Leukotriene B4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of calcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source.

Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 37 degrees C with 0.15 microM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 microM). In neutrophils from allergic patients, the leukotriene B4 (LTB4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 +/- 4.2 and 38.1 +/- 6.8 pmol/5 min per 2 X 10(6) cells in the absence and presence of 10 microM arachidonic acid, respectively; P less than 0.005; n = 8). The LTB4 level in neutrophils from healthy subjects was only 0.97 +/- 0.17 pmol/5 min per 2 x 10(6) cells (n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1-14C]arachidonic acid, released LTB4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentration. Labeled LTB4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1-14C]arachidonate for 1 h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyl-, alkenylacyl- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidylcholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB4 by the 5-lipoxygenase pathway may provide only from 1-O-alkyl-2-arachidonoyl-glycero-3-phosphocholine.     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Effect of cholesterol enrichment on 12-hydroxyeicosatetraenoic acid metabolism by mouse peritoneal macrophages

The metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated in mouse peritoneal macrophages enriched in cholesterol by incubation with acetylated low density lipoproteins. After incubating with labeled arachidonic acid, cholesterol-rich cells released more 12-HETE into the medium than unmodified macrophages. With time, however, 12-HETE decreased in the medium of both cell preparations suggesting re-uptake of this monohydroxyfatty acid and perhaps further metabolism. When control macrophages were incubated with radiolabeled 12-HETE for 2 hr, almost 70% of the cell-associated 12-HETE label was incorporated into phospholipids. In contrast, in cholesterol-rich cells, only 31% of the 12-HETE label was incorporated into phospholipids. Bee venom phospholipase completely hydrolyzed the label, suggesting that the monohydroxyfatty acid was esterified at the sn-2 position of the phospholipid. In cholesterol-rich cells, 69% of the 12-HETE was diverted into neutral lipids. Two major neutral lipids were identified in cholesterol-rich macrophages. One neutral lipid band which migrated with an Rf value of 0.34 contained the hydroxylated fatty acid esterified to a glyceride. The other neutral lipid band having an Rf value of 0.49 contained cholesterol and by further analysis was found to contain predominantly cholesteryl-12-HETE. The labeled fatty acids in these two neutral lipids were mostly oxidized products of 12-HETE in contrast to the native 12-HETE observed in the phospholipids. Cholesterol-rich macrophages released 25% more products of 12-HETE metabolism than control macrophages. Two major products were observed in the medium which eluted in the area of a standard di-HETE, LTB4, on high performance liquid chromatography (HPLC) analysis. We propose that the reincorporation of 12-HETE into these neutral lipids and the increased capacity for further metabolism of this biologically potent hydroxyfatty acid could be a mechanism by which the cholesterol-rich macrophage maintains its membrane function, and regulates the amount of 12-HETE in the pericellular space.     

Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.     

Ultrastructural localization of arachidonic acid in stimulated macrophages

The distribution of 3[H] arachidonic acid incorporated into cultured mouse peritoneal macrophages was assessed upon stimulation of the cells with either the calcium ionophore A23187 or zymosan. After a labeling time of 24 h, cells were stimulated and processed for light and electron microscopic autoradiography. Grains were primarily localized over the plasma membrane and lipid-containing vesicles of both control and stimulated cells. In macrophages stimulated with ionophore, a decreased labeling density was evident in both of these cell compartments. Similar alterations in labeling pattern were observed in zymosan treated cells, although a larger decline in grain density occurred from the plasma membrane compartment. Immunocytochemical localization of PGE2, a major eicosanoid product released upon ionophore stimulation, revealed the presence of the prostaglandin in clear vesicular structures, many of which appear to be continuous with the plasma membrane. These results provide morphological evidence that different cellular pools of arachidonic acid may be differentially mobilized for eicosanoid production as a function of the mode of stimulation.     

Regulation of prostaglandin production in cultured gastric mucosal cells

The aims of this study were to investigate whether exogenous prostaglandin modulates prostaglandin biosynthesis by cultured gastric mucosal cells, and to clarify the role of cyclic nucleotides in the possible modulation of prostaglandin production. After pretreatment for 30 min with buffer alone (control) or 1 to 100ng/ml PGE2, cells were incubated with 4 uM arachidonic acid for 30 min. Pretreatments with greater than 5ng/ml PGE2 inhibited arachidonate-induced PGE2 and PGI2 production in a dose-dependent fashion, as compared with control, with inhibition by 64 +/- 8% and 75 +/- 4% respectively, at 100ng/ml PGE2. PGE2, at 100ng/ml, significantly increased intracellular cAMP accumulation, but pretreatment with dibutyryl cAMP (0.01-mM) did not alter the amounts of arachidonate-induced PGE2 production. Furthermore, while greater than 10ng/ml PGE2 increased cGMP production dose-dependently, preincubation with dibutyryl cGMP (0.001-0.1mM) also failed to affect PGE2 synthesis significantly. In addition, pretreatment with isobutyl-methyl-xanthine, while increasing accumulation of cellular cyclic nucleotides, did not significantly change PGE2 production. Calcium ionophore A23187-induced PGE2 production was also inhibited by pretreatment with PGE2. These results indicate that exogenous PG inhibits subsequent arachidonate or A23187-induced PG biosynthesis in rat gastric mucosal cells, and suggest the possibility that PG regulates its own biosynthesis via feedback inhibition independent of cyclic nucleotides in these cells.     

Influence of arachidonate metabolism on enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages

We examined whether arachidonate metabolism exerted any influence on the enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages. Enhancement of the intracellular transglutaminase activity was observed on stimulation of macrophages with normal sheep red blood cells (SRBC) or immunoglobulin G (IgG)-coated SRBC, and was inhibited by inhibitors of phospholipase A2 and cyclooxygenase. Moreover, prostaglandin E2 (PGE2), a main product of the cyclooxygenase pathway, leukotriene B4 (LTB4), a product of 5-lipoxygenase, and arachidonic acid also could directly induce high levels of intracellular transglutaminase activity without stimulation of macrophages by SRBC or IgG-coated SRBC, but leukotriene C4, prostaglandin D2, and prostacyclin were unable to induce high activity of the enzyme. Enhancement of transglutaminase activity induced by LTB4 was inhibited by cyclooxygenase inhibitor, but the enzyme activity induce by PGE2 was not inhibited. Furthermore, the quantity of PGE2 released into the culture medium of macrophages stimulated with SRBC or IgG-coated SRBC correlated well with the activity of intracellular transglutaminase in macrophages. Moreover, enhancement of transglutaminase activity by treatment of macrophages with SRBC or IgG-coated SRBC was partially suppressed by sodium benzoate, which is a scavenger of hydroxy radical. These findings suggest that arachidonate metabolism, in particular the cyclooxygenase pathway, plays an important role in the enhancement of intracellular transglutaminase activity.     

Inhibition of PGE2 production in macrophages from vitamin E-treated rats.

Rat peritoneal macrophages from vitamin E-treated rats (5 mg per rat for 6 successive days) contained 403.3 +/- 90.7 ng alpha-tocopherol/10(6) cells, whereas control macrophages contained 1.2 +/- 0.4 ng. PGE2 production in the macrophages from vitamin E-treated rats was significantly suppressed when stimulated with PMA and calcium ionophore A23187. The mechanism of vitamin E inhibition of PGE2 production in macrophages was investigated. The release of (14C)-arachidonic acid from pre-labeled macrophages and the conversion of (14C)-arachidonic acid to PGE2 by the homogenate of the cells were remarkably reduced. These results strongly suggested that the inhibition of PGE2 production by vitamin E results from the inhibition of the activities of both phospholipase A2 and cyclooxygenase.     

Decreased arachidonate metabolism in mouse peritoneal macrophages after foam cell transformation with oxidized low-density lipoproteins.

Oxidized low density lipoproteins (LDL) are now considered to be one of the atherogenic lipoproteins in vivo and to play an important role in the pathogenesis of atherosclerosis. We previously demonstrated in mouse peritoneal macrophages that oxidized LDL stimulated prostaglandin (PG) E2 synthesis when incorporated into the cells [Yokode, M. et al. (1988) J. Clin. Invest. 81, 720-729]. In this study, we investigated arachidonate metabolism in macrophages after foam cell transformation. The cells were incubated with 100 micrograms/ml of oxidized LDL for 18 h, then stimulated with zymosan. Lipid-enriched macrophages which had taken up oxidized LDL produced much less eicosanoids, such as PGE2, 6-keto-PGF1 alpha, and leukotriene C4 than control cells. After labeling of the cells with [14C]arachidonic acid, they were stimulated with zymosan and the phospholipase activity was determined. The activity of lipid-enriched cells was about two-thirds of that of control cells. Then we investigated the fatty acid composition of their phospholipid fraction to clarify arachidonic acid content and mobilization. Percent of arachidonic acid of lipid-enriched cells decreased and less arachidonic acid mobilization was observed after stimulation with zymosan. These data suggest that impaired arachidonate metabolism in lipid-enriched macrophages can be explained by their decreased phospholipase activity and changes in their fatty acid composition.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

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.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.     

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.     

Kupffer cell-hepatocyte interactions and the changes in 1-14C-arachidonate incorporation in response to endotoxin in vitro

The present experiments were undertaken to elucidate the effect of either the hepatocyte (HC) or hepatocyte supernatant on prelabeled endotoxin (LPS)-stimulated Kupffer cell (KC) arachidonic acid utilization. HC, KC, or their coculture were incubated for 18 hours with labeled 1-14C- arachidonic acid followed by a 24 hour incubation with 10 micrograms/ml LPS. LPS had no effect on the percent distribution of labeled arachidonate into the HC phospholipid or neutral lipid. KC showed a decreased percent neutral lipid labeled arachidonic acid distribution with generally no effect on the phospholipid. However, KC:HC cocultures or the addition of HC supernatant to KC exposed to LPS dramatically reversed the labeled arachidonate distribution into the KC with an increased incorporation into neutral lipid. Labeled PGE2 and PGD2 were increased in the KC following incubation with HC supernatant while only labeled PGE2 levels were elevated in the cocultures. The changes in the distribution of cell's labeled arachidonate required the addition of LPS. These results suggest that the HC can promote changes in the lipid fraction during sepsis by elaborating a substance that can modulate labeled arachidonate distribution in the KC lipids as well as stimulate prostaglandin production.     

Epidermal growth factor is synergistic with phorbol esters and vasopressin in stimulating arachidonate release and prostaglandin production in renal glomerular mesangial cells.

Epidermal growth factor (EGF) is produced in large quantities by the kidney. We identified EGF-binding sites on cultured rat renal glomerular mesangial cells. These cells serve as a model system for the investigation of renal prostaglandin biosynthesis. Since EGF has been shown to modulate phospholipase activity in other cell lines, we studied the ability of EGF to increase arachidonate release and prostaglandin E2 (PGE2) production in mesangial cells. We found that EGF stimulated arachidonate release and PGE2 production in the presence of the Ca2+ ionophore A23187. This stimulation was markedly potentiated by the addition of phorbol myristate acetate (PMA), which activates protein kinase C. However, down-regulation of protein kinase C by prolonged PMA treatment did not block the ability of EGF to stimulate PGE2 production in the presence of A23187. EGF also markedly potentiated the stimulation of PGE2 production by vasopressin, which increases intracellular Ca2+ and activates protein kinase C in these cells. The stimulatory effects of EGF were not the result of prolongation or enhancement of an increase in intracellular Ca2+ produced by ionophore or vasopressin. Furthermore, the synergistic interaction of EGF with PMA and vasopressin occurred despite the fact that these agents markedly decreased EGF binding in mesangial cells, presumably owing to protein-kinase-C-mediated phosphorylation of the EGF receptor. We conclude that there exists a distinct pathway for EGF-stimulated arachidonate release and PGE2 production in rat renal glomerular mesangial cells, which is synergistic with, but not dependent on, activation of protein kinase C. In contrast with long-term mitogenic responses to EGF, this rapid response may allow delineation of the membrane phospholipid changes and signalling steps involved in this aspect of EGF action.     

Interleukin 1 and tumor necrosis factor potentiate angiotensin II- and calcium ionophore-stimulated prostaglandin E2 synthesis in rat renal mesangial cells

In resting mesangial cells, angiotensin II and the calcium ionophore A23187 stimulated prostaglandin E2 (PGE2) formation. After pretreatment with interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha), which are themselves potent stimuli for PGE2 synthesis, mesangial cells displayed an amplified response to angiotensin II and A23187. The cytokine-induced effects occurred in a time- and dose-dependent manner and were attenuated by actinomycin D, cycloheximide and dexamethasone. IL-1 beta and TNF alpha treatment also increased the amount of arachidonic acid released after stimulation of cells with angiotensin II and A23187. In addition, IL-1 beta but not TNF alpha treatment augmented the formation of PGE2 from exogenous arachidonic acid by mesangial cells. Furthermore, the conversion of prostaglandin H2 to PGE2 was not changed by IL-1 beta and TNF alpha. These results suggest that IL-1 beta and TNF alpha exert a priming effect on PGE2 production in mesangial 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.     

Release of arachidonic acid from human endometrial cells in culture mediated by calcium ionophore (A23187) or fluoride.

Primary cultures of endometrial glands and stromal cells were labelled with [14C]-arachidonic acid for 4 h before exposure to either the calcium ionophore, A23187 (which activates phospholipase A2 (PLA2) by increasing intracellular calcium concentrations) or sodium fluoride (which activates a G-protein). Calcium ionophore (0.5-50 mumol/l) stimulated a dose- and time-dependent release of arachidonic acid from endometrial glands. Incubation with ionophore (10 mumol/l) for 1 h released 22% of the incorporated arachidonic acid. There was a corresponding decrease in phospholipids and no loss from triglycerides. Stromal cells were unresponsive to ionophore. Fluoride (10 mmol/l) stimulated a release of arachidonic acid from stromal cells and endometrial glands (6.5% of the total arachidonic acid incorporated). In stromal cells, arachidonic acid was released from triglycerides in Day-1 cultures and from phospholipids in Day-2 cultures. In both Day-1 and Day-2 cultures of endometrial glands, arachidonic acid was released from phospholipids, but not from triglycerides. Among the phospholipids, phosphatidylcholine was always the major source of arachidonic acid. Arachidonic acid release from endometrial glands and stromal cells may be mediated by activation of PLA2 (or phospholipase C) via a G-protein, but in glands calcium ionophore may have a direct effect on PLA2. The response to calcium ionophore may reflect the differences in calcium requirements of the two endometrial PLA2 isoenzymes.     

Mechanism of arachidonic acid release in human polymorphonuclear leukocytes

Previous studies have demonstrated that [³H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A₂ activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [¹⁴C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A₂ or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [¹⁴C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [¹⁴C]stearate, replaced [¹⁴C]arachidonate, very little [¹⁴C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [¹⁴C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.     

Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2

alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.