3,3',5'-triiodo-L-thyronine reduces efficiency of mRNA knockdown by antisense oligodeoxynucleotides: a study with pyroglutamyl aminopeptidase II in adenohypophysis

The impact of hormones on the efficacy of antisense oligodeoxynucleotides (ASOs) is a poorly analyzed subject. We designed, based on the identification of potentially favorable local elements of mRNA secondary structure, eight phosphorothioate ASOs to knock down the expression of an ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in primary cultures of adenohypophysis. Two of the PPII ASOs were very efficient, sequence-specific, and target-specific. Because the expression of PPII is upregulated by 3,3',5'-triiodo-L-thyronine (T3), we studied the impact of varying the protocol of PPII induction on the knockdown efficacy. Hormone removal at transfection increased markedly the ability of (1) PPII ASOs to reduce PPII mRNA levels or PPII activity in adenohypophyseal cells or in C6 rat glioma cells and (2) a thyrotropin-releasing hormone (TRH) receptor-1 (TRH-R1) ASO to reduce TRH-R1 mRNA levels in adenohypophyseal cells. There was no effect of hormone removal on transfection efficacy and no correlation between target mRNA levels and ASO efficacy. These data demonstrated that ASO efficacy could depend on T3 levels; this might be due to regulation of a step generally critical for ASO efficiency.     

Acute administration of alcohol modulates pyroglutamyl amino peptidase II activity and mRNA levels in rat limbic regions

Released TRH is inactivated by an ectopeptidase, pyroglutamyl aminopeptidase II (PPII). PPII expression and activity are stringently regulated in adenohypophysis, and in rat brain, during kindling stimulation that activates TRHergic neurons. To gain further insight into the possible regulation of PPII, we studied the effect of an acute intraperitoneal ethanol administration that affects TRH content and expression. PPII activity was determined by a fluorometric assay and PPII mRNA levels by semi-quantitative RT-PCR. Activity decreased in frontal cortex 1 h after ethanol injection and, after 6 h, in hippocampus, amygdala and n. accumbens. PPII mRNA levels decreased at 30 and 60 min in frontal cortex and n. accumbens while increased at longer times in these regions and, in hippocampus and hypothalamus. NMDA and GABA(A) receptors' agonists and antagonists were tested at 1 h (+/-ethanol) on PPII activity and mRNA levels, as well as on TRH content and its mRNA. In n. accumbens, PPII mRNA levels decreased by ethanol, MK-801, and muscimol while picrotoxin or NMDA reversed ethanol's inhibition. Ethanol decreased TRH content and increased TRH mRNA levels as MK-801 or muscimol did (NMDA or picrotoxin reverted the effect of ethanol). In frontal cortex, PPII activity was inhibited by ethanol, NMDA and MK-801 with ethanol; its mRNA levels were reduced by ethanol, MK-801 and muscimol (NMDA and picrotoxin reverted ethanol's inhibition). These results show that PPII expression and activity can be regulated in conditions where TRHergic neurons are modulated. Effects of ethanol on PPII mRNA levels as well as those of TRH and its mRNA may involve GABA or NMDA receptors in n. accumbens. Changes observed in frontal cortex suggest combined effects with stress. The response was region-specific in magnitude, tendency and kinetics. These results give further support for brain PPII regulation in conditions that modulate the activity of TRHergic neurons.     

Inhibition of lipoxygenase enzyme in vitro by the cytochrome P-450 inhibitors metyrapone and SKF-525-A

It has recently been demonstrated that agents which block lipoxygenase enzymes (i.e. nordihydroguaiaretic and eicosatetraynoic acid) also block cytochrome P-450 in some $\text{in vitro}$ preparations. In some cases, therefore, results which were based on these lipoxygenase inhibitors could have been produced by the inhibition of cytochrome P-450. We therefore sought a way to distinguish between effects produced by metabolites of arachidonic acid generated by lipoxygenase enzymes and those produced by metabolites of the cytochrome P-450 system. seemed that a straightforward approach might be to administer drugs that inhibit the cytochrome P-450 system first, and then examine effects of lipoxygenase inhibitors. However, it then became necessary to establish the effect of inhibitors of the cytochrome P-450 system on lipoxygenase enzymes. Initial work was carried out using crystalline soybean lipoxygenase enzyme to avoid the complexities involved in isolating enzymes from biological tissues.Enzyme activity was assessed spectrophotometrically by measuring the increase in absorbance at 240 nm produced by the formation of a conjugated triene (15-HETE) from arachidonic acid. Effects of the two most commonly used cytochrome P-450 antagonists, metyrapone and SKF-525A, were determined by Lineweaver-Burke analysis. One major difficulty was that arachidonic acid is soluble in aqueous media at Ph 8 whereas both SKF-525-A and metyrapone are soluble at pH 7. In order to maintain all components in solution at the same time, experiments were carried out at pH 7.35 in the presence of 5% dimethylsulfoxide. There was no observable difference in the activity of 15-lipoxygenase at pH 9 in the absence of DMSO and that observed at pH 7.35 in the presence of DMSO.Studies were carried out in 2 ml quartz spectrophotometer cuvettes. For each experiment, two cuvettes were prepared containing arachidonic acid, DMSO (0.1 ml), SKF-525-A or metyrapone, and Tris buffer (pH 7.35). The cuvettes were placed in a Beckman DB/G spectrophotometer. One cuvette was used as reference and to the other was added 3ug (0.1 ml) of a solution of crystalline soybean lipoxygenase (Sigma). The change in absorbance at 240 nm was recorded and reflected product formation. Concentrations of arachidonic acid ranged from 10–50 uM, each tube contained either 0–50 uM SKF-525-A or 0–200 uM metyrapone.Results show that both SKF-525-A and metyrapone inhibited soybean lipoxygenase. Lineweaver-Burke analysis indicated that both agents acted in uncompetitive fashion. Of particular importance is that these concentrations of SKF-525-A and metyrapone are similar to those routinely used to block cytochrome P-450. From these results, it appears that experiments in which SKF-525-A and metyrapone were employed may need to be reevaluated. Work is underway using platelet-derived 12-lipoxygenase.     

Modulation of the 5-lipoxygenase activity of MC-9 mast cells: Activation by hydroperoxides

In order to identify regulatory steps in leukotriene synthesis, the biochemical characteristics of a 5-lipoxygenase activity in the 100,000 xg supernatant from sonicates of cells of an IL-3 dependent murine mast cell clone, MC-9 were determined. Principal products from exogenous ¹⁴C-arachidonic acid were identified as leukotriene B₄, diastereomeric 5,12-dihydroxy-eicossatetraenoic acids (5.12 diHETEs) 5-hydroperoxy and hydroxyeicosatetraenoic acids (5-HPETE and 5-HEYE) as well as a novel metabolite 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The lipoxygenase activity had a pH optimum of 6.9 and was highly dependent upon added Ca⁺⁺. The effective Ca⁺⁺ concentration for 50 per cent activation (EC₅₀) was 3 uM. Activity was also stimulated by ATP (EC₅₀ = 160 uM). The cytosolic 5-lipoxygenase activity exhibited a biphasic concentration dependence for arachidonic acid with maximum product formation occurring at 35 uM (ca. 20 nmole/mg/4 min). The lipoxygenase activity exhibited apparent lag phase kinetics which were more pronounced at low protein concentrations (0.3 mg/ml). In addition, the lag phase was greatly accentuated by the addition of a hydroperoxide scavenging system consisting of glutathione (1 mM) plus glutathione peroxidase (0.4 unit/ml). In contrast, addition of any several hydroperoxides, i.e. 5-,8-,9- or 15-HPETE (EC₅₀ ca. 1 uM), but not the corresponding alcohols (5-HETE and 15-HETE), shortened the lag phase. These results show that the 5-lipoxygenase requires hydroperoxide for activation and that cellular level of hydroperoxides may be an important factor regulating leukotriene synthesis.     

Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Optimization of an assay for studying the effects of agents on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in washed human platelets

Before one can examine the effects of substances on the metabolism of arachidonic acid (AA) by the cyclooxygenase and lipoxygenase pathways, an assay system which allows one to detect increases or decreases in both pathways in needed. In order to develop such a system, we have examined nonaggregating washed human platelets (10(8) platelets/0.5 ml) incubated for various times with 2 microCi 3H-AA and increasing concentrations of AA. T/B2, HHT, 12-HETE, and AA were extracted and separated using reverse phase-HPLC. We first calculated the mass of AA products formed with 10(-7) to 10(-4) M AA and found that the cyclooxygenase was saturated with 10(-5) M AA whereas the lipoxygenase was not saturated with 10(-4) M AA. Cyclooxygenase products were more prevalent than 12-HETE below 10(-5) M AA, while lipoxygenase products predominated at 3 x 10(-5)-10(-4) M AA. Using 3 microM AA, which does not saturate the cyclooxygenase, we examined the effect of 0.25-10 minute incubation durations on the distribution of AA metabolites and found AA product formation to increase throughout this period without completely depleting the substrate. Since substrate depletion does not occur and further metabolism could be detected for both pathways with a 5 minute incubation with 3 microM AA, these incubation parameters were chosen in order to further test the assay system. Using these parameters, we found that 10(-4) M 5-hydroxytryptamine enhanced platelet 12-HETE formation and decreased T/B2 and HHT formation, thus demonstrating the capacity of this system to simultaneously detect changes in cyclooxygenase and lipoxygenase enzyme metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic ethanol or glucose consumption alter TRH content and pyroglutamyl aminopeptidase II activity in rat limbic regions

Thyrotropin-releasing hormone (TRH), its receptors and inactivating enzyme (PPII) are present in limbic regions. Nutritional changes or acute ethanol administration in male rats differentially modulate TRH or PPII expression. Chronic ethanol effect was studied in male (3, 6 and 8 weeks) and female rats (6 weeks) including naive and pair-fed (glucose) groups. Daily solid food and liquid intake, serum TSH and corticosterone, TRH content and PPII activity in limbic regions, were quantified. Gender differences were found in ethanol and total caloric intake and body weight gain, TSH and corticosterone levels. Ethanol consumption decreased TRH content and PPII activity in frontal cortex of male rats after 3-6 weeks. In contrast, glucose ingestion altered, by the third week, TRH content in amygdala, hippocampus, hypothalamus and nucleus accumbens, PPII activity in hippocampus and frontal cortex; by the sixth week, TRH content in amygdala and n. accumbens of male and females. Withdrawal at 24 h after 3-week ethanol ingestion decreased TRH content in amygdala and PPII activity in n. accumbens, while withdrawal from glucose reverted some of the effects produced by chronic glucose ingestion. Variations in TRH content or PPII activity support a region specific involvement of TRH neurons that depend on the treatment.     

Differential effects of 15-HPETE on arachidonic acid metabolism in collagen-stimulated human platelets

The 15-hydroperoxyeicosatetraenoic acid (15-HPETE) has been shown to affect platelet aggregation induced by collagen, arachidonic acid (AA), and PGH2-analogue. Furthermore, it also inhibits the platelet cyclooxygenase and lipoxygenase enzymes, and prostacyclin synthase. The present study was designed to test the effect of 15-HPETE on the mobilization of endogenous AA in collagen-stimulated human platelets. For this purpose, human platelets pretreated with BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase) were stimulated with collagen in the presence of varied concentrations of 15-HPETE. We observed a significant inhibition of oxygenases at all concentrations of 15-HPETE. In contrast, our results indicate that 15-HPETE at lower concentrations (10 microM and 30 microM) significantly stimulated the collagen-induced release of AA from phospholipid sources. Although higher concentrations of 15-HPETE (50 microM and 100 microM) caused some inhibition of AA accumulation in the free fatty acid fraction (25% and 60%), the degree of inhibition was significantly lower than the inhibition observed for the oxygenases (65% and 88% for cyclooxygenase and 77% and 94% for lipoxygenase respectively). These results provide support that hydroperoxides also regulate phospholipases presumably by a different mechanism, which may be important in the detoxification of phospholipid peroxides.     

In vitro study of frog adrenal function--IX. Evidence against the involvement of lipoxygenase metabolites in the control of steroid production

The possible role of arachidonic acid metabolites of the lipoxygenase pathway in the regulation of steroidogenesis was studied in vitro using perifused frog interrenal (adrenal) glands. Graded doses of arachidonic acid (10(-6)-10(-4)M) increased the production of corticosterone and aldosterone in a dose-dependent manner. In the presence of indomethacin (5 X 10(-6)M), the effect of arachidonic acid on steroid secretion was totally abolished. Nordihydroguaiaretic acid (NDGA: 10(-6)M), a lipoxygenase inhibitor, did not alter the spontaneous secretion of corticosteroids and did not impair the stimulatory effect of arachidonic acid. In the presence of NDGA, both ACTH and angiotensin II were still able to stimulate corticosteroid production. Our data support the view that arachidonic acid metabolites play an important role in the regulation of amphibian steroidogenesis. Moreover, the results show that the lipoxygenase pathway is not involved in the spontaneous secretion of corticosteroids and in angiotensin II- or ACTH-induced steroidogenesis.     

Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: its polymorphism and relation to hemoglobin binding

Haptoglobin (Hp) binds hemoglobin (Hb) specifically and stoichiometrically. Since Hb stimulates prostaglandin (PG biosynthesis), we investigated if Hp effects arachidonic acid (AA) metabolism. The results showed that Hp (50-250 microg protein) inhibited the biosynthesis of PGs via cyclooxygenase (COX) and 12-HETE via lipoxygenase pathway in human platelets. Additional evidence was obtained by the loss of Hp inhibitory activity upon removal of Hp by affinity chromatography on hemoglobin sepharose and by inhibition of AA or bradykinin-induced bronchoconstriction in the guinea pig. Hb reduced the inhibitory effect of Hp in a concentration-related manner such that all its inhibitory activity was lost when completely bound by Hb. Of the three Hp phenotypes, Hp 1-1 showed maximum binding capacity to Hb indicating its greater protective role. These findings implicate Hp in the regulation of COX and lipoxygenase pathways and show Hp involvement in the body's endogenous defense system against inflammation. This indicates that mammals have dual defense system, i.e., a specific immune system and non-specific Hp defense system.     

Amygdala kindling differentially regulates the expression of the elements involved in TRH transmission

Subthreshold electrical stimulation of the amygdala (kindling) activates neuronal pathways increasing the expression of several neuropeptides including thyrotropin releasing-hormone (TRH). Partial kindling enhances TRH expression and the activity or its inactivating ectoenzyme; once kindling is established (stage V), TRH and its mRNA levels are further increased but TRH-binding and pyroglutamyl aminopeptidase II (PPII) activity decreased in epileptogenic areas. To determine whether variations in TRH receptor binding or PPII activity are due to regulation of their synthesis, mRNA levels of TRH receptors (R1, R2) and PPII were semi-quantified by RT-PCR in amygdala, frontal cortex and hippocampus of kindled rats sacrificed at stage II or V. Increased mRNA levels of PPII were found at stage II in amygdala and frontal cortex, and of pro-TRH and TRH-R2, in amygdala and hippocampus. At stage V, pro-TRH mRNA levels increased and those of PPII, decreased in the three regions; TRH-R2 mRNA levels diminished in amygdala and frontal cortex and of TRH-R1 only in amygdala. In situ hybridization analyses revealed, at stage II, enhanced TRH-R1 mRNA levels in dentate gyrus and amygdala while decreased in piriform cortex; those of TRH-R2 increased in amygdala, CA2, dentate gyrus, piriform cortex, thalamus and subiculum and of PPII, in CAs and piriform cortex. In contrast, at stage V decreased expression of TRH-R1 occurred in amygdala, CA2/3, dentate gyrus and piriform cortex; of TRH-R2 in CA2, thalamus and piriform cortex, and of PPII in CA2, and amygdala. The magnitude of changes differed between ipsi and contralateral side. These results support a trans-synaptic modulation of all elements involved in TRH transmission in conditions that stimulate the activity of TRHergic neurons. They show that reported changes in PPII activity or TRH-binding caused by kindling relate to regulation of the expression of TRH receptors and degrading enzyme.     

Comparison of the effect of lipoxygenase metabolites of arachidonic acid and eicosapentaenoic acid on human natural killer cell cytotoxicity

We compared in vitro effect of lipoxygenase (LO) products derived from arachidonic acid (AA) and eicosapentaenoic acid (EPA) on cytotoxic activity of human natural killer (NK) cell against human erythroleukemia cell line K-562. Leukotriene B4 (LTB4) derived from AA was found to significantly augment NK cell activity compared to the control level (in the absence of LTB). LTB5 showed a weak, but not significant, enhancing effect on NK cell activity. LTB4 was significantly more potent than LTB5 in the enhancement of NK cell activity. On the other hand, both 5- and 15-hydroperoxy fatty acids derived from AA and EPA significantly enhanced NK cell activity compared to the control level with similar potencies.     

Inhibition of phospholipase A2 activity in guinea pig eosinophils by human recombinant IL-1 beta.

The effect of human rIL-1 beta on the release of arachidonic acid (AA) and on the phospholipase A2 (PLA2) activity in guinea pig eosinophils was investigated. Stimulation of [3H]AA-labeled eosinophils with the ionophore A23187 resulted in a time and concentration-dependent release of AA in parallel to hydrolysis of endogenous phosphatidylcholine (PC). Both events were abrogated by the chelation of intracellular free calcium, but not by its depletion from the medium, suggesting that the ionophore-induced AA release involves a PLA2 activity dependent on the mobilization of intracellular calcium. Addition of human rIL-1 beta (0.01 to 100 ng/ml) to eosinophils for 15 min had no effect on the release of AA induced by the ionophore. However, prolonged incubation with human rIL-1 beta (30 to 180 min) inhibited in a concentration- and time-dependent manner the release of AA and the hydrolysis of phosphatidylcholine in ionophore-stimulated eosinophils. Our results also showed that eosinophil homogenates contain a calcium-dependent PLA2 whose activity was markedly reduced when eosinophils were pretreated with human rIL-1 beta. The inhibition was time and concentration dependent and was observed in the presence of calcium and phospholipid excess. Finally, studies with Fura-2-loaded eosinophils showed that the ionophore A23187 stimulated an increase in intracellular calcium concentration that was not altered by pretreating the eosinophils with human rIL-1 beta. These results suggest that human rIL-1 beta inhibits the release of AA by eosinophils via the inhibition of a PLA2 activity and through a calcium-independent mechanism. Inhibition by human rIL-1 beta required a prolonged incubation (30 to 180 min) and was observed after its removal from the medium, suggesting that human rIL-1 beta did not interact directly with the PLA2 itself, but with a metabolic process involved with the regulation of its activity in eosinophils.     

Effect of arachidonic acid on duodenal enterocyte ATPases

Duodenal ion transport processes are supported by ATPase enzymes in basolateral membranes of the enterocyte. In vivo studies have shown that long term n-6 poly-unsaturated fatty acid (PUFA) supplementation in rats causes increases in intestinal Ca absorption, coupled with a higher total calcium balance and bone calcium content. The present in vitro study was undertaken to test the effect of arachidonic acid (AA), a highly unsaturated (and thus physiologically potent) member of the n-6 PUFA family, on ATPases in enterocyte basolateral membranes isolated with a sorbitol density gradient procedure. This paper presents results which show that AA inhibits Na+,K+-ATPase in a dose-dependent manner (-67% of basal activity at a concentration of 30 microg/ml, P < 0.005) but that this effect is not mediated by protein kinase C, as shown by the use of the protein kinase C blocker calphostin (0.5 microM). Indomethacin (IDM) at 0.1 mM, a cyclo-oxygenase blocker, could also not reverse the inhibitory effect of AA on Na+,K+-ATPase. Ca2+-ATPase, on the other hand, is not affected significantly (-10%, P > 0.05) by arachidonic acid at 30 microg/ml.     

Altered arachidonate distribution in macrophages from caveolin-1 null mice leading to reduced eicosanoid synthesis

In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E(2) and LTB(4) production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D4 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 microM), and by arachidonic acid (AA), melittin and LTD4. FPL55712 (0.1 and 1 microM) inhibited contractions induced by AA (100 microM) and the phospholipase A2 activator melittin (3 micrograms/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 micrograms/ml) more than by A23187 (1 microgram/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 microM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Inhibition by Salicylhydroxamic Acid, BW755C, Eicosatetraynoic Acid, and Disulfiram of Hypersensitive Resistance Elicited by Arachidonic Acid or Poly-l-Lysine in Potato Tuber

The hypothesis that arachidonic acid (AA) induction of sesquiterpene accumulation and browning in potato (Solanum tuberosum) is mediated by a lipoxygenase metabolite of AA was tested using lipoxygenase inhibitors. Salicylhydroxamic acid (SHAM) and 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) delayed the response to AA. Inhibition by eicosatetraynoic acid (ETYA) was more persistent. These results are consistent with previous reports that SHAM and BW755C are reversible inhibitors of lipoxygenase and easily oxidized by potato while ETYA acts as an irreversible inhibitor. Disulfiram (tetraethylthiuram disulfide) also inhibited AA elicitor activity. SHAM was most effective if applied at the time of AA treatment, having no effect if applied 6 hours afterward. SHAM was effective in the presence of MES or MOPS buffers but not in acetate-buffered or unbuffered solutions; neither BW755C nor ETYA exhibited this restriction. However, SHAM, BW755C, and ETYA also were inhibitors of browning and sesquiterpene accumulation elicited in potato by poly-l-lysine, which, unlike AA, is not a lipoxygenase substrate. SHAM effectiveness also was restricted to 6 hours after treatment with poly-l-lysine. While the results with AA support a role for lipoxygenase, those with poly-l-lysine may be evidence that these compounds are having other effects in potato tissue.     

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.     

Prostaglandin synthesis by enterocyte microsomes of rabbit small intestine

We studied the prostaglandin (PG) synthetic capacity of microsomes of a relatively pure population of rabbit enterocytes and determined ideal conditions for product synthesis. The epithelial cells were freed from the basement membrane by a combination of calcium chelation and mechanical vibration, and 100,000 X g microsomes were prepared. These microsomes were found to synthesize PG from exogenously added arachidonic acid. The ideal conditions for the reaction were a microsomal protein concentration of 1.0 mg/ml, an arachidonic acid concentration of 33 uM, a reaction mixture pH of 8.0-9.5 and with epinephrine 1.5 mM added as a cofactor. The product yields increased linearly with time up to 30 min. of incubation and were inhibited by 100 uM indomethacin. Under the above ideal conditions enterocyte microsomes yielded the following products expressed as pmole/mg protein/20 min. incubation: PGF2 alpha 98 +/- 7, PGE2 48 +/- 9, PGD2 28 +/- 7, TxB2 40 +/- 5, 6 Keto PGF1 alpha 15 +/- 6.     

Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.