Prostaglandins (PGs) have an important physiological role in the modulation of various cell immune functions. The main sources of PGs during immune responses are monocyte cells. We report here the ability of non-stimulated macrophages to synthesize prostanoids and show that peritoneal mouse macrophages synthesize PGE 2 , PGF 2a and thromboxane B 2 , spleen macrophages produce PGE 2 and PGF 2a , and in a fresh medium this synthesis reaches a constant basal level in a few hours. We studied the kinetics of Con A-induced proliferation of murine splenocytes under the influence of a wide range of PGE 2 concentrations (10 14 -10 7 M). The suppressive effect of PGE 2 decreased when its concentration was lowered and disappeared at 10~9 M PGE 2 (this concentration corresponded to the basal level of non-stimulated macrophage synthesis of PGE 2 ). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE 2 caused a suppressive effect comparable with that for 10 8 M PGE 2 . We also found that PGE 2 significantly inhibited cell proliferation when it was added 1 h before the addition of mitogen, as compared with simultaneous mitogen addition. The effect was obtained for both low (
The interest in the prostaglandin (PG) synthesis by animal cells today grows steadily because of the difficulties in obtaining them by any other way. Murine peritoneal macrophages can under certain conditions synthesize large amounts of PGs. The effect of well-known nonsteroidal anti-inflammatory drug ibuprofen on PG synthesis by the cells using a high-performance liquid chromatography (HPLC) method with fluorescence detection of 4-bromomethyl-7-methoxy-coumarin (BrMMC) derivatives was studied. In our case, the main metabolites were PGE2 and PGF2a. The PG synthesis activation effect was shown by ibuprofen concentrations in the 10(-10)-10(-14) M range with the maximum effect of 10(-12)M. In this case, the ibuprofen effect was comparable in value with the effect of well-known cell PG synthesis activator--calcium ionophore A23187. Although the exact mechanism of such an effect is not clear at the moment, at low concentration, ibuprofen itself is able to activate PG synthesis in murine peritoneal macrophages.
The dependence of prostanoid synthesis on the nature of free arachidonic acid (AA) appearance was investigated in mouse peritoneal macrophages. AA delivery from intracellular sources to the constitutive prostaglandin (PG)H synthase was provided by action of calcium-ionophore A23187; and from extracellular sources by AA addition to the culture medium. It was found that the kinetics of prostanoid synthesis dramatically depends on the sources of AA. Free AA concentration used for prostanoid synthesis is either a constant or a variable value depending upon the sources. The kinetics of cellular prostanoid synthesis can be regulated by the following processes: (a) the irreversible inactivation of PGH-synthase in the course of the reaction (k in ), (b) prostanoid metabolism (k r ), and (c) incorporation of exogenous AA into cellular membranes (k a ). From our experiments and mathematical calculation these parameters were found to be k in = 0.20^0.02 min 21 , k r = 0.17^0.03 min 21 in the case of stimulation with A23187, and k in = 0.0156 min 21 , k r = 0.0134 min 21 , k a = 0.0025 min 21 in the case of exogenous AA addition. The studies of prostanoid biosynthesis by macrophage microsomes led to independent determination of k in = 0.20^0.02 min 21 . This value perfectly fits the kinetics of the prostanoid cell synthesis under endogenous AA supply but shows a 10-fold decrease in the case of exogenous AA supply. Our study on the kinetics of prostanoid synthesis by mouse peritoneal macrophages clearly demonstrate that AA is able to regulate cellular prostanoid synthesis in the presence of constitutive PGH-synthase only. A regulation mechanism based on the co-operation of the constitutive PGH-synthase isoform and the availability of free AA is proposed and could be confirmed by mathematical modelling.Keywords: arachidonic acid; mathematical modelling; prostanoids; peritoneal macrophage; prostaglandin-H synthase Prostanoids, the oxygenated metabolites of arachidonic acid (AA), mediate a wide variety of physiological processes, including ovulation, hemostasis, platelet aggregation, kidney water balance and immune responses [1,2]. Prostanoid production is thought to be regulated by both prostaglandin (PG) H synthases and phospholipases, main of them are considered to be phospholipase A 2 (PLA 2 ) [3±5]. To date, more than 10 forms of PLA 2 have been identified in mammals, among which secretory PLA 2 (s-PLA 2 ) and cytosolic PLA 2 (c-PLA 2 ) have been paid considerable attention [6±9]. Phospholipases release AA from membrane phospholipids then PGH-synthase converts free AA to PGH 2 , the common precursor for all prostanoids. PGH 2 is then enzymaticaly transformed into various prostanoid types, such as PGE 2 , PGF 2a , prostacyclins and thromboxanes.PGH synthase, the rate limiting enzyme in the conversion of free AA to prostanoids, has two distinct activities: a cyclooxygenase activity which catalyses the formation of PGG 2 from arachidonate and a peroxidase activity which reduces the hydroperoxide group of PGG 2 to form PGH 2 ....
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