in the class N (harmless or slightly harmful) of IOBC (International Organization of Biological Control) categories for natural enemies, namely spiders. Conclusions Our results indicate that TS is a botanical insecticide that has a good controlling efficacy in E. obliqua larvae, which suggests it has promise as application in the integrated pest management (IPM) envisaged for tea crops.

Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2

1994 ◽  
Vol 131 (5) ◽  
pp. 510-515 ◽  
Author(s):  
Osamu Kozawa ◽  
Haruhiko Tokuda ◽  
Atsushi Suzuki ◽  
Jun Kotoyori ◽  
Yoshiaki Ito ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Phosphoinositide Hydrolysis ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent

Kozawa O, Tokuda H, Suzuki A, Kotoyori J, Ito Y, Oiso Y. Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2. Eur J Endocrinol 1994;131:510–15. ISSN 0804–4643 It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2α stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2α-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2α in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2α- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2α-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2α-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells. Osamu Kozawa, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan

Cinnamomum cassia ameliorates Ni-NPs-induced liver and kidney damage in male Sprague Dawley rats

2020 ◽  
Vol 39 (11) ◽  
pp. 1565-1581
Author(s):  
S Iqbal ◽  
F Jabeen ◽  
C Peng ◽  
MU Ijaz ◽  
AS Chaudhry
Keyword(s):  
Dependent Manner ◽  
Sprague Dawley ◽  
Cinnamomum Cassia ◽  
Preliminary Information ◽  
Sprague Dawley Rats ◽  
Liver And Kidney ◽  
Altered Blood ◽  
Biochemical Analyses ◽  
Dose Dependent ◽  
Different Doses

Nickel nanoparticles (Ni-NPs) have been widely used in various industries related to electronics, ceramics, textiles, and nanomedicine. Ambient and occupational exposure to Ni-NPs may bring about potential detrimental effects on animals and humans. Thus, there is a growing effort to identify compounds that can ameliorate NPs-associated pathophysiologies. The present study examined Cinnamomum cassia ( C. cassia) bark extracts (CMBE) for its ameliorative activity against Ni-NPs-induced pathophysiological and histopathological alterations in male Sprague Dawley rats. The biochemical analyses revealed that dosing rats with Ni-NPs at 10 mg/kg/body weight (b.w.) significantly altered the normal structural and biochemical adaptations in the liver and kidney. Conversely, supplementations with CMBE at different doses (225, 200, and 175 mg/kg/b.w. of rat) ameliorated the altered blood biochemistry and reduced the biomarkers of liver and kidney function considerably ( p < 0.05) in a dose-dependent manner. However, the best results were at 225 mg/kg/b.w. of rat. The study provided preliminary information about the protective effect of C. cassia against Ni-NPs indicated liver and kidney damages. Future investigations are needed to explore C. cassia mechanism of action and isolation of single constituents of C. cassia to assess their pharmaceutical importance accordingly.

scholarly journals Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices

1984 ◽  
Vol 218 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Y Fujimoto ◽  
N Akamatsu ◽  
A Hattori ◽  
T Fujita
Keyword(s):  
Fatty Acids ◽  
Prostaglandin E2 ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Unsaturated Fatty Acids ◽  
Rabbit Kidney ◽  
Dependent Manner ◽  
Kidney Medulla ◽  
Diacylglycerol Lipase ◽  
Dose Dependent

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.

scholarly journals Negative Regulation of Parathyroid Hormone (PTH)-Activated Phospholipase C by PTH/PTH-Related Peptide Receptor Phosphorylation and Protein Kinase A

Endocrinology ◽  
2008 ◽  
Vol 149 (8) ◽  
pp. 4016-4023 ◽  
Author(s):  
Hesham A. W. Tawfeek ◽  
Abdul B. Abou-Samra
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Protein Kinase A ◽  
Phospholipase C ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Receptor Phosphorylation ◽  
Pthrp Receptor ◽  
Dose Dependent ◽  
Kinase A

PTH binding to the PTH/PTHrP receptor activates adenylate cyclase/protein kinase A (PKA) and phospholipase C (PLC) pathways and increases receptor phosphorylation. The mechanisms regulating PTH activation of PLC signaling are poorly understood. In the current study, we explored the role of PTH/PTHrP receptor phosphorylation and PKA in PTH activation of PLC. When treated with PTH, LLCPK-1 cells stably expressing a green fluorescent protein (GFP)-tagged wild-type (WT) PTH/PTHrP receptor show a small dose-dependent increase in PLC signaling as measured by inositol trisphosphate accumulation assay. In contrast, PTH treatment of LLCPK-1 cells stably expressing a GFP-tagged receptor mutated in its carboxyl-terminal tail so that it cannot be phosphorylated (PD-GFP) results in significantly higher PLC activation (P &lt; 0.001). The effects of PTH on PLC activation are dose dependent and reach maximum at the 100 nm PTH dose. When WT receptor-expressing cells are pretreated with H89, a specific inhibitor of PKA, PTH activation of PLC signaling is enhanced in a dose-dependent manner. H89 pretreatment in PD-GFP cells causes a further increase in PLC activation in response to PTH treatment. Interestingly, PTH and forskolin (adenylate cyclase/PKA pathway activator) treatment causes an increase in PLCβ3 phosphorylation at the Ser1105 inhibitory site and that increase is blocked by the PKA inhibitor, H89. Expression of a mutant PLCβ3 in which Ser1105 was mutated to alanine (PLCβ3-SA), in WT or PD cells increases PTH stimulation of inositol 1,4,5-trisphosphate formation. Altogether, these data suggest that PTH signaling to PLC is negatively regulated by PTH/PTHrP receptor phosphorylation and PKA. Furthermore, phosphorylation at Ser1105 is demonstrated as a regulatory mechanism of PLCβ3 by PKA.

scholarly journals Differential pathways (phospholipase C and phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts. Involvement of intracellular Ca2+ oscillations in phosphatidylcholine breakdown

1992 ◽  
Vol 283 (2) ◽  
pp. 347-354 ◽  
Author(s):  
T Fu ◽  
Y Okano ◽  
Y Nozawa
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Dependent Manner ◽  
3T3 Cells ◽  
3T3 Fibroblasts ◽  
Similar Dose ◽  
A Cell ◽  
Nih 3T3 ◽  
Dose Dependent ◽  
Nih 3T3 Cells

Bradykinin (BK) induced a biphasic increase in 1,2-diacylglycerol (DAG) in both K-ras-transformed fibroblasts (DT) and the parent NIH-3T3 cells. The first phase was coincident with the increase in Ins(1,4,5)P3 resulting from PtdIns(4,5)P2 hydrolysis, and the second, sustained, phase was derived from phosphatidylcholine (PtdCho) hydrolysis. In NIH-3T3 cells, stimulation by BK induced greater production of choline than phosphocholine in [3H]choline-labelled cells and appreciable phosphatidylethanol (PtdEtOH) formation in [3H]myristic acid-labelled cells, suggesting that PtdCho was hydrolysed mainly by a phospholipase D (PLD) activity. Pretreatment with propranolol, an inhibitor of phosphatidate phosphohydrolase, markedly diminished the second DAG accumulation, supporting the above notion. In DT cells, BK induced predominantly phosphocholine generation and little PtdEtOH formation, indicating that the PtdCho hydrolysis was due to a phospholipase C (PLC) activity. The BK-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) observed in single DT cells [Fu, Sugimoto, Oki, Murakami, Okano & Nozawa (1991) FEBS Lett. 281, 263-266] were detected as a sustained [Ca2+]i elevation when assayed in a cell suspension. A receptor-operated Ca2+ channel blocker, SK&F 96365, suppressed both the BK-induced phosphocholine generation and the sustained [Ca2+]i elevation in a similar dose-dependent manner. These results thus suggested that oscillations in [Ca2+]i are involved in the activation of PtdCho-specific PLC in DT cells.

scholarly journals AT1 receptor-activated signaling mediates angiotensin IV-induced renal cortical vasoconstriction in rats

2006 ◽  
Vol 290 (5) ◽  
pp. F1024-F1033 ◽  
Author(s):  
Xiao C. Li ◽  
Duncan J. Campbell ◽  
Mitsuru Ohishi ◽  
Shao Yuan ◽  
Jia L. Zhuo
Keyword(s):  
Blood Flow ◽  
Phospholipase C ◽  
Mitogen Activated Protein Kinase ◽  
Classical Type ◽  
Dependent Manner ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Angiotensin Iv ◽  
Ang Iv ◽  
Dose Dependent

Angiotensin IV (ANG IV), an active ANG II fragment, has been shown to induce systemic and renal cortical effects by binding to ANG IV (AT4) receptors and activating unique signaling transductions unrelated to classical type 1 (AT1) or type 2 (AT2) receptors. We tested whether ANG IV exerts systemic and renal cortical effects on blood pressure, renal microvascular smooth muscle cells (VSMCs), and glomerular mesangial cells (MC) and, if so, whether AT1 receptor-activated signaling is involved. In anesthetized rats, systemic infusion of ANG II, ANG III, or ANG IV (0.01, 0.1, and 1.0 nmol·kg−1·min−1 iv) caused dose-dependent increases in mean arterial pressure (MAP) and decreases in renal cortical blood flow (CBF; P < 0.01). ANG II also induced dose-dependent reductions in renal medullary blood flow ( P < 0.01), whereas ANG IV did not. ANG IV-induced pressor and renal cortical vasoconstriction were completely abolished by AT1 receptor blockade with losartan (5 mg/kg iv; P < 0.05). When ANG IV (1 nmol·kg−1·min−1) was infused directly in the renal artery, CBF was reduced by >30%, and the response was also blocked by losartan ( P < 0.01). In the renal cortex, unlabeled ANG IV displaced 125I-labeled [Sar1,Ile8]ANG II binding, whereas unlabeled ANG II (10 μM) inhibited 125I-labeled Nle1-ANG IV (AT4) binding in a concentration-dependent manner ( P < 0.01). In freshly isolated renal VSMCs, ANG IV (100 nM) increased intracellular Ca2+ concentration, and the effect was blocked by losartan and U-73122, a selective inhibitor of phospholipase C/inositol trisphosphate/Ca2+ signaling (1 μM). In cultured rat MCs, ANG IV (10 nM) induced mitogen-activated protein kinase extracellular/signal-regulated kinase 1/2 phosphorylation via AT1 receptor- and phospholipase C-activated signaling. These results suggest that, at nanomolar concentrations, ANG IV can increase MAP and induce renal cortical effects by interacting with AT1 receptor-activated signaling.

scholarly journals Glutathione conjugation of chlorambucil: measurement and modulation by plant polyphenols

1997 ◽  
Vol 325 (2) ◽  
pp. 417-422 ◽  
Author(s):  
Kai ZHANG ◽  
Kim Ping WONG
Keyword(s):  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Glutathione S Transferase ◽  
Plant Polyphenols ◽  
Simultaneous Exposure ◽  
Adenocarcinoma Cells ◽  
Combined Action ◽  
Dose Dependent

Chlorambucil (CMB), an anticancer drug, was cytotoxic at concentrations of 5–20 μM to human colon adenocarcinoma cells. It inhibited [14C]thymidine uptake in a dose-dependent manner. Both effects were potentiated by simultaneous exposure of the cells to 10 μM plant polyphenols. In an attempt to explain the possible mechanism of action of the polyphenols in relation to these observations, an HPLC-radiometric method was developed to measure the conjugation of CMB with glutathione in these cells and to monitor the export of monochloromonoglutathionyl CMB (MG-CMB), its main glutathione conjugate. At micromolar concentrations, five polyphenols, namely quercetin, butein, tannic acid, 2′-hydroxychalcone and morin, inhibited the efflux of CMB significantly; an inhibition of 40% was observed with 10 μM quercetin. The glutathione S-transferase (GST) activity of the cancer cells, measured with 1-chloro-2,4-dinitrobenzene, was also inhibited by the polyphenols. Their combined action on GST and on the efflux of MG-CMB conjugate could provide an enhanced positive modulation of sensitivity of the tumour cells to CMB.

Abstract 16460: Enhanced Trpc Channel-Mediated Ca2+ Influx in the Cells With Phospholipase C-δ1 Overexpression

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Kazuo Murakami ◽  
Tomohiro Osanai ◽  
Makoto Tanaka ◽  
Kimitaka Nishizaki ◽  
Ikuyo Narita ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Transient Receptor Potential ◽  
Coronary Spasm ◽  
Dependent Manner ◽  
Hek 293 ◽  
Trpc Channel ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dose Dependent

Background: We recently showed that ergometrine induces coronary spasm in the mice with enhanced phospholipase C (PLC)-δ1 activity, which was detected in patients with coronary spasm (Circulation 2012), and PLC-mediated Ca2+ entry is involved in the genesis of coronary spams. We examined the role of enhanced PLC-δ1 in extracellular Ca2+ entry and its underlying mechanisms in human embryonic kidney (HEK)-293 cells and human coronary arteries smooth muscle cells (CASMC). Methods and Results: The cells were stimulated with acetylcholine (ACh) in a extracellular Ca2+-free condition, and the increase in intracellular free Ca2+ concentration ([Ca2+]i) after addition of extracellular Ca2+ was defined as Ca2+ influx. [Ca2+]i was measured by fura-2. In HEK-293 cells, ACh-induced Ca2+ influx (nM ) was 21±2 in the control and 52±6 in the cells with PLC-δ1 overexpression (p<0.05). ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by nifedipine in a dose-dependent manner but was partially by 36±13% at 10-5M (p<0.05), thereby even after treatment with nifedipine at 10-5M, ACh-induced Ca2+ influx was increased by 2.9±0.1 times by enhanced PLC-δ1 compared with the control (p<0.05). To clarify the role of diacylglycerol (DAG) in Ca2+ influx, the effect of blockers for DAG-activated transient receptor potential (TRPC) channel was examined. ACh-induced Ca2+ influx in the cells with PLC-δ1 overexpression was suppressed by 2-APB, an inhibitor of non-selective cation channel TRPC, in a dose-dependent manner, and was almost completely blocked by 89±12% at 10-4M (p<0.05). While TRPC3 siRNA did not affect ACh-induced Ca2+ influx (59±27 vs 75±23 nM, p=ns), TRPC6 siRNA suppressed Ca2+ influx to 37±28 nM (p<0.05). In human CASMC, ACh-induced Ca2+ influx was 41±11 in the control and 64±15 in the cells with PLC-δ1 overexpression (p<0.05). Like HEK-293 cells, pretreatment with nifedipine partially suppressed Ca2+ influx, whereas either 2-APB or TRPC6 siRNA almost completely blocked it. Conclusion: ACh-induced Ca2+ influx, mediated by both voltage-gated Ca2+ channels and non-selective cation channels TRPC6, is enhanced by PLC-δ1 overexpression. Inhibition of TRPC may be effective in enhanced PLC-δ1-mediated coronary spasm.

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