Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes

Lung cells have been isolated by enzymatic digestion of guinea pig lungs and mechanical dispersion to obtain a suspension of viable cells (approximately 500 X 10(6) cells). Type II pneumocytes have been purified to approximately 92% by centrifugal elutriation (2000 rpm, 15 ml/min) followed by a plating in plastic dishes coated with guinea pig IgG (500 micrograms/ml). We have investigated the arachidonic acid metabolism through the cyclooxygenase pathway in this freshly isolated type II cells (2 x 10(6) cells/ml). Purified type II pneumocytes produced thromboxane B2 (TxB2) predominantly and to a smaller extent the 6-keto prostaglandin PGF1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) after incubation with 10 microM arachidonic acid. The stimulation of pneumocytes with 2 microM calcium ionophore A23187 released less eicosanoids than were produced when cells were incubated with 10 microM arachidonic acid. There was no additive effect when the cells were treated with both arachidonic acid and the ionophore A23187. Guinea pig type II pneumocytes failed to release significant amounts of TxB2, 6-keto-PGF1 alpha and PGE2 after stimulation with 10 nM leukotriene B4, 10 nM leukotriene D4, 10 nM platelet-activating factor, 5 microM formyl-methionyl-leucyl-phenylalanine, 0.2 microM bradykinin and 10 nM phorbol myristate acetate. Our findings indicate that guinea pig type II pneunomocytes possess the enzymatic machinery necessary to convert arachidonic acid to specific cyclooxygenase products, which may suggest a role for these cells in lung inflammatory processes.     

Release of eicosanoids from cultured rat aortic endothelial cells; studies with arachidonic acid and calcium ionophore A23187

The release of prostanoids from the three different vascular cell types derived from rat aortic explants has been studied in vitro. Under resting conditions and when incubated with exogenous arachidonic acid (AA, 10 microM), the endothelial cells (EC) produced the highest concentration of prostacyclin (PGI2 PGE2 PGF2 alpha TxA2). In contrast, PGE2 was the major prostanoid produced by the smooth muscle cells and fibroblasts. Pretreatment of EC with aspirin (10 microM) or indomethacin (10 microM) effectively inhibited the production of prostanoids by these cells. Incubation with the calcium ionophore A23187 (10 microM) did not stimulate production of PGI2 or leukotriene B4 (LTB4) by EC. However, treatment of EC with a combination of A23187 and AA led to production of amounts of both PGI2 and LTB4 which were greater than the summed values for the different drug treatments. These findings indicate that the concentration of substrate, AA, is a limiting factor in prostanoid formation by these cultured vascular cells but that rat EC are relatively poor in the enzymes required for leukotriene formation.     

Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.     

Metabolism of arachidonic acid by guinea pig Clara cells.

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Examination of the effects of piriprost (U-60,257B) on alkaline phosphatase activity of rat endometrial stromal cells in vitro.

Previously, piriprost (U-60,257B; an inhibitor of leukotriene (LT) synthesis) was shown to increase alkaline phosphatase (ALP) activity in cultured endometrial stromal cells (1). The present study investigated the mechanism of action of piriprost in this system. Sensitized rat endometrial stromal cells were isolated and cultured for up to 72 hr with various treatments. Piriprost (100 microM) was found to decrease 5-hydroxyeicosatetraenoic acid (a 5-lipoxygenase product) by 53% after 72 hr which provided evidence that 5-lipoxygenase was being inhibited by piriprost. Lactate dehydrogenase (LDH) activity confirmed that piriprost was not toxic to the cells. The possibility of piriprost acting in an analogous manner with that of PGs was examined. Three microM PGE2 or 20 microM carba-prostacyclin (CP), an analogue of PGI2, maximally increased (p less than 0.01) ALP activity at 72 hr and the further addition of 100 microM piriprost to PGE2 or CP caused an additional, additive increase in ALP activity. This indicated that the mechanism of action of piriprost was probably different from that of PGE2 or PGI2. The possibility that piriprost was shunting arachidonic acid into PG production was examined. Ten microM indomethacin (an inhibitor of PG synthesis) caused a decrease (p less than 0.01) in ALP activity and a 99% reduction in PGE2 at 72 hr. The effects of the combination of 100 microM piriprost and 10 microM IM were statistically additive, suggesting that the effects of piriprost were not due to an increase in PG production. These studies suggest that the effects piriprost on possible in vitro decidualization may be due to inhibition of 5-lipoxygenase.     

Production of prostaglandins by fetal rat lung type II pneumonocytes and fibroblasts

The output of prostaglandins I2, E2, F2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM) from third passage day 20 rat fetal fibroblasts and type II alveolar pneumonocytes was studied. In 2 h incubations, the output levels for each cell type were: PGI2 greater than PGE2 much greater than PGF2 alpha = PGFM when cells were incubated with Ca2+ ionophore A23187 (10 microM) or arachidonic acid (1 microgram/ml).     

Characterization of the priming effects of human granulocyte-macrophage colony-stimulating factor on human neutrophil leukotriene synthesis

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro and in vivo stimulator of human bone marrow myelomonocytic precursor cells and mature granulocyte and macrophage effector cells. We have compared the effect of GM-CSF on the synthesis of 5-lipoxygenase products induced by the chemotactic peptide fMet-Leu-Phe and the calcium ionophore A23187 in human neutrophils. Although GM-CSF alone did not stimulate detectable synthesis of products of the 5-lipoxygenase pathway, pre-incubation of neutrophils with 200 pM GM-CSF for 1 hour at 23 degrees C enhanced synthesis of leukotriene B4, its all-trans isomers and omega-oxidation products, and 5-hydroxyeicosatetraenoic acid in response to both the calcium ionophore A23187 (1.5 microM), and the chemotactic peptide fMet-Leu-Phe (0.1 microM). This priming effect of GM-CSF was maximal after a 60 min incubation at 23 degrees C, or after a 30 min preincubation at 37 degrees C. The effect of GM-CSF was maximal using a concentration of 1 nM. Enhancement of the leukotriene synthesis stimulated by A23187 was only observed when the cells were stimulated by the ionophore for periods of 3 minutes or less. In contrast, the enhancing effect of GM-CSF was still apparent when cells were exposed to fMet-Leu-Phe for as long as 15 minutes. Furthermore, the enhancing effect of GM-CSF was ablated when neutrophils were stimulated with A23187 and exogenous arachidonic acid. However, co-addition of exogenous arachidonic acid with fMet-Leu-Phe did not entirely mask the effect of GM-CSF. Possible mechanisms of action of GM-CSF are discussed.     

Modulation of alveolar macrophage-derived 5-lipoxygenase products by the sulfhydryl reactant, N-ethylmaleimide

The sulfhydryl reactant N-ethylmaleimide (NEM) stimulates the release and cyclooxygenase metabolism of arachidonic acid in rat alveolar macrophages. Because both 5-lipoxygenation and leukotriene (LT) C4 synthesis represent sulfhydryl-dependent steps in the 5-lipoxygenase pathway, we examined the effect of NEM on 5-lipoxygenase, as well as cyclooxygenase, metabolism in resting and agonist-stimulated cells by reverse-phase high performance liquid chromatography and radioimmunoassay. NEM at 5-10 microM stimulated the synthesis of thromboxane, but not prostaglandin E2 or the 5-lipoxygenase products LTC4, LTB4, or 5-hydroxyeicosatetraenoic acid from endogenously released arachidonate. In the presence of exogenous fatty acid, however, NEM stimulated the synthesis of large quantities of LTB4. The effect of NEM on arachidonate metabolism stimulated by the calcium ionophore A23187 and the particulate zymosan was also investigated. NEM augmented arachidonate release and thromboxane synthesis stimulated by A23187 but inhibited A23187-induced LTC4 synthesis with an IC50 of approximately 4.3 microM. This inhibitory effect closely paralleled the ability of NEM to deplete intracellular glutathione (IC50 approximately 4.3 microM). Preincubation with the intracellular cysteine delivery agent L-2-oxothiazolidine-4-carboxylate augmented intracellular glutathione concentration and A23187-stimulated LTC4 synthesis and attenuated the capacity of NEM to deplete glutathione and inhibit LTC4 synthesis. While LTB4 and 5-hydroxyeicosatetraenoic synthesis were unaffected at these low NEM concentrations, LTB4 synthesis was inhibited at high concentrations (IC50 approximately 210 microM). Zymosan-induced eicosanoid synthesis was modulated by NEM in a similar fashion. Thus, NEM is an agonist of arachidonate metabolism with the capacity to modulate the spectrum of macrophage-derived eicosanoids by virtue of specific biochemical interactions with substrates and enzymes of the 5-lipoxygenase pathway.     

Human peritoneal macrophages synthesize leukotrienes B4 and C4

Macrophages were isolated from the dialysis fluid of patients undergoing continuous ambulatory peritoneal dialysis and separated by gradient centrifugation and purification on 50% Percoll. The cells were prelabeled with [14C]arachidonic acid for 1.5 h. The labeled cells were then incubated with calcium ionophore A23187 (1 microM), serum-treated zymosan (200 micrograms/ml), and a lipoxygenase inhibitor, nordihydroguairetic acid (1 X 10(-5) M). The arachidonate metabolites in the medium were separated on Sep-Pak columns, and finally purified by reverse-phase high-pressure liquid chromatography (HPLC). The labeled products co-chromatographed with authentic leukotriene B4 and leukotriene C4 standards. Serum-treated zymosan and A23187 significantly stimulated and nordihydroguairetic acid significantly inhibited leukotriene synthesis. Leukotriene D4 was not detected, which suggests that these cells contain low gamma-glutamyltranspeptidase or high dipeptidase activity. These results establish, for the first time, that human peritoneal macrophages synthesize the lipoxygenase products, leukotriene B4 and leukotriene C4.     

Calcium ionophore enables soluble agonists to stimulate macrophage 5-lipoxygenase

The regulation of arachidonic acid conversion by the 5-lipoxygenase and the cyclooxygenase pathways in mouse peritoneal macrophages has been studied using particulate and soluble agonists. Particulate agonists, zymosan and latex, stimulated the production of cyclooxygenase metabolites as well as the 5-lipoxygenase product, leukotriene C4. In contrast, incubation with the soluble agonist phorbol myristate acetate or exogenous arachidonic acid led to the production of cyclooxygenase metabolites but not leukotriene C4. We tested the hypothesis that the 5-lipoxygenase, unlike the cyclooxygenase, requires activation by calcium before arachidonic acid can be utilized as a substrate. Addition of phorbol myristate acetate to macrophages in the presence of calcium ionophore (A23187) at a concentration which alone did not stimulate arachidonate metabolism resulted in a synergistic increase (50-fold) in leukotriene C4 synthesis compared to phorbol ester or A23187 alone. No such effect on the cyclooxygenase pathway metabolism was observed. Exogenous arachidonic acid in the presence of A23187 produced similar results yielding a 10-fold greater synthesis of leukotriene C4 over either substance alone without any effects on the cyclooxygenase metabolites. Presumably, calcium ionophore unmasked the synthesis of leukotriene C4 from phorbol myristate acetate-released and exogenous arachidonate by elevating intracellular calcium levels enough for 5-lipoxygenase activation. These data indicate that once arachidonic acid is released from phospholipid by an agonist, it is available for conversion by both enzymatic pathways. However, leukotriene synthesis may not occur unless intracellular calcium levels are elevated either by phagocytosis of particulate agonists or with calcium ionophore.     

Inhibition of leukotriene formation in human leukocytes by halothane

The effects of an inhalation anesthetic, halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the formation of 5-lipoxygenase metabolites such as leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-isomers of leukotriene B4 and leukotriene C4 were studied in human leukocytes stimulated with calcium ionophore A23187. Halothane inhibited the formation of all these metabolites dose dependently and the formation was restored by removal of the drug. The anesthetic also reversibly inhibited the release of [3H]arachidonic acid from neutrophils with a half-inhibition concentration of less than 0.19 mM. The formation of 5-lipoxygenase metabolites was not inhibited by the anesthetic when leukocytes were stimulated with the ionophore in the presence of exogenous arachidonic acid. These observations indicate that the inhibitory effect of halothane on the formation of 5-lipoxygenase metabolites in leukocytes is mainly due to the inhibition of arachidonic acid release.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Leukotriene A4, conversion to leukotriene B4 in human T-cell lines

Human T-cell lines (HSB, MOLT-4 and CCRF-CEM) produced leukotriene B4 when incubated with leukotriene A4. The product was characterized by chromatographic properties, UV-spectroscopy and gas chromatography mass spectrometry. About 10 pmol of leukotriene B4 was obtained per 10(6) cells. When incubated with arachidonic acid plus the calcium ionophore A23187 however, no leukotriene B4 was found, indicating that the T-cell lines lack 5-lipoxygenase yet contain LTA4 hydrolase.     

Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact-inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time-dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Detection of a novel cyclooxygenase metabolite produced by human promyelocytic leukemia (HL-60) cells

Arachidonic acid metabolism via the lipoxygenase pathway was examined in HL-60 cells before and after N,N-dimethylformamide induced differentiation along granulocytic lines. Untreated HL-60 cells produced small amounts of the 5-lipoxygenase products, 5-hydroxy-eicosatetraenoic acid and leukotriene B4 upon stimulation with calcium ionophore A23187. N,N-dimethylformamide treatment, caused a 10 to 20 fold increase in the amount of ionophore A23187-induced 5-lipoxygenase metabolites. An additional, and as yet unidentified arachidonic acid metabolite was routinely observed during reverse-phase high pressure liquid chromatography analyses of lipoxygenase products. Sensitivity to inhibition by less than 10(-7)M indomethacin coupled with other characteristics of its production, strongly suggest the compound is a cyclooxygenase product. The unusual UV absorbance and chromatographic elution pattern, however, suggest that it is not a typical prostaglandin, thromboxane or prostacyclin product.     

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1 alpha production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6-keto PGF1 alpha production; the maximum effects are obtained after 2-4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.