[Some critical points on current liver cancer research].
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Animals↗
Biomedical subjects
Y Ruan
Publications and source records attributed to Y Ruan.
[Morphometric investigation on hypoxic structural remodeling of intraacinar pulmonary arteries].
OBJECTIVE: To observe the inhibitory effects of Radix Astragali (RA) on hypoxic structural remodeling of intraacinar pulmonary arteries (IAPA) and pulmonary hypertension (PHT). METHOD: 60 rats were divided into H (hypoxia) group (n = 20 rats) H + RA group (n = 20) and normal control (C) group (n = 20). The rats of H group and H + RA group were fed in hypoxic environment under normal atmospheric pressure (10% O2, 10 hours/day). On the 15th and 30th day of hypoxia, right ventricular systolic pressure (RVSP) and right ventricle hypertrophy index (RVHI) were measured and pulmonary vessel changes were studied under light and electron microscope with morphometric analysis. RESULT: Compared with H + RA group on 30th day, RVSP in H was 1.8 time as high as that of H + RA(P < 0.05); RVHI in H was 1.3 time as high as that of H + RA (P < 0.05); the media thickness of IAPA in H was 2.3 time as high as that of H + RA (P < 0.05); the density of adventitial fibroblasts of IAPA was 13.1 +/- 0.3 (H) and 7.6 +/- 0.2 (H + RA) (P < 0.05). CONCLUSION: Preserving the IAPA wall cells and dilating IAPA by RA may play an important role in inhibiting remodeling of IAPA and PTH.
Animals↗
[Effect of herbs 814 on tumor necrosis factor a production from alveolar macrophages in vitro].
OBJECTIVE: To study the influence of herbs 814 on the secretion of tumor necrosis factor alpha (TNF-alpha) from hamster alveolar macrophages (AMs) in vitro and elucidate mechanism of herbs 814's prevention of emphysema. METHODS: AMs of hamster were collected by bronchial alveolar lavage (BAL). The lung lavage cells were adjusted to 5 x 10(5)/ml and seeded in each well of a 24-well tissue culture plates. The herbs 814 was added into these wells before or after lipopolysaccharide (LPS) stimulation and then cultured supernatants were collected. TNF-alpha production in the supernatants was tested by ELISA, TNF-alpha cytotoxicity was assayed using L929 cells which were susceptible to TNF-alpha and a monoclonal antibody (MAb) that neutralizes rhTNF-alpha was utilized to identify the cytotoxicity of cultured TNF-alpha. RESULTS: ELISA showed that TNF-alpha production in the supernatants with 814 added was lower than those either in LPS-stimulated or non LPS-stimulated supernatants and there was a significant decrease in the supernatants of higher concentration (1:5) of herbs 814. Cytotoxicity test showed that TNF-alpha cytotoxic activity in the supernatants into which herbs 814 was added was lower than one in the LPS-stimulated supernatants and there was a significant decrease in the supernatants of higher concentration (1:5) of herbs 814. Meanwhile, MAb significantly reduced cytotoxicity of LPS-stimulated culture supernatants. CONCLUSIONS: Herbs 814 could inhibit the secretion of TNF-alpha and prevent the lung damage mediated by TNF-alpha.
Animals↗
Alpha-1A adrenergic receptor stimulation with phenylephrine promotes arachidonic acid release by activation of phospholipase D in rat-1 fibroblasts: inhibition by protein kinase A.
This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modulation by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A > alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR-expressing R-1Fs. 8-(4-chlorophenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, alpha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A.
Adenine↗
Expression of CD40 induces neural apoptosis.
The tumor necrosis factor receptor superfamily includes 12 members, some of which (e.g., tumor necrosis factor receptor I and FAS) induce cell death triggered by ligand binding. Another member of the superfamily, the neurotrophin receptor p75NTR, induces neural apoptosis, with apoptosis being inhibited by binding of ligand to the receptor. As such, it is a candidate for the mediation of neurotrophin dependence. Here, we show that CD40, a superfamily member that is closely related to p75NTR, also induces neural apoptosis, but apoptosis is inhibited by binding of the G28-5 monoclonal antibody to CD40. These results provide further support for a model in which some members of the tumor necrosis factor receptor superfamily induce apoptosis triggered by ligand binding, whereas other members may, at least under certain conditions, induce apoptosis in the absence of ligand binding, with apoptosis being inhibited by binding of ligand or monoclonal antibody.
Animals↗
The relationship between 67KD laminin receptor expression and metastasis of hepatocellular carcinoma.
The 67KD laminin receptor (LN-R) that binds laminin (LN) is involved in the metastasis cascade. Using immunohistochemical technique, in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR), we studied LN-R protein and RNA levels in 30 cases of human hepatocellular carcinoma (HCC) to further understand its role in the metastasis of HCC. In our 14 cases of HCC with metastasis, its positive rates were 71.4%, 57.1%, 85.7% respectively, whereas its positive expression in 16 cases without metastasis were 31.3%, 18.8%, 50.0% respectively. The significant difference was found between these two groups. The results suggest that the 67KD LN-R expression plays a very important role in the metastasis of HCC.
Basement Membrane↗
Mechanism of resistance to alpha-adrenergic receptor antagonists of renal nerve stimulation-induced vasoconstriction at low frequencies.
To determine why renal vasoconstriction elicited by periarterial nerve stimulation (RNS) at lower frequencies (< 4 Hz) is resistant to alpha-adrenergic receptor blockade in the rat kidney, we reevaluated the effect of alpha-receptor antagonists on the vasoconstrictor response to norepinephrine (NE) and to RNS and on the release of adrenergic transmitter. The alpha-receptor antagonist prazosin (PZ) at 0.2 and 7 nM reduced the vasoconstrictor response to NE, and 2.4 microM PZ abolished it. PZ (0.2 or 7 nM) reduced RNS-induced vasoconstriction without altering the fractional tritium overflow. PZ (2.4 microM) enhanced fractional tritium overflow and reduced the vasoconstrictor response to RNS at 2-10 Hz, but not at 0.5 or 1 Hz. The effect of 0.2 nM PZ to reduce RNS-induced vasoconstriction was reversed by increasing the concentration to 2.4 microM. Corynanthine (COR; 2.6 microM), a preferential alpha-receptor blocker, or phenoxybenzamine (PBZ; 30 nM) abolished the vasoconstrictor response to NE but only partially reduced response to RNS and enhanced the fractional tritium overflow. Rauwolscine (RW; 2.5 nM), a preferential alpha 2-receptor antagonist, did not alter the vasoconstrictor response to NE but potentiated RNS-induced vasoconstriction and fractional tritium overflow. RW (7.7 microM) inhibited NE-induced vasoconstriction but potentiated the vasoconstrictor response to RNS and fractional tritium overflow. PZ (7 nM) abolished the potentiation by RW and reduced the vasoconstrictor response to RNS. These data suggest that a component of RNS-induced vasoconstriction in the rat kidney is attributable to co-release of a nonadrenergic transmitter with NE. The diminished effect of alpha-receptor antagonists at higher concentrations (e.g., PZ 2.4 microM) to reduce RNS-induced vasoconstriction is caused by their prejunctional action to enhance co-release of the nonadrenergic transmitter.
Adrenergic Antagonists↗
[Therapeutic effect of radix Astragali on hypoxia pulmonary hypertension in rats].
Radix Astragali could inhibit the type III collagen deposition, smooth muscle cell proliferation in IAPA, and increase the tolerance of myocardium to hypoxia in rats.
Animals↗
Beta adrenergic receptor stimulated prostacyclin synthesis in rabbit coronary endothelial cells is mediated by selective activation of phospholipase D: inhibition by adenosine 3'5'-cyclic monophosphate.
Activation of beta adrenergic receptors in the isolated rabbit heart by catecholamines stimulates prostacyclin (PGI2) synthesis, which is inhibited by adenosine 3'5'-cyclic monophosphate (cAMP). The purpose of this study was to determine if activation of beta adrenergic receptors in cultured coronary endothelial cells (CEC) of rabbit heart with isoproterenol (ISOP) stimulates PGI2 synthesis and if cAMP inhibits the synthesis of this prostanoid and to investigate the underlying mechanism. Incubation of CEC with ISOP increased production of cAMP and PGI2, measured as immunoreactive cAMP and 6-keto-prostaglandin F1alpha, (6-keto-PGF1alpha), respectively. Forskolin, an activator of adenylyl cyclase, increased cAMP accumulation and inhibited ISOP-stimulated 6-keto-PGF1alpha synthesis. 8-(4-chlorophenyl-thio) cAMP also inhibited ISOP-induced 6-keto-PGF1alpha production. However, miconazole, an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-stimulated 6-keto-PGF1alpha synthesis in CEC. ISOP-induced 6-keto-PGF1alpha synthesis was attenuated by C2-ceramide, an inhibitor of phospholipase D (PLD) by propranolol, a beta-AR antagonist that also inhibits phosphatidate phosphohydrolase and by the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267). Acetylcholine (ACh) induced 6-keto-PGF1alpha synthesis was also inhibited by these agents. Both ISOP and ACh increased PLD activity, which was inhibited by C2-ceramide but not by RHC 80267 or propranolol. ACh but not ISOP increased phospholipase A2 activity in CEC. ISOP- but not ACh-induced increase in PLD activity was attenuated by forskolin and 8-(4-chlorophenyl-thio)-adenosine 3'-5'-cyclic monophosphate and augmented by miconazole. These data suggest that beta adrenergic receptors activation promotes PGI2 synthesis in the CEC by selective activation of PLD and that cAMP decreases PGI2 synthesis by decreasing PLD activity. Moreover, beta adrenergic receptors activated PLD appears to be distinct from that stimulated by ACh.
Animals↗
Signal transduction mechanism(s) involved in prostacyclin production elicited by acetylcholine in coronary endothelial cells of rabbit heart.
The purpose of this study was to elucidate the mechanism by which acetylcholine (ACh) promotes prostacyclin (PGI2) production in cultured coronary endothelial cells (CEC) of the rabbit heart. ACh-induced production of PGI2, measured as immunoreactive 6-keto-PGF1alpha, was enhanced by increasing the extracellular calcium (Ca++) concentration and reduced by Ca++ depletion. The receptor-operated Ca++ channel blocker SK&F96365, but not the voltage-dependent Ca++ channel blockers verapamil or nifedipine, attenuated ACh-induced 6-keto-PGF1alpha production and the associated rise in cytosolic Ca++. Thapsigargin, which depleted Ca++ accumulation from the intracellular Ca++ store, did not prevent the ACh-induced rise in cytosolic Ca++. In the absence of extracellular Ca++, ACh and ATP increased cytosolic Ca++ but did not alter 6-keto-PGF1alpha production. In permeabilized CEC, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) but not ACh enhanced 6-keto-PGF1alpha synthesis. ACh increased 6-keto-PGF1alpha production in the presence of GTP-gamma-S. These effects of GTP-gamma-S were attenuated by guanosine 5'-O-(2-thiotriphosphate). In the absence of extracellular Ca++, ACh or ATP increased cytosolic Ca++ in cells permeabilized with beta-escin and loaded with GTP-gamma-S; this effect was attenuated by guanosine 5'-O-(2-thiotriphosphate). The effect of ATP but not ACh to mobilize intracellular Ca++ or increase 6-keto-PGF1alpha was inhibited by pertussis toxin. The phospholipase C inhibitor D609, which attenuated ACh- and ATP-induced mobilization of intracellular Ca++, did not alter 6-keto-PGF1alpha production. The NO synthase inhibitor N-monomethyl-arginine also failed to alter ACh-induced 6-keto-PGF1alpha synthesis. These data suggest that, in CEC of the rabbit heart, ACh stimulates prostacyclin production via a pertussis toxin-insensitive G protein and by increasing the influx of extracellular Ca++ through a G protein-independent receptor-operated Ca++ channel.
Acetylcholine↗
Identification of elements in the PDA1 promoter of Nectria haematococca necessary for a high level of transcription in vitro.
Expression of the PDA1 gene in the ascomycete Nectria haematococca MPVI (anamorph: Fusarium solani) is induced by exposure of mycelium to pisatin, an isoflavonoid phytoalexin produced by its host plant, garden pea. The PDA1 gene encodes a cytochrome P-450 monooxygenase which detoxifies pisatin. Regulatory elements controlling transcription from the PDA1 promoter were identified using a homologous Nectria in vitro transcription system through analysis of 5' deletions, specific oligonucleotide competition, and fusion of upstream segments to a heterologous promoter. A promoter-distal element which provided transcriptional activation was localized to a 35-bp region positioned -514 to -483 upstream of the transcriptional start site. This 35-bp region binds a previously characterized pisatin-responsive DNA-binding factor (PRF) and thus may provide pisatin-responsive control of transcription. A second promoter-proximal positive-acting region was found to be necessary for promoter transcription in both homologous and heterologous extracts, and so is likely to bind less genespecific transcription activator(s). A negative-acting element located between these two positive regions may act to make the positive-acting elements interdependent. The identification of an activator responding to pisatin provides a model for the control of a number of genes and processes controlled by host-specific signals, particularly the flavonoids.
Base Sequence↗
The relationship between mast cell infiltration and the expression of PCNA and EGFr in experimental hepatocellular carcinoma of liver and squamous cell carcinoma of stomach in rats.
The expression of PCNA and EGFr in chemically induced hepatocellular carcinoma of liver and squamous cell carcinoma of stomach in rats were immunohistochemically observed. The results showed that the carcinoma cells of both tumors revealed a positive immunreaction to PCNA and EGFr. The histochemical observation of mast cell (MC) in both tumor tissues showed that the amount of MC in the surroundings of tumor cell nests was markedly different. According to the amount of the surrounding MCs the tumor cell nests could be divided into two groups: Group A with abundant MC infiltration and Group B with only scarce or without MC infiltration. The PCNA-positive cells in the tumor cell nests of both groups were calculated respectively. The results revealed that the amount of PCNA-positive cells in the group B was markedly more than that in the group A. The numerical ratio between two groups was 3:1 in the liver carcinoma and 2:1 in the stomach carcinoma approximately. An overexpression of EGFr was observed in tumor tissues of both groups, but there was also a marked difference in the amount of positively expressed cells and in the intensity of their staining reaction between both groups. The positively expressed cells in group B were much more and their staining intensity was much stronger than those in group A. According to the above mentioned results of observation, the expression state of both factors (PCNA and EGFr) was basically identical, suggesting that the MC may possess some inhibitory effect upon the growth rate of tumor cells of the experimental hepatocellular carcinoma of liver and the squamous cell carcinoma of stomach in rats.
Animals↗
Analysis of determinants of binding and transcriptional activation of the pisatin-responsive DNA binding factor of Nectria haematococca.
Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca mating population VI (anamorph: Fusarium solani) that are highly virulent on pea have been found to possess the PDA1 gene encoding a pisatin detoxifying activity. Expression of PDA1 is specifically and highly induced by exposure of mycelia to pisatin. A pisatin-responsive DNA-binding activity has previously been identified with properties suggestive of a transcriptional regulator of PDA1. In this study, the sequence determinants for binding this pisatin-responsive factor (PRF) were localized to a 14-bp region through analysis of sequence alterations that reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts that shared the sequence specificity characteristic of the PRF, indicating function of the DNA-binding protein in transcriptional control. A 70-kDa protein was shown to be a DNA-binding component of PRF by three independent assays for DNA-binding proteins: Southwestern (DNA-protein) blotting, UV-crosslinking, and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter that is responsive to a host-specific compound and provide insight into the regulation of fungal genes in response to plant flavonoids.
Base Sequence↗
Modulation of beta-adrenergic receptor-stimulated lipolysis in the heart by prostaglandins.
The purpose of the present study was to investigate the contribution of prostaglandins to lipolysis elicited by beta-adrenergic receptor activation in the heart. We have studied the effect of prostaglandin E2 (PGE2), prostaglandin I2 (PGI2), and their precursor arachidonic acid (AA) in the presence and absence of a cyclooxygenase inhibitor, sodium meclofenamate, on glycerol output elicited by stimulation of beta-adrenergic receptors in the isolated rabbit heart with isoproterenol (ISOP). Bolus injections of ISOP (475 pmol) produced a constant increase in glycerol and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) output. Infusion of sodium meclofenamate (16 microM) reduced basal and attenuated ISOP-induced 6-keto-PGF1 alpha output and enhanced glycerol output. During inhibition of endogenous prostaglandin synthesis with meclofenamate, infusion of PGI2 or PGE2 (0.1-1 microM) inhibited ISOP-induced glycerol output. Infusion of AA (0.1-1 microM) increased 6-keto-PGF1 alpha and reduced glycerol output. Infusion of sodium meclofenamate abolished the effect of AA to increase 6-keto-PGF1 alpha and to decrease glycerol output. These data suggest that prostaglandins synthesized in the heart act as an inhibitory modulator of beta-adrenergic receptor-stimulated cardiac lipolysis.
Animals↗
[Expression of PCNA and protooncogenes during experimental hepatocarcinogenesis in rats].
The expression of PCNA and the protooncogenes of ras family (N-ras, H-ras and Ki-ras) in hepatocarcinoma cells and the proliferating liver cells in precancerous, hyperplastic nodules during experimental hepatocarcinogenesis, induced by diethylnitrosamine in rat liver was observed immunohistochemically and by in situ hybridization. The results indicated that there was a positive expression of PCNA in both the carcinomas cells and precancerous liver cells. The amount of PCNA-positive cells exhibited a negative correlation with the amount of infiltrating mast cells surrounding carcinoma cell nests and the hyperplastic, precancerous nodules. These results basically coincided with that of the separate observation of the expression of protooncogenes in the same study.
Animals↗
Modulation by cyclic AMP of beta adrenergic receptor-stimulated prostacyclin synthesis in rabbit ventricular myocytes.
The purpose of the present study was to determine the possible interaction of cyclic AMP (cAMP) and the synthesis of prostacyclin [measured as immunoreactive 6-keto-prostaglandin (PG)F1 alpha] elicited by the beta adrenergic receptor agonist isoproterenol (ISOP), in freshly dissociated rabbit ventricular myocytes. ISOP (10(-13) to 10(-11) M) increased 6-keto-PGF1 alpha synthesis without altering the level of cAMP. Increasing the concentration of ISOP from 10(-10) to 10(-7) M enhanced accumulation of cAMP, which was associated with a decline in 6-keto-PGF1 alpha synthesis. Forskolin (10(-6) M), an activator of adenylyl cyclase, and 3-isobutyl-1-methylxanthine (10(-5) M), an inhibitor of cAMP phosphodiesterase, increased cAMP accumulation and inhibited ISOP-induced 6-keto-PGF1 alpha synthesis. 8-(4-chlorophenylthio) (cpt)-cAMP (10(-7) M) also inhibited ISOP-induced 6-keto-PGF1 alpha production. On the other hand, miconazole (10(-4) M), an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-induced 6-keto-PGF1 alpha synthesis in myocytes. Miconazole also attenuated ISOP-, forskolin- and cpt-cAMP-induced increases in protein kinase A activity. The protein kinase A inhibitor H-89 {N-[2-(p-bromocinnamylamino)ethyl] -5-isoquinolinesulfonamide} attenuated the ISOP (10(-7) M)-induced increase in the activity of this enzyme and minimized the decline in 6-keto-PGF1 alpha synthesis produced by 10(-7) M ISOP and the inhibitory effect of cpt-cAMP and forskolin on 6-keto-PGF1 alpha production. 3-Isobutyl-1-methylxanthine, forskolin and cpt-cAMP did not alter the conversion of exogenous arachidonic acid to 6-keto-PGF1 alpha. These data indicate that beta adrenergic receptor activation promotes prostacyclin synthesis in rabbit ventricular myocytes and that cAMP acts as an inhibitory modulator. This action is mediated via activation of protein kinase A, probably by decreasing the activity of the lipase, involved in beta adrenergic receptor-induced arachidonic acid release.
Animals↗
Localization and characterization of the subtypes(s) of muscarinic receptor involved in prostacyclin synthesis in rabbit heart.
The present study was conducted to localize and characterize the subtype(s) of muscarinic receptor involved in prostacyclin production elicited by the cholinergic transmitter acetylcholine (ACh) in various cell types in the rabbit heart. ACh increased prostacyclin synthesis, measured as 6-keto-prostaglandin(1 alpha) (6-keto-PGF(1 alpha)), in cultured coronary endothelial cells and freshly dissociated ventricular myocytes in a dose-dependent manner, but not in cultured coronary smooth muscle cells of rabbit heart. McN-A-343 {(4-hydroxy-2-butynyl)-1-trimethylammonium-m-chlorocarbanilate chloride}, a selective M1 muscarinic ACh receptor (mAChR) agonist, did not alter 6-keto-PGF(1 alpha) synthesis in these cell types. ACh induced 6-keto-PGF(1 alpha) synthesis in coronary endothelial cells and ventricular myocytes was not altered by a low concentration (0.01 microM) of pirenzepine, an M1 mAChR antagonist, but was reduced by a higher concentration (1 microM). In coronary endothelial cells, ACh-induced 6-keto-PGF(1 alpha) production was reduced by hexahydrosila-difendial (HHSiD), an M3 mAChR antagonist, and in ventricular myocytes by both AF-DX 116 [11-{2-[(diethylamino)methyl]-1-piperidinyl]acetyl-5,11-dihydro-6H- pyrido[2,3-b]-benzodiazepine-6 one}], an M2 receptor antagonist, and HHSiD. The decrease by ACh of isoproterenol-stimulated cAMP accumulation was minimized by AF-DX 116, but not by HHSiD or pirenzepine. Pertussis toxin treatment minimized ACh-induced decrease in isoproterenol-stimulated rise in cAMP, but not ACh-induced 6-keto-PGF(1 alpha) synthesis. These data suggest that ACh stimulates prostacyclin production in coronary endothelial cells via M3 mAChR and in ventricular myocytes via M2 and M3 mAChR, and may contribute to its cardioprotective effects. Moreover, ACh induced decrease in cAMP, but not the increase in 6-keto-PGF (1 alpha) production, is mediated by pertussis toxin-sensitive G(alpha i) proteins in these cells.
Acetylcholine↗
Involvement of mitogen-activated protein kinase and translocation of cytosolic phospholipase A2 to the nuclear envelope in acetylcholine-induced prostacyclin synthesis in rabbit coronary endothelial cells.
We previously showed that acetylcholine (ACh) stimulates production of prostacyclin, measured as immunoreactive 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in coronary endothelial cells (CEC) of rabbit heart by increasing influx of extracellular Ca2+ through a receptor-operated Ca2+ channel and by activating a pertussis toxin-insensitive G protein. The purposes of this study were to elucidate the type of phospholipase A2 (PLA2) involved in 6-keto-PGF1 alpha production and the mechanism(s) by which ACh activates PLA2 in cultured CEC. In CEC transiently transfected with cytosolic PLA2 but not secretory PLA2 antisense oligonucleotide, ACh failed to increase 6-keto-PGF1 alpha; this was prevented by cotransfection with cPLA2 sense oligonucleotide. ACh increased production of prostacyclin and increased protein kinase C (PKC) activity. The PKC inhibitor calphostin C attenuated the ACh-induced increase in PKC activity but not 6-keto-PGF1 alpha production. Phorbol-12-myristate-13-acetate and phorbol-12, 13-dibutyrate increased PKC activity but failed to alter 6-keto-PGF1 alpha production. ACh enhanced the activity of cPLA2 and p42 mitogen-activated protein kinase (MAPK) in cell lysate prepared from CEC. ACh also caused phosphorylation of p42 MAPK and cPLA2, which was inhibited by AG126 ([alpha-cyano-(3-hydroxy-4-nitro)cinnamonitrile]), a tyrosine kinase inhibitor known to decrease MAPK activity. In addition, ACh stimulated translocation of cPLA2 from cytosol to nuclear envelope; the translocation of cPLA2 was prevented by removal of extracellular calcium but not by AG126 treatment. Okadaic acid, a protein phosphatase inhibitor, increased cPLA2 activity in cell lysate prepared from CEC but did not alter basal 6-keto-PGF1 alpha production in intact CEC; however, ACh-induced 6-keto-PGF1 alpha was enhanced by okadaic acid. These data suggest that ACh stimulates prostacyclin synthesis by activation of cPLA2 in a PKC-independent mechanism and that both cPLA2 translocation to nuclear envelope and phosphorylation by MAPK are required for ACh-induced 6-keto-PGF1 alpha synthesis in CEC.
6-Ketoprostaglandin F1 alpha↗