Modulation of PAF biosynthesis in human polymorphonuclear leukocytes. The role of phorbol esters and protein kinases.
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Humans↗
Biomedical subjects
M L Nieto
Publications and source records attributed to M L Nieto.
Occupancy of platelet receptors for platelet-activating factor in patients with septicemia.
The possible involvement of platelet-activating factor (PAF) in the pathogenesis of endotoxemia, was investigated by using a binding assay to patients' platelets, complemented with the extraction and chemical characterization of PAF obtained from patients' platelets. Platelets from 12 human volunteers had 281 +/- 63 freely accessible high affinity binding sites (PAF-receptors) per platelet; whereas this number was of 49 +/- 37 PAF-receptors per platelet, n = 14 samples, P less than 0.01, in a group of 13 patients with positive blood culture. A group of patients with respiratory or cardiovascular disturbances and negative blood culture had 253 +/- 74, accessible receptors per platelet (n = 19 samples from 16 patients, P less than 0.01 as compared to septic patients, which was not significantly different when compared to control individuals). Patients with sepsis possessed significant amounts of PAF associated to their platelets, whereas this mediator could not be isolated from platelets of patients with respiratory or cardiovascular disturbances and negative blood culture, nor from platelets of control individuals. PAF was also assayed in whole blood samples and found at high concentrations in sepsis patients. These data indicate that occupancy of PAF receptors in combination with high amounts of platelet-associated PAF, is a common finding in patients with sepsis.
Adult↗
Modulation of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase in human polymorphonuclears. The role of cyclic AMP-dependent and phospholipid sensitive, calcium-dependent protein kinases.
In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.
Acetyltransferases↗
Biosynthesis of platelet-activating factor in human polymorphonuclear leukocytes. Involvement of the cholinephosphotransferase pathway in response to the phorbol esters.
The generation of platelet-activating factor (PAF) in response to complement-coated zymosan particles, ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate (TPA) was studied in human polymorphonuclear leukocytes (PMN). TPA was an active stimulator of PAF biosynthesis but showed a time course more protracted than that observed in response to other secretagogues. TPA was not found to activate acetyl-CoA:lyso-PAF acetyltransferase activity nor to significantly enhance the incorporation of either [3H] lyso-PAF or [3H]acetate into a lipid fraction co-migrating as PAF. To assess whether TPA could be a secretagogue that promotes the biosynthesis of PAF through the dithiothreitol-insensitive cholinephosphotransferase pathway, this enzymic activity was assayed in homogenates from PMN preincubated in the presence of several secretagogues. None of the agonists was found to enhance this enzyme activity. However, TPA did enhance the incorporation of [methyl-3H]choline and both alkyl- and acetyl-labeled 1-O-hexadecyl-2-acetyl-sn-glycerol into a lipid fraction co-migrating with PAF. This incorporation showed a time course parallel to that of the generation of PAF in response to TPA. Incubation of [methyl-3H]choline-labeled cells in the presence of 1-O-hexadecyl-2-acetyl-sn-glycerol caused an enhanced incorporation of the label into the fraction co-migrating as PAF, and this incorporation was synergistically enhanced by TPA. Pulse-chase experiments with choline showed that TPA caused an early accumulation of choline into phosphatidylcholine and PAF rather than into CDP-choline. The present data indicate that TPA is the only agonist that could initiate the biosynthesis of PAF in human PMN through the cholinephosphotransferase pathway and that this process of biosynthesis is regulated at an enzyme step other than dithiothreitol-insensitive cholinephosphotransferase.
Acetyltransferases↗
Evidence of a role for PAF-acether in the pathophysiology of the shock state.
The pathophysiology of the shock state includes a variety of hemodynamic changes such as systemic hypotension, pulmonary hypertension and increased vascular permeability leading to the extravasation of protein rich plasma. These changes can be initiated by different etiological factors, but many of them have been related to the stimulation of activation systems (complement, kinins, etc.) or to the generation of inflammatory mediators. The purpose of the present study has been to obtain evidence of the involvement of paf-acether in the pathogenesis of the shock state initiated in rat and mouse by Gram-negative bacteria and soluble aggregates of immunoglobulin G. The injection of 1-2 MDa aggregates of immunoglobulin G to normal Sprague-Dawley rats, induced a dose-dependent systemic hypotension which appeared about five minutes after completion of the intravenous challenge. Simultaneously, extravasation of protein-rich plasma occurred as judged from the finding of an increased clearance of 125I-BSA. In similar experiments in mice, a reduction of the vascular volume was observed using 51Cr-labelled homologous red blood cells. Under these conditions, a lipid compound analogous to paf-acether was obtained from the liver and the spleen of these animals. The generation of this compound preceded the development of blood volume depletion and could be suppressed by either quinacrine or depletion of mononuclear phagocytes by total irradiation with 700 rads. The previous treatment of the rats with the compound BN 52021 (a specific antagonist of the paf-acether receptor) at a dose of 5mg/kg, i.v., prevented the appearance of hypotension and extravasation in response to an i.v. challenge with soluble aggregates of immunoglobulin G. Interestingly, the reversal of hypotension was also observed when BN 52021 was infused after the immunoaggregates (5mg/kg). The possible involvement of paf-acether in the hemodynamic changes of Gram-negative sepsis was studied in rats which had received an intraperitoneal inoculation of E. coli. The animals inoculated with the doses of bacteria which produced mortality showed a time- and dose-dependent increase of vascular permeability as judged from the presence of abundant peritoneal exudate and the reduction of the circulating volume. Simultaneously, significant amounts of paf-acether could be obtained from the peritoneal exudate and from the spleen preceding to the development of the circulating volume depletion.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Modulation of lyso-platelet-activating factor: acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions.
The enzyme lyso-platelet-activating factor: acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca2+ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca2+. The effect of Ca2+ was on the apparent Km of the enzyme for the substrate acetyl-CoA without showing any significant effect on the Vmax of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca2+ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca2+, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca2+ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.
Acetyltransferases↗
Inhibition of the vascular actions of IgG aggregates by BN 52021, a highly specific antagonist of paf-acether.
The effect of BN 52021, a selective antagonist of paf-acether (Braquet GB patent 8, 418, 424 July 19, 1984), was studied in normotensive rats challenged with different doses of paf-acether. Sudden death was observed in animals receiving an i.v. dose of 10 micrograms/kg of paf-acether and this was prevented by prior treatment with BN 52021 (5 mg/kg, i.v.). Animals receiving 2.5 micrograms/kg of paf-acether had a fall of mean arterial pressure of 92.5 +/- 4.7 mmHg which recovered to the prechallenge level 20.5 +/- 0.2 min thereafter. Previous treatment with BN 52021 (5 mg/kg, i.v.) reduced the mean arterial pressure fall to 47 +/- 0.9 mmHg and the time of recovery to 5.7 +/- 1.7 min. The extravasation of 125I-bovine serum albumin under the above conditions was reduced by BN 52021 from 36 +/- 3 to 18 +/- 3%. A lower dose of BN 52021 (1 mg/kg, i.v.) was also effective in reducing later extravasation, but was unable to prevent the extravasation which appears up to 10 min after the injection of paf-acether. To extend these findings to a model of endogenous production of paf-acether, other animals were challenged with soluble aggregates of human IgG (40 mg/kg, i.v.; Iñarrea et al., Immunopharmacology 6:7, 1983).(ABSTRACT TRUNCATED AT 250 WORDS)
Anaphylaxis↗
Synthesis of platelet-activating factor from human polymorphonuclear leukocytes: regulation and pharmacological approaches.
Human polymorphonuclears release a platelet-activating factor (PAF-acether) when challenged with various stimuli. PAF-acether is a mediator that is synthesized during cell activation in a process in which a phospholipase A2 and an acetyltransferase take part. These enzymes are finely regulated and accordingly PAF-acether release may be modulated. The authors have studied some of the transductory mechanisms which are triggered during cell stimulation and the effect of their pharmacological modulation on PAF-acether release. Theophylline, methylisobutylxanthine and dipyridamole, which block phosphodiesterase of cyclic nucleotides, induce a dose-dependent inhibition of PAF-acether release without affecting phagocytic uptake. Polyamines (dansylcadaverine, rimantadine and amantadine) reduced PAF-acether release and the phagocytic process in an order of potency similar to their ability to inhibit phospholipid methylation and the cholinephosphotransferase pathway. The calmodulin antagonist trifluoperazine induced a dose-dependent inhibition of PAF-acether release and acetyltransferase at concentrations from 10(-4) to 10(-5) M. Hence it appears that modulation of PAF-acether release can be obtained by different pharmacological blockades: phosphodiesterase of cyclic nucleotides, phospholipid metabolism and calcium-calmodulin.
Acetyltransferases↗
[Bronchogenic carcinoma and human immunodeficiency virus infection].
The coexistence of bronchogenic carcinoma and human immunodeficiency virus is unusual. Patients are usually young former smokers and histopathologic type is most often adenocarcinoma. We describe two cases of bronchogenic carcinoma in HIV positive individuals.
Adenocarcinoma↗