Are blastocyst prostaglandins produced endogenously?

This study was undertaken to determine whether preimplantation mouse and rabbit blastocysts possess cyclooxygenase activity and therefore are able to metabolize arachidonic acid (AA) to prostaglandins (PGs) and thromboxane B2. Single rabbit blastocysts or groups of 100 mouse blastocysts were incubated for 4 h in the presence of 5 muCi/ml [3H] AA (sp. ac. 61 Ci/mmol) or for 24 h and 18 h with 0.2 and 0.1 muCi/ml [14C] AA (sp. ac. 56.5 Ci/mmol), respectively. Incubated blastocysts were subsequently exposed for 10 min to 24 h to the Ca2+ specific ionophore, X-537A (10 microM), to stimulate release of radiolabeled AA from the phospholipid pool. After incubation, incorporation of AA into the phospholipid and neutral lipid pools, and metabolism of the fatty acid to PGs and thromboxane B2, were determined using thin-layer chromatographic (TLC) and liquid scintillation spectroscopic techniques. In addition, spent incubation media were analyzed for radiolabeled PG content. In blastocysts of both species, incorporation of AA into phospholipids was greater than that into neutral lipids (mouse: 1.0 vs. 0.7 pmol/blastocyst; rabbit: 159.7 vs. 56.9 pmol/blastocyst). The ionophore stimulated the release of AA from the phospholipid, and to a lesser extent, from the neutral lipid pool, of both blastocysts. No newly synthesized PGs were detected in either mouse blastocysts or their spent incubation media after stimulation with X-537A. Radiolabeled PGs (PGE2, PGF2 alpha, PGD2, PGA2) and thromboxane B2 were present in the media of rabbit blastocyst incubations, however, but were undetectable in the tissue extracts. Increases in metabolism of AA into each compound were observed with an increase in time of exposure to ionophore, and meclofenamic acid (2 microM) partially inhibited the synthesis of all compounds during a 24-h incubation. The results are discussed with regard to the role of blastocyst PGs in the events of implantation.     

A rapid assay for activity of phospholipase A2 using radioactive substrate

A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.     

Arachidonic acid release in rat peritoneal mast cells stimulated with antigen, ionophore A23187, and compound 48/80

Rat peritoneal mast cells respond to various types of secretagogues, such as antigen (receptor-mediated), A23187 (calcium mobilizing), and compound 48/80 (membrane perturbing), and release arachidonic acid from membrane phospholipids prelabeled with [3H]arachidonate. The rate of arachidonic acid liberation varied from one stimulant to the other. Ionophore A23187 (0.1 micrograms/ml) appeared to be most potent in releasing arachidonate among the three stimulants at which doses each secretagogue caused almost equivalent histamine secretion. However, upon stimulation with these three secretagogues, the radioactivity of phosphatidylcholine (PC) was markedly reduced with a concomitant increase of arachidonate radioactivity. Hydrolysis of PC by phospholipase A2 is likely to be the major route of arachidonic acid liberation in either IgE-mediated or non-IgE activation in mast cells.     

Chloroquine inhibition of cholera toxin

Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA). The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway. Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of [3H]AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml). Inhibition of [3H]AA release was complete when chloroquine was added before or within 30 min after CT. The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug.     

Effects of endometrium on metabolismof arachidonic acid by bovine blastocysts in vitro

The effects of endometrium on metabolism of [³H]-arachidonic acid ([³H]-AA) by bovine blastocysts recovered on day 19 postmating were studied $\text{in vitro}$. Blastocysts (n = 12) and endometrial slices were assigned to four incubation groups. In group 1, blastocysts were incubated alone; group 2, endometrial slices were incubated alone; group 3, blastocysts were incubated with endometrial slices; group 4, blastocysts were incubated in 7.5 ml fresh incubation medium plus 7.5 ml frozen-thawed medium from endometrial incubations. In all groups, tissues were incubated in 15 ml modified minimum essential medium (MEM) containing 5 μCi of [³H]-AA and 200 μg radioinert arachidonic acid for 24 h at 37°C in an atmosphere of 50% N₂:45% O₂:5% CO₂. For incubation controls, 5 μCi of [³H]-AA were added to 15 ml MEM and incubated at the same time as tissues from each cow. To evaluate metabolism of [³H]-AA, [³H]-AA and its metabolites were extracted from aliquots of MEM and separated on columns of Sephadex LH-20. Most (78.3 ± 3.2%) of the radioactivity (dpm) in the incubation controls was recovered as [³H]-AA, indicating that there was little breakdown of [³H]-AA in the absence of tissue. Blastocysts produced compounds that migrated with [³H]-13,14-dihydro-15-keto-PGF_2^α ([²H]-PGFM), [³H]-PGE₂ and [³H]-PGF_2^α. Endometrial slices metabolized very little of the [³H]-AA. Data from groups 1 and 4 were combined (group $\text{1}\text{4}$) for analysis because the distribution of dpm did not differ between the two groups. In group 3, blastocysts and endometrial slices incubated together tended(P<.10) to produced more [³H]-PGE₂ than did group $\text{1}\text{4}$, there tended to be less (P<.10)_[³H]-PGF_2^α, and there was more (P<.05) [³H]-PGFM than in group $\text{1}\text{4}$. Neither endometrial secretions nor endometrial slices altered the proportion of [³H]-AA metabolized by blastocysts. Endometrial slices appear capable of metabolizing [³H]-PGF_2^α synthesized by blastocysts, and capable of directing blastocyst metabolism of [³H]-AA away from synthesis of [³H]-PGF_2^α and toward synthesis of [³H]-PGE₂. It is postulated that the endometrium has an important role in regulating the amounts and ratios of prostaglandins in th uterine lumen during early prenancy in cows.     

Accumulation of oestrone by pig blastocysts and its potential physiological significance for blastocyst development in vitro

Oestrone accumulation of Day-5 pig blastocysts and the potential physiological significance of oestrone and oestradiol-17 beta for blastocyst development were investigated in vitro. After 6 h of in-vitro culture in medium supplemented with 10 nM-[3H]oestrone, the accumulation amounted to 550 +/- 49 d.p.m. (s.e.m.) per 10 blastocysts. The accumulation of [3H]oestrone (or its metabolite(s] was reduced (P less than 0.001) in the presence of a 100-fold excess of unlabelled oestrone or oestradiol-17 beta to 135 +/- 14 d.p.m. or 148 +/- 28 d.p.m. per 10 blastocysts, respectively. The accumulation of [3H]oestrone was not affected in the presence of a 100-fold excess of unlabelled progesterone, testosterone or oestrone sulphate. When blastocysts were post-incubated for 30 or 60 min in [3H]oestrone-free medium, blastocysts retained 74.1 +/- 16.8% and 66.0 +/- 10.4%, respectively of their initial radioactivity. In parallel experiments with [3H]progesterone the respective values were 23.8 +/- 3.0% and 21.7 +/- 2.1%. The presence of the antioestrogen nafoxidine (15 micrograms/ml) in basic culture medium impaired (P less than 0.001) the transformation of morulae to blastocysts (21.5 +/- 8.9%) compared to controls (98.3 +/- 1.7%). The inhibitory effects could be overcome (P less than 0.001) by a supplementation with 1 nM- or 100 nM-oestradiol-17 beta (62.5 +/- 12.8% and 80.0 +/- 6.2% development to blastocysts) but not with 1 nM- or 100 nM-oestrone (30.3 +/- 9.6% and 45.2 +/- 10.5%). Blastocyst expansion was also decreased P less than 0.01) to 61.0 +/- 11.4% of control values in the presence of 15 micrograms nafoxidine ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.     

Formation and metabolism of leukotriene C4 in macrophages exposed to bacterial lipopolysaccharide

Lipopolysaccharide (10 micrograms/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 +/- 1.3 ng/10(6) cells, SEM, n = 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was less than 1.6 ng/10(6) cells. Leukotriene C4 was characterized by reversed-phase high-performance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C4. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5-60 micrograms/ml), on cell counts and on the incubation time (4-16 h). The released leukotriene C4 was converted to a compound identified as a C6-cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins E2 and F2 alpha by radioimmunoassay demonstrated that leukotriene C4 and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.     

Plasma from uninephrectomized rats stimulates phospholipid methylation and arachidonic acid release in renal-cortical slices.

Phospholipid methylation and arachidonic acid release in renal-cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats ('uni-plasma') stimulated phospholipid methylation when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response in phospholipid methylation was obtained. Addition of 50 nM-12-O-tetradecanoylphorbol 13-acetate for 10 min to intact slices also stimulated phospholipid methylation, whereas incubation of slices before addition of 'uni-plasma' with 100 microM-1-(5-isoquinolinylsulphonyl)-2-methylpiperazine prevented it, suggesting that protein kinase C stimulates phospholipid methylation in renal-cortical slices. Plasma from uninephrectomized rats also stimulates [3H]arachidonic acid release from phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) via activation of phospholipase A2. Two mechanisms of phospholipase A2 activation are proposed: first, in which it is activated by protein kinase C and releases 3H radioactivity from PtdCho, and second, in which phospholipase A2 is stimulated by Ca2+ ions and releases 3H radioactivity from PtdEtn.     

A novel labeling procedure of anteiso-fatty acid-containing lipids in staphylococci for investigating the effect of penicillin on lipid release

L-[4,5-3H]isoleucine was introduced to label anteiso-fatty acid (AIFA)-containing lipids in Staphylococcus aureus SG 511. After an overnight incubation in peptone broth in the presence of 37 kBq L-[4,5-3H]isoleucine/ml, 8.5-13% of the total radioactivity applied was found to be incorporated into the cells. 22.4-25.6% of the incorporated radioactivity was found in AIFA-containing lipids extracted by chloroform-methanol-water (2:1:0.2, v/v/v) at pH 2. The interphase contained 70-75% of the incorporated radioactivity. Lipoteichoic acid, extracted by phenol-water (80:20, w/v) contained less than 1% of the incorporated radioactivity, as measured after purification by hydrophobic interaction chromatography on octyl sepharose gel. Within 1 h after addition of 10 micrograms/ml penicillin G to exponentially growing cultures of S. aureus, that led to non-lytic death of the cells, 11.9-18.1% of the incorporated L-[4,5-3H]isoleucine label were released. Lipids containing AIFA were excreted to 5.4-8.4% of total incorporated activity; this amount represents more than 1/4 of the labeled cellular lipids.     

Thrombin-induced transfer of arachidonic acid in human platelets is not inhibited by trifluoperazine

Human platelets have been shown to contain a Ca++- and CoA-independent transacylase enzyme that catalyzes the transfer of arachidonic acid from phosphatidylcholine (PC) to lysoplasmenylethanolamine. It has been suggested that this route may represent a major source for released arachidonic acid in stimulated platelets. In this study, we have shown using arachidonic-labelled human platelets that the thrombin-induced activation of a transacylase reaction was not affected by concentrations of trifluoperazine (TFP) (15 micrograms/2 X 10(9) cells) which abolished the accumulation of free [3H]arachidonic acid in the presence of the cyclooxygenase/lipoxygenase inhibitor BW755C. TFP, at this concentration failed to block the hydrolysis of phosphatidylcholine (PC) completely and had no effect on the increased radioactivity seen in total phosphatidylethanolamine (PE) (160% of control after 4 min of incubation). These results suggest that the transacylase pathway activated in response to thrombin is not likely dependent on calcium. As TFP blocks effectively both the accumulation of free [3H]arachidonic acid and the mass of arachidonic acid without affecting the transfer of this fatty acid from PC to PE in thrombin-stimulated human platelets, it is very unlikely that the transacylation pathway represents a major source of release arachidonic acid. Based on these findings, we conclude that the above pathway may be primarily involved in the turnover of plasmenylethanolamine lipids in stimulated human platelets.     

Nerve Growth Factor Stimulation of Arachidonic Acid Release from PC12 Cells: Independence from Phosphoinositide Turnover

The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.     

Effects of endometrium on metabolismof arachidonic acid by bovine blastocysts

Metabolism of radiolabeled arachidonic acid (^*AA) by blastocysts and endometrial slices recovered from five gilts 16 days after detection of estrus was studies . Blastocysts from each gilt were divided into four 216 ± 18 mg, and each portion was placed into a separate petri dish containing 15 ml modified minimum essential medium (MEM)_. The incubates from each gilt received either 25, 50, 100 or 200 μg radioinert arachidonic acid (AA). Endometrium was dissected from each uterin horn, sliced and duplicate 509 ± 3 mg portions from each gilt were placed into petri dishes containing 15 ml MEM and 200 μm AA. All incubates received 5 νCi of ^*AA (either [¹⁴C]-arichidonic acid or [³H]-arichidonic acid). The incubates were rocked at 37°C for 24 h in an atmosphere of 50% n₂:45% O₂:5% CO₂. After incubation, tissues and MEM were separated by centrifugation. Metabolism of ^*AA was assessed in extracts of MEM and tissue homogenates by separating ^*AA and its metabolites on columns of Sephades LH-20. Blastocysts produced compounds that migrated with [³H]-13,14-dihydro-15-keto-PGF_2^α (^*PGFM), [³H]-PGE₂ (^*PGE₂) and [³H]-PGF_2^α (^*PGF_2^α). The greatest (P<.05) proportion (35.7 ± 1.8%) of the radioactivity in blastocyst MEM was recovered as PGE₂. In blastocyst homogenates, most (66.2 ± 3.3%; P<0.05) of the radioactivity was in a nonporal peak assumed to be arachidonate esters. The concentration of AA ni MEM did not alter metabolism of ^*AA by blastocysts. Endometrial slices produced ^*PGFM and ^*PGE₂ but only in small amounts, and they were capable of producing nonpolar, probably esterified, forms of ^*AA. It was concluded that porcine blastocysts produced and metabolized prostaglandins and that they make a contribution to the uterine milieu during early pregnancy.     

Metabolism of arachidonic acid in vitro by porcine blastocysts and endometrium

Metabolism of radiolabeled arachidonic acid (¹AA) by blastocysts and endometrial slices recovered from five gilts 16 days after detection of estrus was studies $\text{in vitro}$. Blastocysts from each gilt were divided into four 216 ± 18 mg, and each portion was placed into a separate petri dish containing 15 ml modified minimum essential medium (MEM)_. The incubates from each gilt received either 25, 50, 100 or 200 μg radioinert arachidonic acid (AA). Endometrium was dissected from each uterin horn, sliced and duplicate 509 ± 3 mg portions from each gilt were placed into petri dishes containing 15 ml MEM and 200 μm AA. All incubates received 5 νCi of ¹AA (either [¹⁴C]-arichidonic acid or [³H]-arichidonic acid). The incubates were rocked at 37°C for 24 h in an atmosphere of 50% n₂:45% O₂:5% CO₂. After incubation, tissues and MEM were separated by centrifugation. Metabolism of ¹AA was assessed in extracts of MEM and tissue homogenates by separating ¹AA and its metabolites on columns of Sephades LH-20. Blastocysts produced compounds that migrated with [³H]-13,14-dihydro-15-keto-PGF_2^α (¹PGFM), [³H]-PGE₂ (¹PGE₂) and [³H]-PGF_2^α (¹PGF_2^α). The greatest (P<.05) proportion (35.7 ± 1.8%) of the radioactivity in blastocyst MEM was recovered as PGE₂. In blastocyst homogenates, most (66.2 ± 3.3%; P<0.05) of the radioactivity was in a nonporal peak assumed to be arachidonate esters. The concentration of AA ni MEM did not alter metabolism of ¹AA by blastocysts. Endometrial slices produced ¹PGFM and ¹PGE₂ but only in small amounts, and they were capable of producing nonpolar, probably esterified, forms of ¹AA. It was concluded that porcine blastocysts produced and metabolized prostaglandins $\text{in vitro}$ and that they make a contribution to the uterine milieu during early pregnancy.     

Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

Inhibitors of Na+/H+ exchange block stimulus-provoked arachidonic acid release in human platelets. Selective effects on platelet activation by epinephrine, ADP, and lower concentrations of thrombin

We have observed previously that removal of extraplatelet Na+ blocks platelet secretion of dense granule contents in response to epinephrine, ADP, and 0.004 unit/ml thrombin, all agents which must mobilize arachidonic acid for its subsequent conversion to cyclooxygenase products in order to elicit platelet secretion. The present studies demonstrate that removal of extraplatelet Na+ blocks arachidonic acid mobilization in response to epinephrine, ADP, and 0.004 unit/ml thrombin without altering arachidonic acid conversion to thromboxane A2. The data also provide several lines of evidence which suggest that the blockade of arachidonic acid release due to removal of extraplatelet Na+ is a manifestation of blockade of Na+/H+ exchange system. 1) There is a concentration-dependent effect of extraplatelet Na+ (EC50 congruent to 55 mM) on [3H]arachidonic acid release such that mobilization is observed when [Na+]o greater than [Na+]i. 2) Increasing extraplatelet [H+] (i.e. decreasing extraplatelet pH from pH 7.35 to 6.8) causes a concentration-dependent decline in stimulus-provoked [3H]arachidonic acid release. 3) Ethylisopropylamiloride and other potent 5-amino analogs of amiloride block [3H]arachidonic acid release with a potency that parallels their effects on Na+/H+ exchange in other cellular systems. None of the above manipulations alter primary aggregation induced by epinephrine, ADP, or 0.004 unit/ml thrombin, indicating that stimulus-receptor binding, subsequent exposure of fibrinogen receptors, and fibrinogen-mediated platelet-platelet cross-linking are not significantly inhibited by [3H]arachidonic acid release in response to greater than 0.1 unit/ml thrombin, a stimulus that can elicit platelet secretion in the absence of products of the cyclooxygenase pathway. Therefore, Na+/H+ exchange may selectively modulate arachidonic acid mobilization in response to the so-called "weak agonists," agonists that require this mobilization to effect vigorous platelet aggregation and dense granule secretion.     

In-vitro prostanoid production by the rat vas deferens

Prostaglandins (PGs), E-2, F-2 alpha, 6-keto-F-1 alpha and thromboxane B-2 (TXB-2), produced in vitro by the vas deferens of rats at different stages of sexual maturation (10, 35 and 100 days of age), were estimated by radioimmunoassay. Reversed-phase high-performance liquid chromatography was used to evaluate the RIA and to give a more complete profile, after incubation of the vas deferens with [14C]arachidonic acid. The amount of PGE-2 released into the medium after incubation at 37 degrees C for 5 min increased with age, and was the predominant prostanoid in the adult vas deferens. In prepubertal organs, 6-keto-PGF-1 alpha predominated. TXB-2 was always a minor product. The addition of exogenous arachidonic acid (10 micrograms/ml/20 mg tissue), provoked a higher production of the four compounds. PGE-2 and PGF-2 alpha levels were reduced after castration or hypophysectomy and were re-instated after treatment with testosterone propionate. PGE-2 was much more sensitive to hormonal deprivation than PGF-2 alpha. The production of 6-keto-PGF-1 alpha was not significantly affected by the above treatments.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

Radiotoxicities of [3H]thymidine and of [3H]arginine compared in mouse embryos in vitro

Tritium that is bound to organic molecules is of special risk for living systems, in particular when such molecules are components of the cell nucleus. Therefore, [3H]thymidine and [3H]arginine were studied for radiotoxicity in early mammalian embryo development. Starting with the two-cell stage, mouse embryos were incubated in vitro with [3H]thymidine or [3H]arginine at either 370 Bq/ml (10 nCi/ml) or 925 Bq/ml (25 nCi/ml). Development in vitro was followed up to the formation of the inner cell mass at 192 h postconception (p.c.). There was no difference in radiotoxicity of the two substances with respect to cell proliferation; however, formation of blastocysts, hatching of blastocysts, trophoblast outgrowth, and formation of inner cell mass were impaired more strongly by [3H]arginine than by [3H]thymidine when the external exposure concentrations were the same. Similarly, micronuclei were seen in blastocysts at 96 h p.c. at higher frequency after incubation with [3H]arginine. However, uptake of [3H]arginine by the embryos was considerably faster than that of [3H]thymidine, and this most probably accounts for the apparent difference in radiotoxicity.     

Stimulation of arachidonic acid metabolism by a streptococcal preparation (OK-432) in rat peritoneal macrophages.

A streptococcal preparation OK-432 is reported to be an immunopotentiator and a potent antitumor agent. In order to elucidate the mechanism of biologic action, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages were investigated. Prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were found to be stimulated by OK-432 in a concentration-dependent manner (5 to 80 micrograms/ml). Heat-treatment of OK-432 further stimulated its effects. These stimulative effects on arachidonic acid metabolism by OK-432 were not observed in MDCK cells that have no phagocytotic activity. Furthermore, cytochalasin B treatment completely suppressed the stimulative effects induced by OK-432 in macrophages. These results strongly indicate that the stimulative effects by OK-432 on arachidonic acid metabolism are dependent on phagocytosis of OK-432 particles. Significance of stimulation of arachidonic acid metabolism in macrophages by OK-432 for its biological effects is discussed.