RHC 80267 does not inhibit the diglyceride lipase pathway in intact platelets

RHC 80267 inhibits diglyceride lipase activity in microsomes from canine platelets (1). Chau and Tai (2) reported that RHC 80267 prevents the transient accumulation of monoglyceride in thrombin-stimulated human platelets, while leaving arachidonate release unimpaired. In contrast, we find that while the drug inhibits both diglyceride lipase (I₅₀=15 μM) and monoglyceride lipase (I₅₀=11 μM) activities in platelet microsomes, it is ineffective when added to intact platelets. The transient intermediates in the diglyceride lipase pathway, 1,2-diglyceride and 2-monoglyceride, both accumulated after thrombin stimulation of intact platelets treated with RHC 80267, and arachidonate release was not inhibited. We conclude that RHC 80267 cannot be used to evaluate the diglyceride lipase pathway in intact platelets.     

Arachidonate activation of protein kinase C may be involved in the stimulation of protein synthesis by insulin in L6 myoblasts

Insulin stimulated protein synthesis in L6 myoblasts but did not increase the labelling of DAG or the release of phosphocholine from phosphatidylcholine. The DAG lipase inhibitor, RHC 80267, more than doubled the amount of label appearing in DAG but did not stimulate protein synthesis. Even in the presence of the DAG lipase inhibitor insulin failed to have any effect on DAG labelling, and conversely RHC 80267 did not modify the insulin-induced increase in protein synthesis. These results suggest that endogenous DAG production is not involved in the stimulation of protein synthesis by insulin. However, exogenous diacylglycerols (1-oleoyl-2-acetyl glycerol and 1-stearoyl-2-arachidonoyl glycerol) both stimulated protein synthesis in L6 myoblasts. The efficacy of the former (arachidonatefree) DAG suggested that their action was by activation of protein kinase C rather than by arachidonate release and prostaglandin formation. Ibuprofen, an inhibitor of cyclo-oxygenase failed to block the effects of insulin whereas a second cyclo-oxygenase inhibitor, indomethacin had only a partial inhibitory effect. The protein kinase C (PKC) inhibitor, RO-31-8220, totally blocked the effect of insulin. Since indomethacin is also recognised to inhibit phospholipase A₂, the data suggests that insulin acts on protein synthesis in myoblasts by arachidonate activation of PKC.     

Diacylglycerol Enrichment of Endoplasmic Reticulum or Lipid Droplets Recruits Perilipin 3/TIP47 during Lipid Storage and Mobilization

Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3- and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking membrane trafficking with AlF₄⁻ during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF₄⁻ activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF₄⁻ perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF₄⁻ redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor {"type":"entrez-protein","attrs":{"text":"RHC80267","term_id":"1470879788","term_text":"RHC80267"}}RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with {"type":"entrez-protein","attrs":{"text":"RHC80267","term_id":"1470879788","term_text":"RHC80267"}}RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell.     

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.     

Angiogenin activates phospholipase C and elicits a rapid incorporation of fatty acid into cholesterol esters in vascular smooth muscle cells

Angiogenin activates the phosphoinositide-specific phospholipase C (PLC) in cultured rat aortic smooth muscle cells to yield a transient (30 s) peak of 1,2-diacylglycerol (DG) and inositol trisphosphate. Within 1 min, the DG level falls below that of the control and remains so for at least 20 min. A transient increase in monoacylglycerol indicates that depletion of DG may be the consequence of hydrolysis by DG lipase. In addition to these changes in second messengers, a rapid increase in incorporation of radiolabeled tracer into cellular cholesterol esters is observed. Stimulated cholesterol ester labeling is inhibited by preincubation with either the DG lipase inhibitor RHC 80267 or the acyl coenzyme A:cholesterol acyltransferase inhibitor Sandoz 58035. Cells prelabeled with [3H]arachidonate show a sustained increase in labeling of cholesterol esters following exposure to angiogenin. In contrast, cells prelabeled with [3H]oleate show only a transient elevation that returns to the basal level by 5 min. This suggests initial cholesterol esterification by oleate followed by arachidonate that is released by stimulation of the PLC/DG lipase pathway.     

Inhibitors of diacylglycerol lipase and diacylglycerol kinase inhibit carbamylcholine-stimulated responses in guinea pig pancreatic minilobules

We earlier showed that the diacylglycerol (DG) lipase inhibitor, RHC 80267, increased the steady-state level of DG and inhibited the release of arachidonic acid (AA) in carbamylcholine (CCh)-stimulated pancreatic minilobules (J. F. Dixon and L. E. Hokin, (1984) J. Biol. Chem. 259, 14418-14425). There was no effect on phospholipid metabolism. We have now investigated the effect of RHC 80267 on CCh-stimulated formation of inositol monophosphate formation, cGMP formation, and amylase release. CCh (10 microM) increased cGMP formation by approximately 20-fold, and this response was inhibited 55-75% by RHC 80267 (75-100 microM). RHC 80267 had no effect on either nitroprusside- or calcium ionophore-stimulated cGMP formation, arguing against a direct inhibition of guanylate cyclase by RHC 80267. Arachidonic acid, the release of which is inhibited by RHC 80267, neither stimulated cGMP formation nor reversed the effect of RHC 80267 on CCh-stimulated cGMP formation. This suggests, but does not prove, that the rise in cGMP in response to CCh is not due to an increase in AA as has been suggested. Both phorbol myristate acetate (25 nM) and the DG kinase inhibitor R 59022 (10 microM) inhibited CCh-stimulated cGMP formation by 40%. RHC 80267 also inhibited CCh-stimulated inositol phosphate accumulation and amylase release by 60 and 40%, respectively. The data suggest that the inhibition of CCh-stimulated cGMP formation and other muscarinic responses by RHC 80267 is probably the result of feedback inhibition of the cholinergic receptor via activation of protein kinase C by the elevated DG.     

Prostaglandins and myogenic control of tension in lower esophageal sphincter in vitro

Active tension is produced by the lower esophageal sphincter (LES) of North American opossum $\text{in vitro}$ by a myogenic mechanism. Strips of LES, but not those from the esophageal body, contracted to prostaglandin (PG)F_2α, stable expoxymethano derivatives of PGH₂ and to thromboxane B₂. Stable endoperoxides were more than 500 times more potent than PGF_2α. PGI₂ and 6-keto PGF_1α were weak relaxants of LES strips. LES strips transformed arachidonic acid into contractile substances. This transformation was prevented by agents which interfere with PG synthesis by inhibiting cyclo-oxygenase [indomethacin (IDM), 5,8,11,14-eicosatetraynoic acid (ETA) or thromboxane synthetase [imidazole]. Tranylcypromine 500 μg/ml also inhibited contractions to arachidonic acid. These agents also reduced muscle tone, so that endogenous PG formation may contribute to active tension in the LES. ETA and IDM increased tone before inhibiting it, and this effect was prevented by prior treatment with ETA or imidazole. There may also be an endogenous PG which inhibits LES tone. The possibility that this may be PGI₂ is discussed.     

Quantitative studies on prostaglandin synthesis in man. II. Determination of the major urinary metabolite of prostaglandins F1 alpha and F2 alpha

A method was developed for quantitative determination of 5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α in man. The method was based on the use of the O-methyloxime derivative of [5β-³H; 10,10,12,12-²H₄]5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid as internal standard and determination of ratios between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Excretion values found for healthy human subjects were: males, $\text{10.8–59.0 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 24.0 ± 17.2 (SD) μg) and females, $\text{7.6–13.6 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 10.5 ± 2.1 (SD) μg).     

Paradoxical endogenous synthesis of a coronary dilating substance from arachidonate

Isolated bovine, canine, and human coronary arteries exhibited dose dependent contractions to prostaglandin (PG) E₂ and F_2α (50 ng/ml to 10 μg/ml). The ED₅₀ value for both PGE₂ and PGF_2α was 500 ng/ml in the bovine and human coronary arteries. Paradoxically, although PGE₂ and PGF_2α are vasoconstrictors, administration of their precursor, arachidonate (100 ng/ml to 10 μg/ml) caused relaxation of the bovine, canine and human coronary arteries. This observation suggests that arachidonate is not being converted by the coronary PG synthetase to PGE₂ or PGF_2α. However, the arachidonate induced coronary relaxation was inhibited by pretreatment with PG synthetase inhibitors, indomethacin, meclofenemate and aspirin. Indomethacin addition to the strips previously relaxed by arachidonate caused contraction. In contrast to other PGs (E₂ and F_2α), PGE₁ (10 ng/ml to 10 μg/ml) caused dose dependent relaxation of the bovine coronary arteries (ED₅₀ = 100 ng/ml). Indomethacin induced further relaxation of the blood vessels previously relaxed by PGE₁. Since PGE₁ cannot arise from arachidonate, the arachidonate coronary dilation and reversal by indomethacin must be independent of PGE₁ formation. Linolenate (100 ng/ml to 10 μg/ml) and oleate (100 ng/ml to 10 μg/ml) also caused relaxation of the bovine coronary blood vessels both before and after indomethacin, thereby eliminating a direct non-specific fatty acid effect as the cause of the arachidonate relaxation. These results suggest that in isolated coronaries, arachidonate undergoes a novel conversion, possibly by PG synthetase, to a dilating substance which exerts different contractile effects than exogenously administered PGE₂, PGF_2α and PGE₁.This work was supported by (USPHS) training grants NS 05221, RCDA (P.N.) HL-19586, HL-11771A, HL-14397 and SCOR grant HL-17646, HL-17646-0.     

Indomethacin but not aspirin inhibits basal and stimulated lipolysis in rabbit kidney

The concurrent effect of indomethacin or aspirin on prostaglandins (PGs) biosynthesis and on cellular fatty acid efflux were compared. Studies with rabbit kidney medulla slices and with isolated perfused rabbit kidney showed a marked difference between the two non-steroidal anti-inflammatory drugs, with regard to their effects on fatty acid efflux from kidney tissue. While aspirin effect was limited to inhibition of PGs biosynthesis, indomethacin also reduced the release of free fatty acids. In medullary slices, indomethacin inhibited the Ca²⁺ stimulation of phospholipase A₂ activity and the resulting release of arachidonic and linoleic fatty acids. In the isolated perfused rabbit kidney, indomethacin inhibited the basal efflux of all fatty acids as well as the angiotensin II — induced selective release of arachidonate. Indomethacin also blunted the angiotensin II — induced temporal changes in the efflux of all other fatty acids. Neither indomethacin nor aspirin affected significantly the uptake and incorporation of exogenous (¹⁴C)-arachidonic acid into kidney total lipid fraction.Our tentative conclusion is that indomethacin inhibits basal as well as Ca²⁺ or hormone stimulated activity of kidney lipolytic enzymes. This action of indomethacin reduces the pool size of free arachidonate available for conversion to oxygenated products (both prostaglandin and non-prostaglandin types). The non-steroidal anti-inflammatory drugs can therefore be divided into two groups: a) $\text{aspirin-type compounds}$ which inhibit PGs formation only by interacting with the prostaglandin endoperoxide synthetase and b) $\text{indomethacin-type compounds}$ which inhibit PG generation by both reduction in the amount of available arachidonate and direct interaction with the enzyme.     

The effect of α1-acid glycoprotein (orosomucoid) on triglyceride metabolism in the nephrotic syndrome

We have identified α₁-acid glycoprotein as a new co-factor in the lipoprotein lipase reaction. We isolated an active form of the compound from nephrotic urine that is effective both $\text{in vitro}$ and $\text{in vivo}$. α₁-acid glycoprotein increased lipolysis 100% in the presence of C-II apolipoprotein in a lipoprotein lipase assay system. Rats with induced nephrotic syndrome showed a decrease in triglyceride clearance. $\text{T}\text{1}\text{2}$ was increased from 14 min to 43 min. The injection of α₁-acid glycoprotein restored the lipid clearance to normal. These findings suggest that elevated plasma triglycerides in human nephrotic patients is the direct result of excessive loss of α₁-acid glycoprotein from plasma into urine. We propose that replacement therapy may be possible.     

Guanosine 5'-(gamma-thio)triphosphate stimulates arachidonic acid liberation in permeabilized rat peritoneal mast cells

The exocytotic histamine secretion from ATP-permeabilized and Mg-resealed rat peritoneal mast cells is markedly enhanced by the addition of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) at a concentration of 100 uM. GTP gamma S also caused a great enhancement of arachidonic acid liberation from these cells. The level of released arachidonic acid in permeabilized cells enhanced by GTP gamma S in the absence of Ca2+ was nearly equal to the level of permeabilized cells incubated in the presence of Ca2+ but without GTP gamma S, suggesting the Ca2+ sparing effect of GTP gamma S. From the time sequential changes in the [3H]arachidonate radioactivities in various phospholipids, it is conceivable that nucleotide-dependent arachidonic acid release was mediated via phospholipase A2 pathway. The entrapment of a diacylglycerol (DG) lipase inhibitor, RHC 80267, caused suppression of both Ca2+- and guanine nucleotide-dependent arachidonic acid liberation in mast cells, indicating contribution of DG lipase pathway for arachidonic acid generation.     

The effects of mepacrine and p-bromophenacyl bromide on arachidonic acid release in human platelets

Two inhibitors of thrombin-stimulated arachidonic acid release from platelets, p-bromophenacyl bromide and mepacrine, were examined for their ability to inhibit the phospholipase C-diglyceride lipase pathway. This pathway involves hydrolysis of phosphatidylinositol to diglyceride, followed by release of arachidonate from diglyceride, and has been proposed as an alternative or addition to phospholipase A₂ as a mechanism for arachidonate release. p-Bromophenacyl bromide, a potent alkylating agent, was shown to cause a time-dependent inhibition of phosphatidylinositol-specific phospholipase C activity in crude platelet extracts; the inhibition was >90% after 15 min incubation with 100 μmp-bromophenacyl bromide. However, p-bromophenacyl bromide was also shown to destroy about one-half of the titratable sulfhydryl groups in whole platelets under similar conditions. The lack of specificity of p-bromophenacyl bromide was further demonstrated by our finding that thrombin-stimulated serotonin release was also inhibited by conditions inhibiting arachidonate release and that diglyceride lipase activity was decreased by higher levels of p-bromophenacyl bromide. Mepacrine was found to inhibit the activity of phosphatidylinositol-specific phospholipase C and had a greater effect at low substrate concentrations. The loss of [¹⁴C]arachidonate from both endogenous phosphatidylinositol and phosphatidylcholine in intact platelets was also inhibited. Thrombin-stimulated serotonin release was impaired by mepacrine also but only at a concentration 10-fold greater than that required to prevent arachidonate release. Thus we have shown that these two agents which inhibit arachidonate release are inhibitors of the phosphatidylinositol-specific phospholipase C-diglyceride lipase pathway. The multiple effects produced by both compounds limit their utility as agents to examine the source and mechanism of arachidonate release.     

Characterization of a partially purified diacylglycerol lipase from bovine aorta

A partially-purified diacylglycerol (DG) lipase from bovine aorta has been characterized with respect to the effects of lipid metabolites and two lipase inhibitors, phenylboronic acid and tetrahydrolipstatin (THL). DG lipase activity was determined by the hydrolysis of the sn-1 position of 1-[1-⁴C]palmitoyl-2-oleoyl-sn-glycerol. The products of the lipase reaction, 2-monoacylglycerol (2-monoolein) and non-esterified fatty acids (oleate, arachidonate) produced a concentration-dependent (20–200 μM) inhibition of DG lipase activity. Oleoyl-CoA and dioleoylphosphatidic acid also inhibited aortic DG lipase activity, but lysophosphatidylcholine had little or no effect. The inhibition of aortic DG lipase by phenylboronic acid was competitive, with a K_i of approx. 4 mM. THL was a very potent inhibitor of aortic DG lipase; the concentration required for inhibition to 50% of control was 2–6 nM. THL was a very potent inhibitor of concentration of substrate in the assay was increased. Attempts to identify the aortic DG lipase by covalent-labelling with [¹⁴C]THL were unsuccessful. Immunoblotting experiments revealed that hormone-sensitive triacylglycerol lipase (HSL) could not be detected in bovine aorta.     

The relationship between 13, 14-dihydro-15-keto PGF2α and LH secretion in bulls

Half-life ($\text{t}\text{1}\text{2}$), volume of distribution (Vd)_and total body clearance (TBC) of 13, 14-dihydro-15-keto PGF_2α (PGFM) were measured in order to determine optimal sampling frequency for accurate measurement of PGFM. Three yearling Holstein bulls (349.2 ± 6.7 kg) and 3 yearling Holstein steers (346.7 ± 7.0 kg) were utilized in a 3 × 3 Latin square design. Animals were given 0, 25 or 50 μg PGF_2α I.V.; blood samples collected every 2 min and plasma PGFM determined. The $\text{t}\text{1}\text{2}$, Vd and TBC of PGFM were 2.3 ± .2 min, 43.3 ± 3.3 liters and 13.7 ± 1.9 liters/min, respectively and were similar for 25 and 50 μg doses. To determine the relationship between endogenous PGFM and LH secretion in bulls, blood samples were collected every 2 min for 12 h in 4 yearling Angus bulls (489.1 ± 11.6 kg). All animals elicited at least one LH surge and PGFM concentrations were measured in samples coincident with the LH surge. Mean plasma PGFM concentrations were greater prior to the LH surge than during the LH surge. In addition, mean plasma PGFM concentration and frequency of PGFM peaks appeared to increase prior to the LH surge suggesting an association between PGFM and pulsatile LH secretion in the bull.     

Prostaglandins of the F series are extremely powerful growth factors for primary neonatal rat hepatocytes

At low concentrations (i.e., 10^?12–10^?9 mol/l), PGF_1α and PGF_2α very intensely stimulated both the DNA-synthetic and mitotic activities of hepatocytes in 4-day-old primary cultures of neonatral rat liver. DNA replication was more intensely enhanced by PGF_2α than by PGF_1α, whereas mitotic activity was nearly equally affected by the two prostaglandins. On the whole, the growth-promoting activity of PGF_1α used by itself or in equimolar mixtures with other prostaglandins ($\text{e}$. $\text{g}$., A₁, E₁, $\text{etc}$.) mimicked that of arachidonic acid we previously reported (1). On a molar basis, PGF_2α by itself stimulated hepatocytes′ DNA synthesis is more powerfully than arachidonate did, and when used in equimolar mixtures with other prostaglandins was at least as potent as arachidonic acid. These observations establish prostaglandins of the F series as quite powerful commitment factors and, though by a lesser degree, also intracycle regulators for neonatal rat hepatocytes in primary culture. However, the understanding of the role(s) of prostaglandins of F and other series in the physiological control of hepatocytes′ proliferative activation must wait the clarification of their interaction(s) with other arachidonate derivative(s) and polypeptide growth factor(s) which also may be involved in the process.     

Prostaglandin synthesis in rat embryo tissue: The effect of non-steroidal anti-inflammatory drug in vivo and ex vivo

Aspirin and salicylate are well-known but poorly understood teratogens in laboratory animals. Because aspirin inhibits PG synthesis, we systematically examined PG synthesis in rat embryo homogenates, the inhibition of PG synthesis $\text{in vivo}$ and $\text{ex vivo}$ by various non-steroidal anti-inflammatory drugs, and tested the hypothesis that the inhibition of PG synthesis is responsible for aspirin-induced limb defects in rats. We report that embryonic rat homogenates synthesis 6-keto-PGF_1α, PGE, and PGF in large amounts from endogenous substrate, that aspirin and other non-steroidal anti-inflammatory drugs inhibit PG synthesis $\text{in vitro}$ but not necessarily $\text{in vivo}$, and that contrary to our original hypothesis, the inhibition of PG synthesis is likely not responsible for aspirin-induced limb defects in rats.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and $\text{1}\text{20}$ of that of bradykinin (BK) on a weight basis. The activity of PGF_1α and PGF_2α was only $\text{1}\text{20}$ of that of PGE₁ or PGE₂.In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only $\text{1}\text{3}$–$\text{1}\text{8}$ of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).     

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1α on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI₂ directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI₂-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF_1α were without effect on plasma progesterone levels. The luteotrophic effect of PGI₂ was not due to alterations in circulating LH concentrations. An $\text{in vitro}$ experiment assessed the effects of either PGI₂ alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 μg PGI₂, 10 μg PGI₂, and 10 μg PGI₂ plus 5 ng LH averaged 99 ± 42, 353 ± 70, 152 ± 35, 252 ± 45, and 287 ± 66 ng/ml (n=4), respectively. Thus PGI₂ has luteotrophic effects on the bovine CL both $\text{in vivo}$ and $\text{in vitro}$.