Prostacyclin production after treatment of rat liver cells with tosyl-L-phenylalanine chloromethyl ketone and 12-O-tetradecanoylphorbol-13-acetate is mediated by a phorbol-ester-stimulated intermediate

The prostacyclin (PGI₂) production in rat liver cells by treatment by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is not dependent upon the simultaneous presence of both ligands. Preincubation of the rat liver cells with TPA followed by addition of TPCK, as well as preincubation of the cells with TPCK followed by addition of TPA, results in PGI₂ production. Maximum production is found after a 10 min incubation with TPA or after a 120 min incubation with TPCK. Preincubation with TPA for longer than 10 min or preincubation with TPCK for longer than 2 h results in reduced stimulation of PGI₂ production. Dexamethasone does not eliminate the effects of either preincubation or simultaneous addition of TPCK and TPA. EGTA does not affect either preincubation reaction but does completely inhibit PGI₂ production after simultaneous addition of the agents. Preincubation of the cells for 30 min with aspirin completely inhibits the TPCK-TPA-stimulated PGI₂ synthesis. The PGI₂ production following exogenous addition of arachidonic acid to the cells is unaffected by prior treatment of the cells with TPA, TPCK, or TPA plus TPCK. Taken together the data suggest that TPA stimulates the production of an intermediate which activates a Ca²⁺-dependent phospholipase activity. The intermediate is inactivated by a protease which is inhibited by the SH-reactive agent TPCK. The released arachidonic acid is oxygenated by the constitutively expressed cyclooxygenase (prostaglandin H synthase-1).     

Effect of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line)

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI₂ production (measured as 6-keto-PGF_1α in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI₂ production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca²⁺-ionophore a-23187, but not the PGI₂ synthesis stimulated by exogeneous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.     

Effect of protease inhibitors on protein degradation in rat hepatoma cells II. Effects on ornithine decarboxylase and tyrosine aminotransferase

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Expression of phospholipases A2 in primary human lung macrophages: Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA₂) and secreted phospholipases A₂ (sPLA₂) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA₂s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA₂ (iPLA₂). Two structurally-divergent inhibitors of group IV cPLA₂ completely block arachidonic acid release by macrophages in response to non-physiological (Ca²⁺ ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA₂s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA₂s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA₂ inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA₂-alpha, iPLA₂ and several sPLA₂s. Cytosolic PLA₂-alpha is the major enzyme responsible for lipid mediator production in human macrophages.     

Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells

Histamine stimulates the production of prostacyclin (PGI₂) in cultured human endothelial cells. We have examined the cell specificity of histamine-mediated PGI₂ synthesis in primary and subcultured human cells. Venous and arterial smooth muscle cells and skin fibroblasts synthesized PGI₂ from exogenous arachidonic acid, but they did not synthesize a significant amount of PGI₂ when treated with histamine. Endothelial cells, however, produced similar amounts of PGI₂ in response to histamine and arachidonic acid. Thrombin also stimulates PGI₂ production in endothelial cells. Histamine and thrombin yielded an additive production of PGI₂ when added simultaneously to endothelial cells. When histamine and thrombin were added sequentially, the amount of PGI₂ produced was not additive but equaled the amount characteristic of the first agonist alone. Following an initial treatment with histamine, endothelial cells were unable to respond to histamine for 3 hr, after which the PGI₂ biosynthetic response rapidly returned to normal by $\text{4}\text{1}\text{2}$ hr. When the initial histamine treatment was carried out under mildly alkaline conditions, the complete return of activity was delayed to 8 hr after treatment. The synthesis of PGI₂ from exogenous arachidonic acid was unaffected by prior treatment with histamine. Recovery of histamine-mediated PGI₂ production was not dependent on protein synthesis but required a component of fetal calf serum that is nondialyzable and moderately heat stable. Thus endothelial cell PGI₂ synthesis in response to a physiologic agonist is subject to several levels of regulation, reflecting not only intracellular events but also the extracellular environment.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

Oxysterol activation of arachidonic acid release and protaglindin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase activity

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.     

Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I₂. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI₂ synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (≥ 50% of control) and inhibiting basal and agonist-stimulated PGI₂ synthesis. This reduced PGI₂ synthesis preceded ⁵¹ chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI₂ synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI₂ synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

Regulation of prostaglandin production by osteoblast-rich calvarial cells

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were also shown to stimulate PGE₂ and 6-keto-PGF_1α (the hydration product of PGI₂) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE₂ and PGI₂ (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occuring at approximately 10⁻⁷ M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments which have indicated that the compounds stimulated PGI₂ production. Finally, since the osteoblasts is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early time EGF stimulated PGE₂ release, however, the predominant effect of the growth factor was an inhibition of both PGE₂ and PGI₂ production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the esteoblast and that any increase in PG production by these cells may be in response to vascular agents     

Role of arachidonic acid release in the G2 delay induced by tumor promoter TPA in HeLa cells

The tumor promoter TPA2 (12-O-tetradecanoylphorbol-13-acetate) has been shown to exhibit a radiomimetic activity on the cell cycle of HeLa cells (V. Kinzel, J. Richards, and M. St?hr (1980) Science 210, 429). The response includes a delay of cells in G2 phase. The relation between TPA-induced release of arachidonic acid (AA) and the inhibition in G2 phase was studied. Exogenous AA (greater than 10(-4) M; in presence of 10% serum) is shown to delay HeLa cells in G2 and to enhance the effectiveness of TPA in this respect. The inhibition of the TPA-induced AA liberation by fluocinolone acetonide, however, does not influence the TPA-effected G2 delay. The diacylglycerols 1,2-dioctanoyl-glycerol and 1-oleoyl-2-acetylglycerol delay HeLa cells in G2 but without major stimulation of AA liberation. On the basis of the data it is concluded that AA released from HeLa cells due to the action of TPA is not involved in the TPA-induced delay of cells in G2 phase.     

Actions of vanadate of arachidonic acid metabolism by cells in culture

Sodium vanadate (11 μM) amplified the PGI₂ production of rat liver cells (the C-9 cell line) incubated with thrombin, platelet activating factor, lysine-vasopressin, the Ca²⁺-ionophore A-23187, interleukin-1ß, 12-tetradecanoylphorbol-13-acetate, teleocidin, epidermal growth factor, palytoxin, thapsigargin and colchicine but not that stimulated by exogenous arachidonic acid. Sodium vanadate (2.2 μM) also amplified PGF_2α production of dog kidney cells (the MDCK cell line) incubated with norepinephrine and, at 0.4 μM, PGI₂ production of bovine aorta smooth muscle cells stimulated by serotonin. Sodium vanadate (55 μM) did not affect production of PGE₂ and PGF_2α in rat basophil leukemia cells (the RBL-1 cell line) stimulated by the Ca²⁺-ionophore A-23187, but did inhibit synthesis of peptide-containing leukotrienes and 12-hydroxyeicosatetraenoic acid. When used with cultured cells at micromolar concentrations, vanadate is known to inhibit protein tyrosine-phosphate phosphatases. These results suggest that in some cells deesterification of lipids is positively regulated, at least in part, by phosphorylation of tyrosine whereas in leukocytes, lipoxygenase activities are negatively regulated, at least in part, by phosphorylation of tyrosine.     

The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis

Cultured pulmonary artery endothelial cells produce PGI₂ as their primary prostaglandin. Conditions which inhibit cell division have been shown to accelerate the synthesis of this compound. Exposure of endothelial cells to γ raidation results in an irreversible cessation of growth and enhanced production of PGI₂. The level of PGI₂ measured after radiation exposure exceeds that observed in cultures rendered quiescent by serum reduction. This indicates a role for γ radiation in the elevation of PGI₂ levels which is distinct from its effect on cell division. Result presented indicate that exposure to γ radiation does not, in and of itself, elevate PG levels but capacitates cells for enhanced production when presented with appropriate stimuli. Increased PGI₂ synthesis appears to be a result of an observed increase in arachidonic acid release and an activation of cyclooxygenase.     

The Characterization of Cell Death Induced by 1-(3-C-Ethynyl-β-D-ribo-Pentofuranosyl)Cytosine (ECyd) in FM3A Cells

Abstract

The characterization of cell death induced by 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine(ECyd), a potent inhibitor of RNA synthesis, was performed using mouse mammary tumor FM3A cells in vitro. Accompanied with the cell death induced by ECyd (3.0 μM)-treatment, about 100–200 kbp-sized and internucleosomal DNA fragmentation were observed by orthogonal-field-alternation gel electrophoresis (OFAGE) and conventional gel electrophoresis, respectively. Protease inhibitors, carbobenzoxy-L-aspart-1-yl[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH₂-DCB), Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), effectively blocked the cell death, suggesting that the proteases inhibited by Z-Asp-CH₂-DCB, TLCK or TPCK were involved in the process of the cell death.     

Suppression of phorbol ester-enhanced radiation-induced malignancy in vitro by protease inhibitors is independent of protein kinase C

X-irradiation and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act in a synergistic manner to increase the yield of transformed C3H10T1/2 cells in vitro. TPA modulated both translocation from the cytosol to the plasma membrane, and down regulation of protein kinase C (PKC) after prolonged (48 h) TPA exposure. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), antipain, and soybean-derived Bowman-Birk inhibitor, protease inhibitors that suppress transformation of C3H10T1/2 cells, had no effect on these TPA-mediated alterations of PKC activity, suggesting that protease inhibitors suppress TPA-stimulated promotion in vitro via a PKC-independent pathway. Several experiments were performed to determine whether non-toxic concentrations of the PKC inhibitors, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), TPCK, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), or 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine (H-7), modulated the movement of cells from a quiescent state into the cell cycle. TPCK and the combination of H-7 and W-7 lowered DNA synthesis when cells were stimulated to divide by TPA. Because other protease inhibitors that slow transformation in vitro did not have the same suppressive effect on DNA synthesis, the inhibitory pathway that suppresses carcinogenic activity is likely to be different from the suppression of DNA synthesis.     

Prostacyclin (PGI2) Synthase Is a Constitutively Expressed Enzyme in Human Endothelial Cells

Biogenesis of prostanoids is under the control of some polypeptide growth factors. Cytosolic phospholipase A₂, a form specific for arachidonic acid containing phospholipids, is activated by a translocation mechanism regulated by growth factors, while prostaglandin H synthase isoforms are induced de novo in several cell types. No information is available as far as PGI₂ synthase is concerned. Human umbilical vein endothelial cells were cultured under conditions favoring proliferation or differentiation or capillary-like network formation in the presence of collagen gels. Basic fibroblast growth factor (bFGF 0.5-4 ng/ml) was used as a mitogen, interleukin-1α (IL-1α 10-60 UI/ml) as a differentiating agent, and prostacyclin (PGI₂) biosynthesis was evaluated. Under the first condition, basal PGI₂ production was unaffected while, in the presence of IL-1α, a marked stimulation of PGI₂ synthesis was observed. It is known that IL-1α is a potent inducer of PGH synthase, while it is not known whether PGI₂ synthase is also induced. Two lines of evidence indicate that PGI₂ synthase is a constitutively expressed not inducible enzyme: (a) proliferating nonproducing cells when added with PGH₂ produce an amount of PGI₂ not different from the amount produced by cells stimulated with IL-1α; (b) under this condition PGI₂ synthase was immunodetectable either by immunofluorescence detected by confocal microscopy or by ELISA and, on microsomes isolated from endothelial cells, by Western blotting. It is concluded that the limiting step in the conversion arachidonate-PGI₂ is represented solely by the level of PGH synthase. These results strongly suggest, but do not prove, the constitutive nature of the enzyme. The final demonstration requires the availability of a probe to detect mRNA level, a trial we are carrying out at the moment.     

Aggregation and thromboxane B2 formation in platelets and vascular prostacyclin production from genetically obese rats

Obesity, diabetes, hyperlipidaemia and age are conditions predisposing to atheroscleorosis and arterial occlusion. Recently it has been claimed that increased synthesis of thromboxane A₂ by platelets and decreased synthesis of prostacyclin (PGI₂) by blood vessels play an important role. The “Zucker” rat, a genetically obese animal with hyperlipidaemia, hyperinsulinaemia and normoglycaemia was used to study platelet aggregation, thromboxane (TXB₂) production and aortic PGI₂ synthesis. Two age groups (6–8 months and 14–16 months old) and their homozygote lean controls were used. In the obese rats no increased aggregation was found with ADP, arachidonic acid and collagen. On the contrary platelets from young fatty rats were less sensitive to ADP than platelets from lean young animals. An increase in platelet sensitivity to aggregating agents with age was observed, especially in the obese rats. TXB₂ measured in platelet rich plasma after exposure to ADP, arachidonic acid, arachidonic acid plus ADP and collagen was similar in the fatty and lean animals.Production of PGI₂ from incubated aortic rings was lowest in young lean animals. No differences existed between the other groups of rats studied. Insulin added to aortic rings had no influence on PGI₂ production. It is concluded that age rather than obesity, hyperlipidaemia or hyperinsulinaemia may cause platelet hyperresponsiveness to aggregating agents. Thromboxane and plateletaggregation do not closely correlate. PGI₂ production is not reduced by metabolic alterations, thought to predispose to atherosclerosis.     

Low prostacyclin synthetase activity of fetal rat aorta. Progressive increase after birth.

Aortae from fetal or 3 weeks old rats produced very small amounts of PGI₂, prostacyclin. This production increased from 4 weeks on, reaching adult values at about ten weeks. This maturation seemed to be predominantly determined by a change in the PGI₂ synthetase system, rather than in arachidonic acid availability, phospholipase or cyclo-oxygenase activity. The anti-oxidant ascorbic acid stimulated prostacyclin production more strongly in adult than in young rat aortae. This finding suggests that the lower production of PGI₂ by young tissues is not due to an enhanced inhibition of prostacyclin synthetase by lipid peroxides.     

Endogenous factors affecting arachidonic acid metabolism: I. Biosynthesis of prostacyclin and prostaglandins by carrageenin granulomas of rats

Arachidonic acid was converted by incubated slices of the rat carrageenin granuloma to prostacyclin (PGI₂), prostaglandins (PGs) E₂ and F_2∞ as detected by bioassay and radiochemical assay. PGI₂ was the major product of arachidonic acid metabolism in the granuloma slices. PGI₂ and PGE₂ formation was dependent on the concentration of the substrate and on the age of the granuloma. Slices obtained from 5-day old granulomas produced significantly more PGI₂ than slices prepared from 3-day old or 8- to 9-day old granulomas while PGE₂ generation was not dependent on the stage of the development of the granuloma. Homogenates of granuloma tissue hardly converted arachidonic acid to PGI₂ at all. This was probably due to the presence of an non- dialysable and heat labile material which, when partially isolated, inhibited PGI₂ production by bovine aortic microsomes.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.