Okadaic acid and dinophysistoxin-1, non-TPA-type tumor promoters, stimulate prostaglandin E2 production in rat peritoneal macrophages

Okadaic acid and dinophysistoxin-1 isolated from a black sponge, Halichondria okadai are non-12-O-tetrade-canoylphorbol 13-acetate (non-TPA)-type tumor promoters of mouse skin. Okadaic acid at concentrations of 10-100 ng/ml stimulated prostaglandin E2 production in rat peritoneal macrophages. Dinophysistoxin-1 (35-methylokadaic acid) stimulated prostaglandin E2 production as strong as okadaic acid, but okadaic acid tetramethyl ether, an inactive compound as a tumor promoter, did not. Okadaic acid at 10 ng/ml (12.4 nM) stimulated prostaglandin E2 production as strongly as TPA at 10 ng/ml (16.2 nM) 20 h after incubation. Unlike TPA-type tumor promoters, okadaic acid required a lag phase before stimulation. The duration of this lag phase was dependent on the concentration of okadaic acid. Indomethacin inhibited okadaic acid-induced preostaglandin E2 production in a dose-dependent manner, and its inhibition was more strongly observed in okadaic acid-induced prostaglandin E2 production. Cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages and prostaglandin E2 production dose dependently, suggesting that protein synthesis is a prerequisite for the stimulation of arachidonic acid metabolism. These results support our idea that tumor promoters, at very low concentrations, are able to stimulate arachidonic acid metabolism in rat peritoneal macrophages.     

Combinations of palytoxin or 12-O-tetradecanoylphorbol-13-acetate and recombinant human insulin growth factor-I or insulin synergistically stimulate prostaglandin production in cultured rat liver cells and squirrel monkey aorta smooth muscle cells

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) or the non-TPA-type tumor promoter, palytoxin, recombinant human insulin growth factor-I (IGF-I) and insulin synergistically stimulate prostaglandin production in rat liver cells (the C-9 cell line). Combinations fo palytoxin or TPA with recombinant human IGF-I or insulin also synergistically stimulate deesterification of c ellular lipids in C-9 cells prelabelled with [³H]arachidonic acid. With both types of stimulations, prostaglandin production or deesterification, the synergistic response of the IGF-I and insulin is greater with palytoxin than with TPA. Production of prostaglandin E₂ and F_2α by squirrel monkey smooth muscle cells incubated in the presence of TPA and insulin also is greater than the sum of the effects taken independently.     

Combinations of palytoxin or 12-O-tetradecanoylphorbol-13-acetate and recombinant human insulin growth factor-I or insulin synergistically stimulate prostaglandin production in cultured rat liver cells and squirrel monkey aorta smooth muscle cells

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) or the non-TPA-type tumor promoter, palytoxin, recombinant human insulin growth factor-I (IGF-I) and insulin synergistically stimulate prostaglandin production in rat liver cells (the C-9 cell line). Combinations of palytoxin or TPA with recombinant human IGF-I or insulin also synergistically stimulate deesterification of cellular lipids in C-9 cells prelabelled with [3H]arachidonic acid. With both types of stimulations, prostaglandin production or deesterification, the synergistic response of the IGF-I and insulin is greater with palytoxin than with TPA. Production of prostaglandin E2 and F2 alpha by squirrel monkey smooth muscle cells incubated in the presence of TPA and insulin also is greater than the sum of the two effects taken independently.     

Stimulation of arachidonic acid metabolism: differences in potencies of recombinant human interleukin-1 alpha and interleukin-1 beta on two cell types

Recombinant human interleukin-l (rIL-1) alpha and beta, which have 26% homology in their amino acid sequence, stimulated arachidonic acid metabolism by squirrel monkey smooth muscle cells and rat liver cells; their relative effectiveness, however, varied with the two cells. Recombinant IL-1 alpha was 3 times more effective than rIL-1 beta at stimulating arachidonic acid metabolism by the primate smooth muscle cells. Recombinant IL-1 alpha was 3 times less effective than rIL-1 beta when measured by their capacity to synergistically stimulate arachidonic acid metabolism of rat liver cells in the presence of palytoxin and anti-diuretic hormone (ADH). The rIL-1 alpha and rIL-1 beta also stimulated the release of radiolabelled arachidonic acid from the smooth muscle cells prelabelled with [3H]arachidonic acid. The two recombinant IL-1s have different heat stabilities, again when measured by their capacity to stimulate arachidonic acid metabolism; IL-1 alpha was more heat stable than IL-1 beta.     

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.     

Amplification of arachidonic acid metabolism in rat liver cells (the C-9 cell-line) by tosyl- -phenylalanine chloromethyl ketone and phenylmethyl-sulfonyl fluoride

In the presence of the protease inhibitors phenylmethyl-sulfonyl fluoride and N-tosyl- -phenylalanine chloromethyl ketone, prostacyclin (PGI₂) production by rat liver cells treated with epidermal growth factor, platelet-activating factor, 12-O-tetradecanoylphorbol-13-acetate (TPA), and TPA-type tumor promoters (teleocidin and aplysiatoxin) or 1-oleoyl-2-acetylglycerol is amplified. The PGI2 production stimulated by thapsigargin or exogenous arachidonic acid is not amplified. N-Tosyl- -phenylalanine chloromethyl ketone also amplifies TPA's release of radioactivity from cells isotopically labeled with [³H]arachidonic acid. Indomethacin inhibits the amplification of PGI₂ production but not the release of radioactivity. The presence of the protease inhibitors is not required for the amplification of PGI₂ production. Prior incubation of the cells with these inhibitors, followed by their removal, still results in amplified PGI₂ production by cells subsequently treated with TPA, 1-oleoyl-2-acetylglycerol, or platelet-activating factor but not that stimulated by exogenous arachidonic acid. While phenylmethyl-sulfonyl fluoride's amplification of PGI₂ production by cells treated with TPA was blocked by prior incubation with TPA for 20 h, a similar block of amplification in EGF-treated cells was not observed.     

Oxysterol activation of arachidonic acid release and prostaglandin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase C activity [corrected]

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.     

Effects 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-PGF1 alpha) 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 exogenous 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.     

Stimulation of prostaglandin E2 production by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters in macrophages and its inhibition by cycloheximide

The effects of TPA (12-O-tetradecanoylphorbol 13-acetate)-type and non-TPA-type tumor promoters on prostaglandin E2 production by peritoneal macrophages of rats were examined. Among the TPA-type tumor promoters, aplysiatoxin was most potent in stimulating prostaglandin E2 production followed by dihydroteleocidin B, teleocidin, TPA and debromoaplysiatoxin. Prostaglandin E2 production by aplysiatoxin treatment was stimulated at doses up to 0.1 ng/ml. Palytoxin, a non-TPA-type tumor promoter, also stimulated both prostaglandin E2 production and the release of radioactivity from [3H]arachidonic acid-labeled macrophages. However, the dose required for the expression of these effects by palytoxin was up to 3 pg/ml. It was suggested that the tumor promoters are associated with the activity to stimulate arachidonic acid metabolism, irrespective of their type. Cycloheximide, a protein synthesis inhibitor, inhibited both prostaglandin E2 production and the release of radioactivity from prelabeled macrophages stimulated either by the TPA-type tumor promoters or by the non-TPA-type tumor promoter. It is possible that the tumor promoters may induce the synthesis of some proteins responsible for the stimulation of arachidonate metabolism.     

Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.     

Stimulation of DNA synthesis by tumor promoters in primary rat hepatocytes is not mediated by arachidonic acid metabolites

Studies in vivo using inhibitors of eicosanoid synthesis suggested that prostaglandins may play a role in mediating tumor promotion in liver by agents such as phenobarbital (PB). However, it is not clear whether any stimulation of arachidonic acid metabolism/prostaglandin formation results directly from the action of tumor promoters on hepatocytes or indirectly from effects of promoters on Kupffer cells or other non-hepatocytes. Our laboratory has been utilizing relatively pure populations of rat hepatocytes under the defined conditions of primary cultures, to investigate growth-stimulatory actions of tumor promoters, an important element in the promotion stage of carcinogenesis. It has been shown that most if not all liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with factors such as EGF, insulin, and glucocorticoid. In the present study, we sought evidence for a role of prostaglandins (PGs) in the direct growth-stimulatory actions of tumor promoters on hepatocytes. PGE(2), PGF(2 alpha), and PGD(2) cause concentration-dependent stimulation of hepatocyte DNA synthesis, while arachidonic acid was without any effect. PGE(2) and PGF(2 alpha) required the presence of dexamethasone to exert significant effects. These PGs did not further augment the stimulatory effect of EGF. In contrast, PGD(2) stimulated DNA synthesis in the presence or absence of insulin, dexamethasone, or EGF. The effect of tumor promoters on arachidonic acid metabolism, as measured by [(3)H]arachidonic acid release and PGE(2) production, was determined. The phorbol ester TPA significantly increased [(3)H]arachidonic acid release as well as PGE(2) formation in hepatocytes in line with known effects in other cell types. However, liver tumor promoters phenobarbital (PB), alpha-hexachlorocycohexane (HCH), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pregnenolone-16 alpha-carbonitrile (PCN) were without effects. Finally, inhibitors of arachidonic acid metabolism were tested for effects on the ability of TPA or liver tumor promoters to stimulate DNA synthesis by direct action on cultured hepatocytes. In all cases, lack of selective inhibition was observed. Taken together, the results show that while prostaglandins may directly stimulate DNA synthesis in hepatocytes, they are unlikely to mediate the direct growth-stimulatory actions of liver tumor promoters.     

Synergistic stimulation of luteinizing hormone (LH) release by protein kinase C activators and Ca2+-ionophore

When cultured pituitary cells were stimulated with synthetic diacylglycerol such as 1-oleoyl-2-acetylglycerol (OAG), or with a potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which are known stimulators of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), enhanced release of luteinizing hormone (LH) was observed. Similarly, LH release was also stimulated by the Ca2+-ionophore, A23187. Simultaneous presence of A23187 and OAG or TPA resulted in a synergistic response that mimicked the full physiological response to gonadotropin releasing hormone (GnRH). Removal of extracellular Ca2+ only slightly affected the stimulatory action of TPA and OAG on LH release, but completely blocked the effect of GnRH. The results suggest that the stimulatory effect of GnRH on LH release may be mediated by two intracellular pathways involving Ca2+ and diacylglycerol as second messengers.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

Differential effect of diacylglycerols and phorbol ester on arachidonic acid metabolism in bone marrow-derived macrophages

Murine bone marrow-derived macrophages were induced to prostaglandin synthesis by activators of protein kinase C, the phorbolester TPA and the diacylglycerols dioctanoylglycerol (diC8) and diolein (diC18:1). As short term stimulation of prostaglandin synthesis is mainly dependent on the availability of free arachidonic acid, the modulation of arachidonic acid liberation and reacylation was investigated. DiC8 inhibited the reacylating enzyme lysophosphatide acyltransferase in the in vitro assay, but there was no evidence for an inhibitory effect of TPA or diacylglycerols on the activity of the lysophosphatide acyltransferase in whole cells. The release of arachidonic acid from prelabelled cells was stimulated by TPA and the diacylglycerols even in the presence of an inhibitor of reacylation, indicating an activation of phospholipase A2. An activation of phospholipase A2 was measured in membranes derived from TPA-stimulated macrophages. These data indicate that the enhanced pool of free arachidonic acid, which drives prostaglandin synthesis, is primarily due to a stimulation of the liberation of arachidonic acid from membrane phospholipids.     

Regulation of arachidonic acid metabolism in Madin-Darby canine kidney cells. Comparison of A23187 and 12-O-tetradecanoyl-phorbol-13-acetate

Challenge of Madin-Darby canine kidney (MDCK) cells with the divalent cation ionophore A23187 caused a marked increase in the deacylation of [3H]arachidonic acid but not of [14C]palmitic acid. When the cells were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and A23187, there was an additional increase in the deacylation of [3H]arachidonic acid compared to that observed with either agent alone. In contrast to deacylation, the stimulation of prostaglandin production by A23187 was small compared to the stimulation by TPA. Cycloheximide inhibited synthesis of prostaglandins in TPA-treated cells, but did not block the stimulated deacylation caused by either TPA or A23187. These data indicate that, while both TPA and A23187 stimulated the deacylation of [3H]arachidonic acid, TPA had an additional, cycloheximide-sensitive effect that was required for efficient conversion of the release fatty acids to prostaglandins. Thus, although required, deacylation appeared to be independent of and insufficient to stimulate maximum prostaglandin synthesis in these cells.     

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.     

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.     

Interaction of IL 1 and TPA in modulation of eosinophil function

The tumor co-promotor TPA is believed to enhance a wide variety of cellular processes by interacting with protein kinase C. Interleukin (IL 1) is a family of highly active molecules which augments the host response to infection. We have explored the interactions of these activators of cell function on the modulation of selected eosinophil functions. The effects of purified monocyte-derived IL 1 on the eosinophil functions of oxidative metabolism (as measured by superoxide anion production) and degranulation (as measured by release of the granular enzymes arylsulfatase and beta-glucuronidase) have been examined. Superoxide anion production by eosinophils stimulated with standard doses of the stimulant phorbol myristic acetate (TPA) (1 microgram/ml) was augmented approximately 20% by preincubation with IL 1. However, IL 1 alone had no effect on superoxide anion production. At suboptimal doses of TPA, there was a dose-dependent inhibition of superoxide anion production in the presence of IL 1. Calcium ionophore (2 X 10(-7) M) markedly enhanced superoxide anion production elicited by 0.1 ng/ml of TPA, but had only modest effects in the absence of TPA. When IL 1 was added to eosinophils stimulated by TPA in the presence of calcium ionophore, there was a dose-dependent increase in superoxide anion production. In contrast to other cell types, degranulation as measured by the release of arylsulfatase and beta-glucuronidase was not elicited by the addition of TPA (1 microgram/ml). Although calcium ionophore (2 X 10(-6) M) caused enzyme release (24.2% release of beta-glucuronidase, 29.4% release of arylsulfatase), this release was inhibited by the addition of TPA. The addition of IL 1 alone caused an approximate twofold increase in enzyme release, but pretreatment with IL 1 (1 U) reduced ionophore-mediated degranulation (p less than or equal to 0.05). Studies employing purified monocyte IL 1 were confirmed by recombinant IL 1-beta. These studies demonstrate for the first time that eosinophil function is modulated by IL 1. IL 1 may also modify the response of eosinophils to other stimuli such as ionophore and TPA. Because TPA is known to act by direct binding to protein kinase C, these studies also demonstrate that, in eosinophils, activation of protein kinase C by phorbol esters may augment one cellular function (oxidative metabolism) while inhibiting another cellular function (degranulation). Similarly, phorbol esters may act synergistically with calcium ionophore in regulation of one function (oxidative metabolism) and act antagonistically with another function (degranulation). The concept that IL 1 uniformly enhances cell function may need to be re-evaluated.     

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.     

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.