Kinetics of prostanoid synthesis by macrophages is regulated by arachidonic acid sources

Abstract

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 ....

Pathway-oriented profiling of lipid mediators in macrophages

“…3). In the previous report by Gonchar et al [21], the contribution of COX self-inactivation in the kinetics of PG production has been discussed. Their observation that PG production plateaus by 5-10 min agrees with our results.…”

Strategy for NSAID administration to aspirin-intolerant asthmatics in combination with PGE2 analogue: a theoretical approach

“…Two main metabolic pathways are taken into account; cyclooxygenase and lipoxygenase pathway [1,26]. In the cyclooxygenase pathway, AA is transformed into anti-(aiPGs) and pro-(piPGs) inflammatory prostaglandins by the enzymes prostaglandin H synthase 1 (PGHS1) and prostaglandin H synthase 2 (PGHS2) [5,14,27]. Along the lipoxygenase pathway, AA is first transformed into leukotriene A 4 (LTA 4 ) by the enzyme 5-lipoxygenase .…”