1-14C]Arachidonic acid incorporation into glycerolipids and prostaglandin synthesis in peritoneal macrophages: effect of chloramphenicol

Peritoneal macrophages from normal mice were labelled with [1-14C]arachidonic acid after 2 h culture. The uptake of arachidonic acid into cellular lipids was rapid, time-dependent and can be represented within the limit of the studied times by a parabolic regression. Indomethacin decreased the kinetics of uptake; this inhibition is dose-dependent. Chloramphenicol had no effect on macrophage [1-14C]arachidonic acid uptake. After 3 h, the radioactivity was recovered in phosphatidylcholine (38.6%), phosphatidylserine-phosphatidylinositol (8.5%), phosphatidylethanolamine (22.1%), diacylglycerol (2.9%), triacyglycerol (2%) and cholesteryl ester (11.8%). Chloramphenicol and indomethacin inhibited the labelling of phospholipids and stimulated the labelling of neutral lipids and cholesteryl ester. Studies on arachidonic acid release from glycerolipids of prelabelled 2-h cultured macrophages showed that phosphatidylcholine and phosphatidylserine-phosphatidylinositol are the major source of arachidonic acid in prostaglandin synthesis in macrophages stimulated or not by zymosan. Chloramphenicol inhibited release of fatty acid from phosphatidylcholine and phosphatidylserine-phosphatidylinositol; indomethacin had no effect. Both drugs inhibited prostaglandin synthesis in stimulated or non-stimulated macrophages. In the culture medium, indomethacin increased the release of free arachidonic acid by stimulated macrophages. Possible explanations for the mechanisms underlying these effects are presented. It is concluded that indomethacin and chloramphenicol exert profound effects on the metabolism of phospholipids and its zymosan activation. Chloramphenicol appears to impair prostaglandin synthesis through several mechanisms and especially through phospholipase inhibition.     

Incorporation of [14C] arachidonic acid into ovine conceptus and endometrial lipids.

The purpose of this study was to quantitate conceptus and endometrial incorporation of [14C]arachidonic acid (AA) into individual neutral and polar lipids. Endometrium and conceptuses from pregnant ewes and endometrium from nonbred ewes were collected 14 and 16 d after onset of estrus (d 0). Tissues were incubated for 8 h at 37 degrees C in medium containing 1 microCi of [14C]AA. Thin-layer chromatographic procedures were used to separate 12 lipids. Radioactivity was measured in each lipid, and the amount (ng) of [14C]AA incorporated into each lipid was calculated. Conceptuses and endometrium incorporated more [14C]AA into triacylglycerols than into any other lipid. Day and tissue type affected differentially (i.e., day X tissue interaction) the incorporation of [14C]AA into several lipids; d-14 conceptuses incorporated [14C]AA more actively than did any other day-tissue combination. Results indicate that triacylglycerols may be an important reservoir for conceptus and endometrial AA. The remarkable ability of d-14 conceptuses to incorporate [14C]AA into various lipids may be important for their accelerated elongation and active prostaglandin synthetic system.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Endogenous inhibition of arachidonic acid metabolism in the endometrium of the sheep

Arachidonic acid is metabolised via the cyclo-oxygenase pathway to several biologically active metabolites. These metabolites control important reproductive functions like luteolysis of the corpus luteum. The metabolism of arachidonic acid was studied by the enzymatic conversion of [1-14C]-labelled arachidonic acid in sheep endometrial tissue. The inhibitory capacity of sheep endometrial tissue was measured by the enzymatic conversion of [1-14C]-arachidonic acid by sheep seminal vesicular gland microsomes. Endometrial microsomes converted arachidonic acid into different prostaglandins and monohydroxy acids but at a low rate. A factor(s) inhibiting both prostaglandin and monohydroxy acid synthesis was found in both the microsomal and cytosolic fractions of endometrial tissue. A very high inhibitory potency of prostaglandin and monohydroxy acid synthesis, calculated as IC50 values, was found in cytosolic fractions. For comparison IC50 values of indomethacin, mefenamic acid, carprofen and acetylsalicylic acid were also calculated in this in vitro system. These data indicate that both prostaglandin and monohydroxy acid synthesizing capacities and an inhibitory factor(s) are present in sheep endometrium and possibly regulate arachidonic acid metabolism in this tissue.     

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.     

Hormonal stimulation of arachidonate release from isolated perfused organs. Relationship to prostaglandin biosynthesis

The lipids of isolated Krebs perfused rabbit kidneys and hearts were labelled with [14C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [14C]prostaglandins; little or no release of the precursor [14C]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [14C]arachidonic acid was detected following hormonal stimulation. The release of [14C]arachidonic acid was dose-dependent and greater than 3 fold that of [14C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [14C]prostaglandins and only slightly inhibited release of [14C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.     

Labelling of lipids and phospholipids with [3H]arachidonic acid and the biosynthesis of eicosanoids in U937 cells differentiated by phorbol ester

Phorbol esters induce morphologic and biochemical differentiation in U937 cells, a monocyte/macrophage-like line derived from a human histiocytic lymphoma. We are interested in the phorbol ester-stimulated release of arachidonic acid from cellular membranes and the subsequent synthesis of eicosanoids, as it may prove to correlate with the induced cellular differentiation. Undifferentiated log-phase U937 cells released little recently incorporated [3H]arachidonic acid, but phorbol 12-myristate 13-acetate increased its apparent rate of release to that of cells differentiated by exposure to phorbol myristate acetate for 3 days. Exposure of washed differentiated cells immediately prelabelled with [3H]arachidonic acid to additional phorbol myristate acetate did not augment the release of [3H]arachidonic acid. The basal release of nonradioactive fatty acids from differentiated cells was 5-10 times that of undifferentiated cells, and phorbol myristate acetate increased their release from both types of cell 2- to 3-fold. Differentiated cells immediately prelabelled with [3H]arachidonic acid exhibited greater incorporation into phosphatidylinositol and phosphatidylcholine, and contained more radioactive free arachidonic acid, compared with undifferentiated cells. Undifferentiated cells contained more radioactivity in phosphatidylserine, phosphatidylethanolamine and neutral lipids. Phorbol myristate acetate caused differentiated cells to release [3H]arachidonic acid from phosphatidylinositol, phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine, but release from neutral lipids was reduced, and the content of [3H]arachidonic acid increased. In undifferentiated cells incubated with phorbol myristate acetate, radioactivity associated with phosphatidylserine, phosphatidylethanolamine and neutral lipid was reduced and [3H]arachidonic acid was unchanged. Synthesis of cyclooxygenase products exceeded that of lipoxygenase products in both differentiated and undifferentiated cells. Phorbol myristate acetate increased the synthesis of both types of product, cyclooxygenase-dependent more than lipoxygenase-dependent, especially in differentiated cells. The biological significance of these changes in lipid metabolism that accompany phorbol myristate acetate-induced differentiation are yet to be established.     

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.     

Transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine during storage of human platelets for 5 days

Human platelets are routinely stored for 5 days prior to transfusion, but they deteriorate during storage. Since very little information is available concerning the effect of storage on platelet phospholipid metabolism, the biosynthesis and remodelling of platelet phospholipids were studied. Platelets were incubated separately with [14C]glycerol, [14C]arachidonic acid, or a mixture of [14C]glycerol and [3H]arachidonic acid, and stored in a platelet storage medium at 22 degrees C. Maximum glycerol uptake (20%) was attained after 6 h. [14C]Glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, and to a much lesser extent phosphatidylserine, under storage conditions for 5 days. The distribution of the initial arachidonic acid uptake was not as would be expected based on the molar composition of endogenous phospholipids. The arachidonic acid (75%) which was taken up within 10 min of incubation distributed 55% into the phosphatidylcholine and only 14% into the phosphatidylethanolamine; the molar composition is actually 18% phosphatidylcholine and 47% phosphatidylethanolamine. During storage, there was a continuous transfer of the radiolabelled arachidonic from phosphatidylcholine to phosphatidylethanolamine until, after 5 days, the distribution of arachidonic acid was identical to the endogenous distribution. In contrast, no change in the glycerol incorporation pattern was detected during storage. This suggested that the mechanism for arachidonic acid redistribution was not through exchange of polar head groups, but through acyl transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine.     

Stimulation of deacylation in Madin-Darby canine kidney cells. Specificity of deacylation and prostaglandin production in 12-O-tetradecanoylphorbol-13-acetate-treated cells

Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced.     

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.     

Hormonal stimulation of arachidonic release from isolated perfused organs relationship to prostaglandin biosynthesis

The lipids of isolated Krebs perfused rabbit kidneys and hearts were labeled with [¹⁴C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [¹⁴C]prostaglandins; little or no release of the precursor [¹⁴]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [¹⁴C]arachidonic acid was detected following hormonal stimulation. The release of [¹⁴C]arachidonic acid was dose-dependent and >;3 fold that of [¹⁴C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [¹⁴C]prostaglandins and only slightly inhibited release of [¹⁴C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacrylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

Phospholipase A2 in macrophage plasma membrane releases arachidonic acid from phosphatidylinositol

A high level of arachidonic acid release from [2-14C]arachidonylphosphatidylinositol (PI) was observed at neutral pH (6.0-7.0) in the presence of purified plasma membranes of guinea pig peritoneal macrophages. This activity was at least 10-fold higher than that with arachidonylphosphatidylcholine (PC) or phosphatidylethanolamine (PE) as substrate. The accumulation of [14C]diacylglycerol and [14C]phosphatidic acid was not detected at any time, and arachidonic acid release from [14C]arachidonyldiacylglycerol was not detectable either. The data suggest that arachidonic acid release from PI may not occur via the phospholipase C pathway. In this paper, we demonstrate the possibility that arachidonic acid release from PI at neutral pH in the macrophage plasma membrane is dependent on the action of phospholipase A2 (EC 3.1.1.4) -like activity. The maximum arachidonic acid release was dependent upon both pH and substrate. Particularly, the activity of arachidonic acid release from PI at neutral pH was very high compared with that from PC or PE. We suggest that phosphatidylinositol phospholipase A2 (EC 3.1.1.52) may play an important role in providing arachidonic acid for subsequent metabolic activity in the macrophages.     

Effect of ethinylestradiol on arachidonic acid incorporation, release, and conversion to prostaglandins in rabbit platelets

Intramuscular administration to female rabbits of 2 mg/kg ethinylestradiol every other day for 10 days increased the uptake and incorporation of [14C]arachidonic acid into platelet lipids, and increased the proportion of [14C]arachidonic acid released from platelets after stimulation by thrombin. The conversion of [14C]arachidonic acid to thromboxane B2 did not differ between the control and ethinylestradiol-treated groups. Thus, the results of this study indicate that the major site in the prostaglandin metabolic pathway influenced by estrogen is the incorporation and release of arachidonic acid in platelet phospholipids.     

Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line

The effect of sodium n-butyrate on prostaglandin synthesis in cultured cells was examined. Exposure of BC-90 cells, a clone of an epithelial rat liver cell line, to 1 mM sodium n-butyrate for 40 h induced prostacyclin production. Prostacyclin synthesis was proved by demonstrating: (1) production of labeled 6-ketoprostaglandin F1 alpha by treating [14C]arachidonic acid pre-labeled cells with calcium ionophore A23187, (2) production of unstable substance that inhibited adenosine diphosphate-induced platelet aggregation, and (3) conversion of [14C]arachidonic acid to 6-ketoprostaglandin F1 alpha in homogenates of n-butyrate-treated cells. Untreated control cells showed negligible prostaglandin synthesis. Untreated cell homogenates did not convert [14C]arachidonic acid to any prostaglandins, but they converted [14C]prostaglandin H2 to prostacyclin. Induction of prostacyclin production by n-butyrate was also demonstrated with cells that had been treated with acetylsalicylic acid before n-butyrate treatment in acetylsalicylic acid-free medium. Incorporation of [3H]acetylsalicylic acid by sodium n-butyrate-treated cells increased in accordance with treatment time, while that of untreated cells did not change during culture. There was no difference in the phospholipase A2 activities of n-butyrate-treated and -untreated cells. From these findings, the possibility that n-butyrate induced prostacyclin in BC-90 cells through induction of fatty acid cyclooxygenase activity is discussed.     

Phospholipase C activity in rat kidney. Effect of deoxycholate on phosphatidylinositol turnover

Rat renal cortical and medullary slices incorporate [14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [14C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [14C]phosphatidylinositol, with a concomitant increase in 1,2-[14C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A2. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.     

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.     

Effect of essential polyunsaturated fatty acid modifications on prostaglandins production by MDCK canine kidney cells

Supplementation of growing MDCK canine kidney tubular epithelial cultures with linoleic acid produced a 3.6- to 4.9-fold increase in bradykinin-stimulated PGE₂ release as measured by radioimmunoassay. Under these conditions the cell phospholipids contained 3.9-times more linoleic acid and 5.6-times more arachidonic acid, with the inositol, ethanolamine and choline phosphoglycerie fractions becoming enriched in arachidonic acid. By contrast, supplementation with arachidonic acid did not enhance bradykinin-stimulated PGE₂ release even though the arachidonic acid content of the cell phospholipids was increased 8.8-fold. The distribution of radioactive prostaglandin products was unchanged by these fatty acid enrichments, with PGE₂ accounting for 55 to 68% of the total output from [1-¹⁴C]arachidonic acid. Linoleic acid supplementation also produced a 2.5-fold increase in PGE₂ formation stimulated by extracellular arachidonic acid, whereas supplementation during culture with arachidonic acid caused a 55 to 80% inhibition. This difference cannot be accounted for by changes in the ability of the cells to incorporate extracellular arachidonic acid. it is suggested that at least some of the effects of linoleate supplementation on prostaglandin production are due to the resulting enrichment of the intracellular phospholipid substrate pools with arachidonic acid. In addition, it appears that prolonged exposure to arachidonic acid during culture has an overriding inhibitory effect on prostaglandin production even though the total cell lipids bocome highly enriched in arachidonate.     

Phosphatidylethanolamine turnover is an early event in the response of NB2 lymphoma cells to prolactin

The effect of prolactin on phospholipid metabolism in the prolactin-dependent rat lymphoma cell line Nb2 was investigated in cells prelabeled with [3H]arachidonic acid or [3H]ethanolamine. Prolactin (20 ng/ml) caused (a) a 20-60% loss of radiolabeled phosphatidylethanolamine within 0.5 to 2 min, (b) a loss of [3H]ethanolamine-labeled phosphatidylethanolamine from crude membranes, (c) a rapid accumulation of [3H]phosphoethanolamine and [3H]ethanolamine, and (d) a transient increase (15 s to 2 min) in prostaglandin F2 alpha and E2. Arachidonic acid (1-2 micrograms/ml) induced Nb2 cell growth but prostaglandin F2 alpha, E2, ethanolamine, and phosphoethanolamine did not. Prostaglandin E2 inhibited while prostaglandin F2 alpha enhanced growth in the presence of prolactin or arachidonic acid. These results suggest that stimulation of Nb2 cell growth by prolactin is linked to activation of a phosphatidylethanolamine-specific phospholipase C. Arachidonic acid and prostaglandin F2 alpha may participate in regulating the mitogenic action of prolactin.