Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles.

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.

Ultraviolet B irradiation induces changes in the distribution and release of arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture.

There is increasing evidence that derivatives of 20-carbon polyunsaturated fatty acids, the eicosanoids, play an important role in the inflammatory responses of the human skin. To better understand the metabolic fate of fatty acids in the skin, the effect of ultraviolet B (UVB) irradiation (280-320 nm) on the distribution and release of 14C-labeled arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture was investigated. Ultraviolet B irradiation induced the release of all three 14C-labeled fatty acids from the phospholipids, especially from phosphatidylethanolamine, and this was accompanied by increased labeling of the nonphosphorus lipids. This finding suggests that UVB induces a significant liberation of eicosanoid precursor fatty acids from cellular phospholipids, but the liberated fatty acids are largely reincorporated into the nonphosphorus lipids. In conclusion, the present study suggests that not only arachidonic acid but also dihomo-gamma-linolenic acid, and eicosapentaenoic acid might be involved in the UVB irradiation-induced inflammatory reactions of human skin.

Endogenously generated 5-hydroperoxyeicosatetraenoic acid is the preferred substrate for human leukocyte leukotriene A4 synthase activity.

A single protein from human leukocytes possesses both 5-lipoxygenase and leukotriene A4 (LTA4) synthase activities. It has been reported that LTA4 production is more efficient when the enzyme utilizes arachidonic acid, than when 5-HPETE is exogenously supplied as substrate. In the present study, human leukocyte homogenate 100,000 X g supernatant was incubated with 100 microM octadeuterated arachidonic acid and exogenous 5-HPETE (0-80 microM), and the isotopic composition of LTA4 hydrolysis products was determined by gas chromatography-mass spectrometry. Even though 100 microM deuterated arachidonic acid results in 20-30 microM deuterated 5-HPETE, 80 microM exogenous 5-HPETE in the incubation could reduce the amount of deuterated LTA4 by only approx. 20%. The present study would thus indicate that the arachidonic acid moiety is preferentially converted to LTA4 in a concerted reaction without dissociation of a 5-HPETE intermediate.

Evidence for a 5(6)-epoxytetraene intermediate in the biosynthesis of lipoxins in human leukocytes. Conversion into lipoxin A by cytosolic epoxide hydrolase.

The existence of a 15(S)-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid intermediate in the biosynthesis of lipoxins A and B has recently been proposed. In the present study, human leukocytes were exposed to 15-HETE and the divalent cation ionophore A23187 and alcohol trapping studies were performed. The products containing alkyltetraenes were isolated and characterized. HPLC analysis, UV spectroscopy and GC/MS of the products showed that 5,15-dihydroxy-14-O-alkyleicosatetraenoic acids were formed, indicating that 5(6)-epoxytetraenes (precursor of the trapping product) were formed in human leukocytes. To gain further evidence for the role of 5(6)-epoxytetraene intermediate in the biosynthesis of lipoxins, (15)-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid was prepared by total chemical synthesis. When added to purified human liver cytosolic epoxide hydrolase, the epoxide was rapidly and quantitatively converted into LXA. The results provide further evidence for the role of a 5(6)epoxytetraene intermediate in the biosynthesis of lipoxins.

Effects of repeated oral doses of fenflumizole on platelet aggregation and thromboxane formation in man ex vivo.

Anti-platelet effects of fenflumizole, a new cyclo-oxygenase inhibitor, were studied in man ex vivo. Fenflumizole was given to male volunteers at the oral doses of 25, 50 or 100 mg per day, each dose for a period of seven days. The formation of thromboxane B2 (TXB2) during whole blood clotting, platelet aggregation induced by arachidonic acid and ADP, the formation of TXB2 during aggregation as well as serum concentration of fenflumizole were measured repeatedly during drug administration and for a fortnight after drug discontinuation. TXB2 formation during whole blood clotting was decreased dose-dependently by fenflumizole. The degree of inhibition of TXB2 formation was proportional to fenflumizole concentration in serum within each individual. The lag phase of platelet aggregation induced by arachidonic acid was prolonged and the formation of TXB2 during aggregation decreased by fenflumizole. No total inhibition of either TXB2 synthesis or platelet aggregation was caused by the fenflumizole doses used. The results show that the degree of inhibition of platelet thromboxane forming capacity by repeated doses of fenflumizole is closely related to the concentration of the drug in blood. Platelet aggregation however is less sensitive to changes in fenflumizole levels and cannot be assessed solely on the basis of cyclo-oxygenase activity.

UVB irradiation induces changes in the distribution and release of 14C-arachidonic acid in human keratinocytes in culture.

Following labeling of human keratinocytes in culture for 48 h with 14C-arachidonic acid (800,000 cpm), 86.8 +/- 0.5% (mean +/- SEM) of the radioactivity was incorporated into the cells. Two hours after exposure to UVB irradiation at doses up to 392 mJ/cm2 of erythemally effective (EE) UVB irradiation, only slight changes in the distribution of arachidonic acid could be detected. However, 24 h after irradiation the release of arachidonic acid into the culture medium was significantly increased. The distribution of arachidonic acid was also changed: there was a considerable loss in the amount of radioactivity associated with phosphatidylethanolamine. With doses up to 174 mJ/cm2 (EE) of UVB, the decrease in the labeling of phospholipids was accompanied by an increased arachidonic acid content in the nonphosphorus lipids, especially in the triacylglycerols. Following a high dose of UVB (392 mJ/cm2, EE), a substantial release of label was detected, but the labeling of triacylglycerols was unaltered. The present study suggests that in human keratinocytes UVB irradiation induces the release of arachidonic acid from the cellular lipids and that the major source of the released arachidonic acid is phosphatidylethanolamine.

The antipsoriatic drug metabolite etretin (Ro 10-1670) alters the metabolism of fatty acids in human keratinocytes in culture.

We have studied the effect of etretin (Ro 10-1670), the active metabolite of the widely used antipsoriatic drug etretinate (Ro 10-9359), on the incorporation and release of arachidonic acid in human skin keratinocytes. During 24-h culture, radioactive 14C-arachidonic acid was avidly incorporated into the cellular lipids of the keratinocytes. When the cells were cultured for another 48 h in fresh medium, 8.8% +/- 0.3% of the incorporated radioactivity was released from the cells. The presence of etretin (10(-8) M to 10(-5) M) in the medium stimulated the release of radiolabel. With 10(-5) M etretin in the culture medium, 13.0% +/- 0.4% of the incorporated radioactivity was released, and this was accompanied by decreased labelling of phosphatidylethanolamine. This suggests that phosphatidylethanolamine may be an important source of the released arachidonic acid. Etretin pretreatment reduced the incorporation of 14C-arachidonic acid into diacylglycerols, triacylglycerols, and cholesteryl esters. Pretreatment for 48 h with 10(-5) M etretin reduced subsequent 14C-arachidonic acid incorporation into nonphosphorus lipids from a mean total of 8.2% +/- 0.2% to 3.2% +/- 0.1% (p less than 0.001). These findings suggest that etretin interferes with the esterification of arachidonic acid into nonphosphorus lipids. Etretin was also found to cause changes in the fatty acid composition of keratinocytes. Following 48 h culture with etretin, the percentage amount of the fatty acids belonging to the n3 series was increased whereas that of palmitic acid (16:0) and palmitoleic acid (16:1n7) was decreased. In conclusion, our study suggests that etretin in therapeutical concentrations affects fatty acid metabolism in human keratinocytes in culture.

Time course of incorporation of 20-carbon polyunsaturated fatty acids in a human keratinocyte cell line.

Human keratinocytes (NCTC 2544) in culture were labeled with equal amounts of either 14C-arachidonic acid, 14C-dihomo-gamma-linolenic acid or 14C-eicosapentaenoic acid. At various time points, the incubations were stopped and the distribution of the 14C-fatty acids was analyzed. All these eicosanoid precursor fatty acids were effectively incorporated into the cellular lipids of the keratinocytes, and the major radiolabeled individual lipid fraction was phosphatidylethanolamine. The distributions of arachidonic acid and dihomo-gamma-linolenic acid within cellular lipids were rather the same. However, less eicosapentaenoic acid than either arachidonic acid or dihomo-gamma-linolenic acid was incorporated into the phospholipids and, correspondingly, more eicosapentaenoic acid was incorporated into the nonphosphorus lipids. In the phosphatidylinositol + phosphatidylserine fraction, there was significantly less eicosapentaenoic acid than either arachidonic acid or dihomo-gamma-linolenic acid. The present study suggests that these eicosanoid precursor fatty acids are effectively incorporated into the human keratinocytes and that the pattern of incorporation and distribution of eicosapentaenoic acid appears to differ slightly from that of either arachidonic acid or dihomo-gamma-linolenic acid.

Albumin stimulates the release of arachidonic acid from phosphatidylcholine in hamster lungs.

14C-Arachidonic acid injected into the pulmonary circulation of isolated hamster lungs was effectively incorporated into lung lipids. Once retained the radiolabel was relatively stable but the release of radioactivity increased up to 10-fold when bovine serum albumin (1%) was added to the perfusate. This efflux of radioactivity was not blocked by quinacrine, a phospholipase A2 inhibitor. In albumin experiments the released 14C-arachidonate originated mainly from the phospholipid fraction in which phosphatidylcholine was the main source of the released radioactivity. Pulmonary infusion of albumin had no significant effect on the amount of 14C-arachidonic acid in the neutral lipid or free fatty acid fractions of perfused lungs. In experiments with albumin about 80% of the released radioactivity co-chromatographed with unlabelled arachidonic acid whereas in the absence of albumin only about 20% of the released radioactivity was unmetabolized arachidonic acid. This study indicates that albumin stimulates the release of arachidonic acid from isolated hamster lungs and that the release is increased mainly from the phosphatidylcholine fraction.

Interference with the distribution and release of arachidonic acid in human keratinocytes by bradykinin, histamine and phosphatidic acid.

The effects of bradykinin, histamine, phosphatidic acid and leukotrienes B4 and C4 on the distribution and release of 14C-arachidonic acid in human keratinocytes in culture were investigated. Bradykinin, histamine, and phosphatidic acid were found to liberate 14C-arachidonic acid from membrane phospholipids, whereas leukotrienes B4 and C4 were ineffective in this respect. The decrease in the labeling of phospholipids was accompanied by increased labeling of the non-phosphorus lipids. The present study suggests that bradykinin, histamine, and phosphatidic acid may interfere with the distribution and release of arachidonic acid in human keratinocytes in culture.

The effect of selenium and vitamin E deficiencies on the fate of arachidonic acid in rat isolated lungs.

The fate of exogenous 14C-arachidonic acid (14C-AA) was investigated in the isolated lungs of rats fed selenium and vitamin E deficient diet or diets supplemented with selenium and/or vitamin E. When 80 nmol of 14C-AA was infused into the pulmonary circulation most of the infused 14C-AA was found in different phospholipid and neutral lipid fractions of the perfused lungs. Only less than ten percent of the infused radioactivity was recovered in the perfusion effluent. The amount of arachidonate metabolites in the perfusion effluent was negligible, and most of the radioactivity in the perfusion effluent consisted of unmetabolized arachidonate. Selenium deficiency had no significant effect on the distribution of 14C-AA in different lung lipid fractions. However, in the lungs of vitamin E deficient rats the amount of radioactivity was slightly increased in the neutral lipid fraction, which was due to the increased amount of 14C-AA in the diacylglycerols. The amount of radioactivity was increased especially in the 1,3-diacylglycerols. The amount of radioactivity was increased especially in the 1,3-diacylglycerols. The amount of 14C-AA in the triacylglycerols and in different phospholipids was not significantly changed. The present study might indicate that selenium deficiency has no significant effect on the fate of exogenous arachidonic acid in isolated rat lungs, and that vitamin E deficiency would slightly increase the amount of arachidonic acid in the diacylglycerols.

The amount of arachidonic acid in the triacylglycerols of perfused hamster lungs is increased by prednisolone.

Following the injection of 14C-arachidonic acid (4.1 nmol) into hamster isolated lungs about 80% of the administered radioactivity was retained by the lungs. During subsequent perfusion only a small amount of radioactivity was released to the perfusion effluent. This release was not affected by pulmonary infusion of prednisolone at 20 microM or 100 microM. In control lungs 84 +/- 1% (+/- SEM) of the retained radioactivity was recovered in the phospholipid, 13 +/- 1% in the neutral lipid and 3 +/- 1 in the free fatty acid fraction. Pulmonary infusion of prednisolone increased the amount of radiolabel in the neutral lipids. This was due to the increased amount of 14C-arachidonic acid in triacylglycerols. Prednisolone had no significant effects on the amount of 14C-arachidonate in diacylglycerols or in different phospholipids. Neither was the amount of free 14C-arachidonate in the lungs changed by prednisolone. The present study indicates that the release of arachidonic acid from triacylglycerols may be inhibited by prednisolone in hamster lungs.

Dipyridamole interferes with the incorporation of arachidonic acid and stimulates prostacyclin production in rat lungs.

Following the injection of 4 nmol of 14C-arachidonic acid into the pulmonary circulation of rat isolated lungs more than 90% of the radioactivity was retained by the lung tissue. When dipyridamole (20 microM) was infused into the pulmonary circulation during 14C-arachidonate injection the amount of radiolabel was increased in diacylglycerols as well as in phosphatidylinositol and phosphatidylserine of the perfused lungs whereas the amount of radioactivity was decreased in phosphatidylethanolamine. When dipyridamole was infused into the lungs prelabelled with 14C-arachidonic acid the distribution of radiolabel in different lung lipid fractions was not changed significantly. However, dipyridamole seemed to stimulate the formation of prostacyclin in rat lungs as the amount of 6-keto-PGF1 alpha was increased in the perfusion effluent. The present study indicates that dipyridamole interferes with the incorporation of arachidonic acid into different lipids in rat lungs. In addition, the release of prostacyclin seems to be stimulated by dipyridamole.

The effect of bradykinin, histamine, and leukotrienes B4, C4 and D4 on the formation of 6-keto-prostaglandin F1 alpha and thromboxane B2 in hamster lungs.

Bradykinin, histamine, leukotriene B4, C4, D4 (LTB4, LTC4, LTD4), and bradykinin + LTC4 were injected as a bolus into the pulmonary circulation of hamster isolated lungs, and the amounts of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TXB2) in the perfusion effluent were determined by radioimmunoassay. LTD4, bradykinin, and bradykinin + LTC4 stimulated 6-keto-PGF1 alpha formation whereas TXB2 formation was stimulated only by LTB4 and histamine. The detected changes in the activation of arachidonate metabolism were, however, relatively small. The present study indicates that bradykinin, histamine, LTB4, and LTD4 have only a minor stimulating effect on arachidonic acid metabolism in hamster lungs.

The effect of hydrocortisone on the metabolism of PGE2 in rat lungs.

Hydrocortisone pretreatment of male rats daily for five days (2 or 10 mg/kg, s.c.) had no significant effect on the total inactivation of prostaglandin E2 in isolated perfused rat lungs or on the activity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase in the 100.000 x g supernatant fraction of homogenized lungs. Nor did pulmonary infusion of hydrocortisone (0.1 or 1 microM in the perfusion medium) have any effect on the metabolism of PGE2 in isolated rat lungs. The present study indicates that pulmonary inactivation of PGE2 is not changed by hydrocortisone in rat lungs.

Thromboxane formation in human polymorphonuclear leukocytes is inhibited by prednisolone and stimulated by leukotrienes B4, C4, D4 and histamine.

Human polymorphonuclear leukocytes (PMNL) were incubated for 60 min at 37 degrees C with 20 microM or 100 microM prednisolone, and stimulated thereafter for 1 min with 10 nM LTB4, 10 nM LTC4, 10 nM LTD4 or 10 microM histamine. The amount of thromboxane B2 (TXB2) formed by PMNLs was measured by radioimmunoassay. PMNLs spontaneously released TXB2 during 60 min incubation, and the rate of formation was significantly reduced in the presence of 20 microM or 100 microM prednisolone. LTB4, LTC4, LTD4, and histamine stimulated the rate of TXB2 production during 1 min incubation to 93-, 49-, 60-, and 55-fold, respectively. Preincubation with prednisolone for 60 min had a slight inhibitory effect on the stimulated TXB2 formation but TXB2 production still remained many fold as compared to its spontaneous rate of formation. The present study indicates that human PMNLs are capable of synthetizing TXB2, and its spontaneous rate of formation is inhibited by a synthetic glucocorticoid, prednisolone. The great stimulatory effect of LTB4, LTC4, LTD4, and histamine suggests that these agents may activate phospholipases or other acylhydrolases which liberate arachidonate for eicosanoid biosynthesis.

Carbon monoxide is not responsible for the cigarette smoke-induced changes in the pulmonary metabolism of arachidonic acid and prostaglandin E2.

Cigarette smoke is known to interfere with the pulmonary metabolism of arachidonic acid and prostaglandin E2 (PGE2). We investigated the possible role of carbon monoxide in these cigarette smoke-induced alterations. 14C-Arachidonic acid (50 nmol) was infused into the pulmonary circulation of isolated perfused hamster lungs, and the radioactive metabolites in the perfusion effluent, as well as the distribution of incorporated radioactive arachidonic acid within the lung lipids, were analysed. Carbon monoxide, added into the ventilatory air, had no effect on the oxidative metabolism of arachidonic acid or on the distribution of radioactive arachidonic acid within the lung. In addition, carbon monoxide had no effect on the metabolism of PGE2 following infusion of 100 nmol of 14C-PGE2 into the rat pulmonary circulation. The present study suggests that carbon monoxide is not responsible for the cigarette smoke-induced changes in the pulmonary metabolism of arachidonic acid and PGE2.

The fatty acid composition of 12 North-European fish species.

Cardiovascular diseases among Greenland Eskimos are rare because their diet is rich in fatty fish and marine mammals. The beneficial effect of the fish diet appears to be mediated, at least in part, by the high amount of eicosapentaenoic acid in fish. We investigated the total lipid amount and fatty acid composition of 12 commonly eaten North-European fish species. Most of the detected fatty acids were unsaturated, and the content of eicosapentaenoic acid varied usually between 6 and 16%. The amount of total lipid varied between 3.5 and 216 mg/g wet tissue. The total amount of lipid in different fish species seems to be more important than the respective fatty acid composition when considering which fish should be especially beneficial in the diet. Herring, salmon, Baltic herring, turbot and trout seem to contain most abundantly eicosapentaenoic acid.