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The kinetics of 3H-labeled arachidonic acid (AA, 10—10-10—5 M) incorporation into murine peritoneal macrophages was investigated. During the incorporation of AA into the cells, the steady state was reached at 10 h. The level of incorporation consisted of 48-50% for nanomolar concentrations and 28-30% for micromolar concentrations of AA. Exogenous AA in micromolar but not nanomolar concentrations stimulated [3H]AA release from intracellular stores of pre-labeled cells. A mathematical model fitting the behavior of the experimental system is proposed. The difference in the level of uptake of AA in nanomolar and micromolar concentrations is explained by the activation of AA release from intracellular stores at high concentrations of exogenous AA.

The role of serum fatty acid binding proteins (FABPs) in arachidonic acid (AA) uptake by murine peritoneal macrophages has been studied. The kinetics of [3H]arachidonic acid uptake by the cells was investigated over a wide range of AA concentration (10−10–10−5 M). It was shown that these putative fatty acid transporters dramatically change the uptake processes. In the presence of FABPs, the time-course curves of AA uptake exhibited two distinct periods: one with a rapid AA uptake during the first hour with an equilibrium in 1–2.5 h and another with an equilibrium reached in 20 h, whereas in the absence of FABPs the uptake curves were smooth without kinks and with the equilibrium reached in 10 h. In addition, it was shown that the amount of incorporated AA was linearly dependent on the concentration of AA over the range of 10−10–10−6 M in the presence of serum FABPs and 10−10–10−7 M in their absence. We assume that the changes in the character of AA uptake by macrophages in the presence of FABP soccur due to the interaction of FABPs with the cell plasma membrane.

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 (kin), (b) prostanoid metabolism (kr), and (c) incorporation of exogenous AA into cellular membranes (ka). From our experiments and mathematical calculation these parameters were found to be kin = 0.20 +/- 0.02 min-1, kr = 0.17 +/- 0.03 min-1 in the case of stimulation with A23187, and kin = 0.0156 min-1, kr = 0. 0134 min-1, ka = 0.0025 min-1 in the case of exogenous AA addition. The studies of prostanoid biosynthesis by macrophage microsomes led to independent determination of kin = 0.20 +/- 0.02 min-1. 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.

To check the proposed hypothesis that the relative content of individual polyunsaturated fatty acids (PUFAs)--substrates and inhibitors of prostanoid synthesis--in plasma can be regarded as a quantitative risk factor of blood clotting, a test was conducted on free fatty acids content in blood plasma of healthy people (group 0) and patients with heart ischemia before (group 1) and after (group 2) they were treated for a month with a food additive called "Eiconol," enriched with PUFA omega 3. Different proportions of PUFAs have been calculated in all cases, accounting for the contribution of each acid to the process of primary clotting. Comparison of PUFA rations among the three groups showed significant differences of means between groups 0 and 1 and also group 1 and 2 for 6 out of 7 proposed coefficients, which disappeared after "Eiconol" treatment (comparison of groups 0 and 2). The results led to the conclusion that out of the proposed PUFA proportions, the coefficients describing the relative content of arachidonic acid in blood plasma may be the most informative for diagnosis and treatment efficiently in evaluation of heart and vascular diseases.

It was shown that cytosol of primary sheep vesicular gland cells inhibits peroxidase activity of prostaglandin H synthase (PGHS). The degree of the enzyme inactivation depends on cytosol concentration. It was established that cytosol contains glycoprotein haptoglobin that is one of the cytosol basic components responsible for its property to inhibit PGHS. Haptoglobin is supposed to participate in endogenous regulation of PGHS activity in sheep vesicular glands.

The changes in AA incorporation and release as well as prostanoid synthesis upon differentiation of human premonocytic cell line, U937, induced by three functionally diverse agents—phorbol ester (TPA), dimethyl sulfoxide (DMSO), and retinoic acid (RA) have been investigated. The rate of AA incorporation into the cells remained unchanged whereas a 3- to 6-fold increase in AA release upon stimulation with Ca 2+ -ionophore A23187 as compared to undifferentiated cells was observed. While undifferentiated cells were incapable to metabolise AA via the cyclooxygenase pathway all three types of differentiated U937 cells produced TxB 2 and PGE 2 . Only TPA-differentiated cells responded with a 6-fold increase of prostanoid synthesis after A23187 stimulation, whereas in DMSO-differentiated cells prostanoid synthesis was slightly stimulated by A23187 and in RA-differentiated cells it was not stimulated at all. Thus, agonist-induced prostanoid synthesis in differentiated cells is dependent on the nature of differentiating agent and does not correlate with AA liberation.

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 effect of 10 −14 − 10 −4 M ibuprofen and aspirin both on arachidonic acid metabolism in peritoneal murine macrophages and on the concanavalin A-induced proliferation of murine splenocytes were investigated. It was shown that 10 −7 − 10 −4 M ibuprofen inhibits the arachidonic acid metabolism. On the other hand, 10 −12 −10 −11 M ibuprofen causes pronounced activation of arachidonic acid metabolism. The low concentration (10 −14 − 10 −10 M) effects also take place when non-steroidal anti-inflammatory drugs influence other functions of the immune system: that is, they activate the splenocyte mitogen-induced proliferative response. These results are in accord with our suggestion that the low concentration effects of these drugs do not depend upon cell types and may have an important physiological significance.

Physiological concentrations of arachidonic acid in deproteinized serum can be determined amperometrically in the presence of prostaglandin endoperoxide synthetase and potassium hexacyanoferrate(II). A determination takes 1.5–2 min, by measuring the current at a glassy carbon electrode at 0.0 V vs. SCE.

A new method is proposed for assaying transketolase activity. The method is based on determining erythrulose, the product of the transketolase reaction, by its optical activity.

Thiamine pyrophosphate (TPP), when bound with transketolase (TK) induces some changes in the absorption of the enzyme and coenzyme which can be registered by difference spectrophotometry. The binding of a donor substrate to the binary complex give rise to changes in the absorption region of the TPP thiazolium ring and in the charge transfer spectrum. With low concentrations of hydroxypyruvate, the kinetics of these changes may be revealed. The possibility is discussed of the charge transfer complex (CTC) being involved in the catalytic reaction.

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 PGE2, PGF2a and thromboxane B2, spleen macrophages produce PGE2 and PGF2a, 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 PGE2 concentrations (10(-14)-10(-7) M). The suppressive effect of PGE2 decreased when its concentration was lowered and disappeared at 10(-9) M PGE2 (this concentration corresponded to the basal level of non-stimulated macrophage synthesis of PGE2). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE2 caused a suppressive effect comparable with that for 10(-8) M PGE2. We also found that PGE2 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 (10(-12) M) and high (10(-8) M) PGE2 concentrations. This phenomenon of PGE2 biphasic control of lymphocyte proliferation may play an important role in cellular homeostasis, in particular in immune cell function regulation.

Prostaglandin H synthase (PGHS) as apo-PGHS, holo-PGHS, and holo-PGHS, inactivated in the course of catalysis was studied using chemical modification with diethyl pyrocarbonate (DEPC). The exhausted reaction with DEPC corresponded to the modification of 7 histidine residues in apo-PGHS and 4 in holo-PGHS. All 18 histidine residues became accessible for modification with DEPC in the enzyme, inactivated in the course of catalysis. The velocities of tryptic cleavage of all the three forms into two fragments were fairly different but independent of modification. Based on the results we hypothesize fast and dramatic changes in the protein structure in the course of the substrate conversion.

The influence of 10−10–10−6 M morphine on the release of [3H]arachidonic acid and its metabolites ([3H]AAM) from prelabeled resident peritoneal murine macrophages was investigated. Morphine enhanced [3H]AAM release from A23187- and LPS-stimulated macrophages, as well as the basal release of [3H]AAM. Dose-response curves showed a maximum at 10−8 M morphine. Naloxone had no effect on morphine enhancement of [3H]AAM release. These results are in agreement with the hypothesis that [3H]AAM may be involved in the effects of morphine.