Tapio Puustinen

Incorporation and distribution of dihomo-γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

The effects of aspirin and dipyridamole on the metabolism of arachidonic acid in human platelets

The effects of aspirin and dipyridamole on the metabolism of exogenous 14C-arachidonic acid were investigated in intact human platelets in vitro. The formation of thromboxane B2 (TXB2) and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was inhibited dose dependently by aspirin but not by dipyridamole. Neither was the aspirin caused inhibition in TXB2 and HHT formation modified by dipyridamole. At high concentrations aspirin caused a slight increase in the amount of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) and a corresponding decrease in that of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). This was seen also when aspirin was combined with dipyridamole. At high concentrations (100 microM and 1 mM) dipyridamole caused an increased formation of 12-HETE and an unidentified metabolite group. The present study indicates that dipyridamole has no effect on the formation of the cyclo-oxygenase metabolites in human platelets but the lipoxygenase pathway seems to be stimulated by dipyridamole.

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.

Sodium salicylate interferes with the inhibitory effects of aspirin and indomethacin on human platelets.

The interference of sodium salicylate with the effects of acetylsalicylic acid (ASA, aspirin) and indomethacin on arachidonic acid-induced platelet aggregation and thromboxane formation was studied in human platelet rich plasma. ASA and indomethacin suppressed both aggregation and the concomitant formation of thromboxane B2 whereas sodium salicylate alone had no significant effect on these parameters of platelet function. It did, however, partially prevent the inhibitory effects of ASA and indomethacin on platelet aggregation when it was added to platelet rich plasma before ASA or indomethacin. The inhibition of thromboxane formation by indomethacin was also prevented by sodium salicylate. When sodium salicylate was added to platelet rich plasma after ASA or indomethacin it did not modify the effects of these drugs. The present study indicates that sodium salicylate interferes with the effects of ASA and indomethacin on human platelet cyclo-oxygenase in vitro.

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

SummaryFollowing 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 effect of bradykinin, histamine, and leukotrienes B4, C4 and D4 on the formation of 6-keto-prostaglandin F1α 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.

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 arachidonic acid on the aggregability of human platelet rich plasma

Addition of arachidonic acid to human platelet rich plasma caused a reversible aggregation, which was greatly decreased after aspirin ingestion. ADP induced a greater aggregation, which was only slightly decreased after aspirin ingestion. When PRP was incubated with arachidonic acid for 2 or 6 min before the addition of ADP, the ADP-induced aggregation was greatly decreased. This decrease was not changed by aspirin ingestion. The present study indicates that arachidonic acid is metabolized in human platelets not only to aggregatory compounds but also to anti-aggregatory compound(s). The formation of the latter compound is not inhibited by aspirin.

Glucocorticoids do not decrease thromboxane and prostacyclin levels in boom blood

Abstract Substrate availability is the rate-limiting step in the formation of oxygenated derivatives of arachidonic acid with a wide variety of biological effects. Glucocorticoids are known to induce the synthesis of phospholipase inhibitory peptides and thus prevent eicosanoid synthesis by cells (cf. 1), including prostacyclin generation by the vessel wall (2,3). Therefore, we have investigated the effect of glucocorticoids on the plasma levels of prostacyclin and thromboxane as well as on thromboxane formation during blood clotting.

The distribution of 14C-arachidonic acid in hamster lungs is sensitive to quinacrine

Isolated hamster lungs were labelled with 14C-arachidonic acid. When the lungs were ventilated with a respirator only a small amount of radioactivity was released to the perfusion effluent. This release was not changed significantly by pulmonary infusion of quinacrine (0.5 mM), a known inhibitor of phospholipase A2. After the perfusion about 75% of the radioactivity in the lungs was in phospholipids, mainly in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol and to a lesser degree in phosphatidylserine and phosphatidic acid. About one fourth of the radioactivity was in neutral lipids (tri- and diacylglycerols) and as free unmetabolized 14C-arachidonic acid. Pulmonary infusion of quinacrine increased the amount of radioactivity in diacylglycerols and phosphatidylinositol but had no effect on that in phosphatidylcholine, phosphatidylserine, phosphatidic acid and triacylglycerols. The amount of radioactivity in phosphatidylethanolamine was decreased by quinacrine and increased in the vicinity of an unidentified phospholipid-quinacrine complex. The present study indicates that the distribution of 14C-arachidonic acid in hamster lung lipids is sensitive to quinacrine. The detected changes can, however, not be explained by an overall inhibition of phospholipase A2 activities.