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Z A Cohn

Publications and source records attributed to Z A Cohn.

At least 127 records · Page 7Linked to original sources
PubMed1982

Characterization of a membrane pore-forming protein from Entamoeba histolytica.

We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica. The formation of ion channels by PFM was examined in three systems. (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by [3H]TPP+ uptake. (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles. (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl. Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer. Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8. PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E. coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min. High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000. This protein may play a role in the cytotoxicity mediated by E. histolytica.

Animals↗
PubMed1982

Secretion of leukotriene C and other arachidonic acid metabolites by macrophages challenged with immunoglobulin E immune complexes.

Resident mouse peritoneal macrophages release the slow-reacting substance leukotriene C (LTC) on exposure to particulate IgE immune complexes. Because these cells lose their responsiveness to an IgE stimulus after 4 h in culture, maximum release of 20:4 metabolites is observed before this time. However, a similar diminution in 20:4 metabolism was not observed with a zymosan stimulus. Freshly explanted cells are deficient in intracellular glutathione (GSH) (12.4 +/- 0.4 pmol/micrograms cell protein), but GSH increases to a steady state value of 30-35 pmol/micrograms of cell protein between 3 and 9 h of culture. Because GSH is required for the synthesis of LTC and prostaglandin (PG)E2, cultures challenged immediately after explanation have a diminished capacity to synthesize these 20:4 metabolites and release prostacyclin as the major product. By 4-5 h in culture, macrophages form significant amounts of LTC and PGE2. Under optimum conditions of maximum responsiveness to an IgE stimulus and GSH content (after 4 h of culture), macrophages challenged with latex beads coated with IgE immune complexes synthesize 1.0 +/- 0.3 pmol of LTC/microgram cell protein (60 +/- 18 pmol/10(6) cells) in addition to prostacyclin (8.2 +/- 0.8 pmol/micrograms cell protein) and PGE2 (4.7 +/- 1.5 pmol/micrograms cell protein). These amounts are quantitatively similar to the arachidonic acid metabolites produced by macrophages challenged with IgG immune complex-coated latex beads or zymosan. These data demonstrate that macrophages produce large quantities of LTC and other 20:4 metabolites in response to particle-bound IgE and antigen, provided that the appropriate in vitro conditions are met. The macrophage might, therefore, be a major source of slow-reacting substance and other 20:4 metabolites generated during IgE-mediated reactions in vivo.

Animals↗
PubMed1982

Uptake and metabolism of monohydroxy-eicosatetraenoic acids by macrophages.

Within 5 min, resting macrophages metabolize microM quantities of exogenous arachidonic acid (20:4) to cyclooxygenase and lipoxygenase products. Mono-HETEs represent a major class of metabolites recovered from the medium. However, the quantity of mono-Hetes progressively decreases over a 60-min incubation period, with a concomitant increase in more polar lipoxygenase products, suggesting additional metabolic fates for these hydroxy acids. This was directly confirmed by exposing resident macrophage cultures to radiolabeled 15-, 12-, and 5-HETEs (1 microM). 12-30% of the recovered HETEs were cell-associated and predominantly esterified into phospholipid. High pressure liquid chromatography analyses of medium extracts indicated that 50% of each HETE was also converted to 10 or more metabolites over a 60-min time-course, a rate slower than for 20:4. The major metabolite generated from each mono-HETE had the elution characteristics of a di-HETE. The 5-HETE product has a triene spectrum similar to that of 5(S), 12(S)-di-HETE, whereas the 15- and 12-HETE products exhibited single ultraviolet absorption maxima, indicating a metabolic pathway for 5-HETE distinct from the other mono-HETEs. None of the stable cyclooxygenase products of 20:4 (6-keto PGF1 alpha, PGF2 alpha, PGE2, TXB2) nor polar metabolites of mono-HETEs are either incorporated or metabolized. The results indicate that macrophages have the capacity to specifically metabolize 20:4 and mono-HETEs to polar oxygenated products in the absence of a discernible trigger.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1982

Regulation of arachidonic acid metabolism by macrophage activation.

Levels of zymosan-induced arachidonic acid (20:4) metabolism by peritoneal macrophages elicited with inflammatory agents and resident macrophages were similar. Thyioglycollate (THIO)-elicited macrophages represented the exception; however, the diminished metabolism by these cells was reproduced by exposing resident cells to 5 mg/ml THIO broth in vitro. In contrast, reduced prostaglandin synthesis by macrophages from mice variously treated with the immunologic agents, Corynebacterium parvum or Bacille Calmette Guérin (BCG), closely correlated with enhanced antitoxoplasma activity, one measure of macrophage activation. This relationship, although not causative, suggested that the capacity for 20:4 metabolism is a function of the macrophage activation state. Modulation of macrophage 20:4 metabolism in vivo apparently required factors in addition to lymphocyte-derived products. Treatment of resident macrophages in vitro with BCG lymphokine was without effect on 20:4 release or prostaglandin synthesis. Activated macrophages from animals inoculated i.p. with C. parvum exhibited reduced 20:4 release and also failed to metabolize 70% of the 20:4 released in response to a zymosan stimulus. Consequently, the quantities of 20:4 metabolites formed were significantly less than expected from 20:4 release. These activated macrophages displayed greatly reduced synthesis of prostacylcin and leukotriene C compared with other 20:4 metabolites. It appeared that factors that regulate macrophage 20:4 metabolism influence the level of the inducible phospholipase and synthetic enzymes for specific 20:4 oxygenated products.

Animals↗
PubMed1982

Synthesis of leukotriene C and other arachidonic acid metabolites by mouse pulmonary macrophages.

Mouse resident pulmonary macrophages were subdivided into alveolar (PAM) and interstitial (PTM) populations on the basis of accessibility to pulmonary lavage, and zymosan-induced arachidonic acid (20:4) metabolism was examined in both populations labeled with [3H]20:4. Maximal phagocytic doses of unopsonized zymosan induced the specific release of 11% of phospholipid 20:4 by PTM and 4.6% by PAM. Direct fatty acid analysis of [3H]20:4-labeled PTM cultured in the presence or absence of zymosan indicated that the specific activity of the [3H]20:4 in cell phospholipid provided an accurate measure of 20:4 released by the cells, and could therefore be used to quantitate the synthesis of 20:4 metabolites by PTM in vitro. The single major 20:4 metabolite of PTM was the slow-reacting substance leukotriene C, which was synthesized in quantities of 3-4 pmol/microgram cell protein (280-370 pmol/10(6) cells), and comprised 20-25% of the released 20:4. PTM also synthesized prostaglandin E2, prostacyclin, thromboxane A2, and hydroxyeicosatetraenoic acids. In contrast, PAM produced leukotrienes D and E in addition to leukotriene C, prostaglandin E2, thromboxane A2, and hydroxyeicosatetraenoic acids. Prostacyclin formation by PAM was not observed. These studies define a set of experimental conditions for the study of 20:4 metabolism by pulmonary macrophages, and demonstrate that these cells are rich sources of LTC as well as other 20:4 oxygenated products.

Animals↗
PubMed1982

Glutathione metabolism in resting and phagocytizing peritoneal macrophages.

The steady state GSH content of cultured mouse resident peritoneal macrophages was 34 +/- 5 pmol/microgram of cell protein. Intracellular GSH content decreased concomitantly with zymosan ingestion. The half-life of GSH decreased from 1.9 h in resting cells to 0.58 h during phagocytosis as determined by inhibition of GSH synthesis with buthionine sulfoximine. The decrease in GSH half-life was directly related to the extent of particle uptake. In cytochalasin D-treated cells, attachment of zymosan to the macrophage plasma membrane in the absence of particle interiorization was sufficient to stimulate GSH turnover. Efflux was the major route of GSH loss in [35S]cystine-labeled macrophages, and was enhanced 3-fold by a zymosan challenge. GSH was lost intact since resident macrophages lack gamma-glutamyl transpeptidase (less than 1 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of protein . h). Macrophages obtained from mice challenged in vivo with Corynebacterium parvum maintained higher intracellular GSH levels (50 +/- 5 pmol/microgram of cell protein) than did resident cells. The half-life of GSH in buthionine sulfoximine-treated C. parvum-elicited macrophages was 3.8 +/- 0.2 h while resting and 1.3 +/- 0.2 h during phagocytosis. C. parvum-elicited macrophages, in contrast to resident cells, contained sufficient levels of gamma-glutamyl transpeptidase activity to hydrolyze 55 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of cell protein . h. These studies indicate that phagocytosis and cellular activation have profound effects on GSH metabolism in macrophages.

Animals↗
PubMed1982

Resting macrophages produce distinct metabolites from exogenous arachidonic acid.

Resident mouse peritoneal macrophages rapidly metabolize free arachidonic acid (20:4) in the absence of a discernible trigger. After a 20-min incubation in serumless medium, one-third of the fatty acid was found esterified in cell phospholipid and two-thirds was metabolized to oxygenated products which were recovered in the culture medium. The 20:4 oxygenated metabolites were identified by reverse-phase high performance liquid chromatography as hydroxyeicosatetraenoic acids (HETEs) and 6-keto prostaglandin F(1a) (6-ketoPGF(1a)), the stable form of prostacyclin, together with prostaglandin E(2) (PGE(2)) in proportions of 67:24:9. Inhibitor studies using indomethacin, nordihydroguaiaretic acid, and 5,8,11,14-eicosatetraenoic acid confirmed these metabolites to be lipoxygenase and cyclo-oxygenase products. The proportion of products differs considerably from those generated from phospholipid 20:4 in response to a phagocytic stimulus (HETEs:6-ketoPGF(1a):PGE(2):leukotriene C, 15:25:40: 15-20). Cornyebacterium parvum-elicited macrophages incorporated a higher percentage (70 percent) of exogenously supplied 20:4 and converted less than 20 percent of the fatty acid to oxygenated metabolites. Cyclo-oxygenase products (PGE(2), PGF(2a), TXB(2), and 6-ketoPGF(1a)) represented the major 20:4 metabolites (74 percent) synthesized by these activated macrophages. Esterification of 20:4 into cell phospholipids appeared not to be an initial obligatory step for synthesis of 20:4 oxygenated products by this route. To the contrary, incorporation of 20:4 into cell lipids and metabolism via the cyclo-oxygenase and lipoxygenase pathways represent distinct metabolic fates of exogenously supplied 20:4. These observations establish that resting macrophages contain high levels of cyclo-oxygenase and lipoxygenase activity and suggest macrophages can synthesize lipid mediators of inflammation in the absence of an inflammatory stimulus.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1982

Phagocytosis and fluid-phase pinocytosis.

The generation, flow, directionality and fusion of phagocytic and fluid-phase pinocytic vesicles in cultured macrophages and fibroblasts are reviewed. Specific plasma membrane (PM) receptors, receptor mobility, contractile cytoplasmic elements and lipid composition of the PM serve to regulate the flow of large phagosomes into the perinuclear zone. Fluid-phase vesicles are constitutively generated and carry large quantities of PM, fluid and solutes into the cytoplasm. Quantitative information is cited on the rates of vesicular generation, fusion with other members of the vacuolar system, fluid and solute uptake, and digestion and solute release. The nature and composition of fluid-phase vesicles, phagocytic vacuoles and PM are compared. Once interiorized, PM and its component polypeptides rapidly cycle back to the cell surface. The flow rates of both the centrifugal and the centripetal compartments as well as the fate of a minor degradation pool are illustrated and compared to the turnover of individual membrane polypeptides. Implications of membrane flow for cell shape, motility and new PM insertion are discussed.

Animals↗
PubMed1982

IgE immune complexes stimulate arachidonic acid release by mouse peritoneal macrophages.

Resident mouse peritoneal macrophages were labeled with [3H]arachidonic acid and challenged with Sephadex beads coated with immune complexes of IgE and antigen. Arachidonic acid release by the cells was assessed by the quantity or radiolabel recovered from the culture medium. Freshly isolated macrophages responded to IgE immune complexes with a release of [3H]arachidonic acid that was linear for 1-2 hr. The magnitude of the response was dependent on both the number of immune complex-coated beads and on the degree of opsonization of the beads. Under conditions of maximal stimulation, macrophages challenged with IgE immune complex-coated Sephadex released 23 +/- 4.5% of their incorporated radiolabel. This is compared to values of 34.2 +/- 0.5% and 38.1 +/- 3.3% for cultures that received IgG immune complex-coated Sephadex or zymosan, respectively. Macrophages did not release arachidonic acid upon exposure to soluble IgE and antigen given sequentially or simultaneously, and soluble IgE did not inhibit the cells' response to IgE immune complexes. Incubation of macrophages for longer than 3 hr prior to challenge resulted in a selective loss in the cells' ability to respond to IgE immune complexes. After 16 hr of culture, macrophages released only 3.9 +/- 0.3% of their incorporated 3H on exposure to IgE immune complexes; however radiolabel release in response to zymosan (42.0 +/- 0.8%) was identical to that of freshly isolated cells. These data indicate that macrophages are capable of releasing arachidonic acid in response to preformed particulate immune complexes of IgE and antigen. Because Sephadex beads are too large to be interiorized by the cells, this response results from the interaction of the immune complexes with the macrophage plasma membrane.

Animals↗
PubMed1982

Human alveolar macrophages produce leukotriene B4.

Human alveolar macrophages obtained by bronchoalveolar lavage were labeled overnight with [3H]arachidonic acid. The cells were stimulated with calcium ionophore A23187, and the 20:4 oxygenated metabolites released into the culture medium were identified by reverse-phase HPLC. Leukotriene B4 was the major 20:4 metabolite produced by these cultures. Leukotriene B4 was identified by its reverse-phase HPLC elution time, its UV spectrum, and its chemotactic and chemokinetic activities for neutrophils. In addition, the macrophage- and neutrophil-derived leukotriene B4 free acids and methyl esters were found to have identical HPLC retention times.

Arachidonic Acid↗
PubMed1982

Arachidonic acid metabolism in glutathione-deficient macrophages.

Mouse resident peritoneal macrophages were treated with the glutathione (GSH) synthesis inhibitor buthionine sulfoximine to deplete intracellular GSH. The arachidonic acid metabolites released by the GSH-depleted macrophages in response to a zymosan challenge were analyzed by HPLC. Buthionine sulfoximine treatment resulted in inhibition of both prostaglandin E2 and leukotriene C synthesis that was directly related to the degree of GSH depletion. Macrophages in which GSH levels were reduced to 3% of normal exhibited reductions to 4% and 1%, respectively, in PGE2 and LTC formation. The total quantity of cyclooxygenase metabolites secreted by GSH-deficient macrophages was identical to that of control cells as a result of increased synthesis of prostacyclin and, to a lesser extent, 12-L-hydroxy-5,8,10-heptadecatrienoic acid. Total lipoxygenase products were decreased, however; increased formation of hydroxyicosatetraenoic acids only partially compensated for the deficit in leukotriene C production. These findings extent our earlier observations on the inhibition of leukotriene C synthesis in GSH-depleted macrophages and confirm with intact cells the previously suggested role of GSH in prostaglandin E2 formation.

Arachidonic Acid↗
PubMed1982

Intralysosomal accumulation of polyanions. I. Fusion of pinocytic and phagocytic vacuoles with secondary lysosomes.

The long-term exposure of macrophages to low concentrations of a number of polyanions leads to their accumulation in high concentration within secondary lysosomes. This was associated with enlargement of the lysosomes, the presence of membranous whorls, and intense toluidine blue staining of the organelles at pH 1.0. After the ingestion of a particulate load by these cells, newly formed phagocytic vacuoles failed to fuse with polyanion-laden lysosomes. The lack of fusion was evident in both fluorescence and electron micrographic studies which followed the transfer of acridine orange or Thorotrast from 2 degrees lysosomes to phagosomes. Agents that inhibited phagosome-lysosome (P-L) fusion included molecules containing high densities of sulfate, sulfonate, or carboxylate residues. Dextran sulfate (DS) in microgram/ml quantities was an excellent inhibitor, whereas nonsulfated dextran (D) was without effect at 1,000-fold higher concentrations. In contrast to their effects on P-L fusion, polyanions failed to influence the fusion of pinocytic vesicles with 2 degrees lysosomes. The uptake, intravacuolar distribution, and intralysosomal digestion of fluid-phase pinocytic markers were unaltered in lysosomes containing either D or DS. Furthermore, subcellular fractionation studies showed that the fluid-phase pinocytic marker HRP was efficiently transferred from pinosomes to large, dense 2 degrees lysosomes containing DS.

Animals↗
PubMed1982

Intralysosomal accumulation of polyanions. II. Polyanion internalization and its influence on lysosomal pH and membrane fluidity.

Dextran sulfate (DS) was previously shown to inhibit phagosome-lysosome (P-L) fusion whereas dextran (D) of equivalent size was ineffective. The uptake and interiorization of DS were examined with a tritiated product over the course of 4 d in culture. The exposure of macrophages to 20 micrograms/ml of 3H-DS led to linear uptake for 4 d, at which time fusion was inhibited. Macrophage interiorization of 3H-DS was greatly increased by forming insoluble complexes with either serum lipoproteins or purified human low density lipoproteins (LDL). Under these conditions fusion was inhibited within 4 h. The uptake of large quantities of acetylated LDL in the absence of DS was not associated with the inhibition of fusion. Lipoproteins therefore served as the DS carriers and were not themselves inhibitory. The intralysosomal pH of control and D-treated macrophages was 4.76 (+/-0.06) and 4.68 (+/-0.02), respectively. Storage of DS was associated with a decreased pH to 4.36 (+/-0.14). Increasing the intralysosomal pH with either NH4Cl or chloroquine failed to modify inhibited P-L fusion. Hydrogen ion concentration was therefore not an important factor in DS inhibition. Secondary lysosomes were isolated from D- and DS-loaded cells and exhibited excellent latency. These lysosomes were exposed to the membrane probes, alpha- and Beta-parinaric acid, and compared in fluorescence polarization measurements. The results with the Beta isomer consistently indicated that the membranes of DS lysosomes were more rigid than the D samples. It is suggested that high intralysosomal concentrations of DS interact directly with either lipid and/or polypeptide moieties of the luminal face of the membrane, thereby decreasing its fluidity and fusibility.

Animals↗
PubMed1982

T. cruzi: sensitization to macrophage killing by eosinophil peroxidase.

In this study, we report that trypomastigotes of T. cruzi coated with eosinophil peroxidase (EPO) become sensitized to killing by normal macrophages that are unable to kill uncoated organisms. EPO bound to the surface of the organisms without affecting their extracellular viability. The intracellular killing of EPO-coated trypomastigotes could be inhibited by catalase and azide, suggesting that toxicity was mediated through the small amounts of hydrogen peroxide generated by the phagocytic event in normal macrophages and the peroxidatic activity of EPO. EPO-coated organisms could be killed in a cellfree system by the addition of H2O2 and either iodide, bromide, or chloride. Omission of H2O2 decreased but did not prevent the killing of trypanosomes by the cellfree system and this residual toxicity was abolished by catalase. This suggests that H2O2 generated by trypanosomes contributes to the death of EPO-coated organisms. EPO-coated organisms could also be killed extracellularly when exposed to normal macrophages at high parasite to cell ratios or when a high phagocytic load of another particle was given simultaneously. This effect could be inhibited by both azide and catalase, but not by superoxide dismutase. This suggests that enough H202 is released by phagocytosis of a high number of organisms to generate toxic concentrations of H2O2 outside the confines of the vacuolar system.

Animals↗
PubMed1982

Trypanosoma cruzi: induction of microbicidal activity in human mononuclear phagocytes.

Antigen-stimulated peripheral blood mononuclear cells from 14 patients with chronic Chagas' disease were examined for their ability to generate soluble factor(s) capable of activating human macrophages to a microbicidal state. Mononuclear cell factors (MCF) from all but one patient were capable of inducing macrophages to a state where they were able to kill trypomastigotes of Trypanosoma cruzi. Macrophage microbicidal activity against this organism was nonspecific, because it could be induced by lymphokine from PPD-positive subjects exposed to heat-killed BCG or by concanavalin A stimulation of normal donors cells. A factor(s) generated by the stimulation of mononuclear cells from normal donors by T. cruzi antigen did not induce macrophage microbicidal activity. Opsonization of the organisms with specific IgG did not alter their fate in normal macrophages, but enhanced their killing in MCF-activated cells. Induction of macrophage activation in the human system differed from the results previously described in mice in a few features: 1) Optimal microbicidal activity did not require daily addition of the soluble factors. 2) The MCF dose-response curve was shifted to lower concentrations. 3) MCF activity generated by antigen-stimulated peripheral blood lymphocytes correlates with their proliferative responses to antigen. Half of the patients showed low proliferative responses and correspondingly lower MCF activity. Mitogen responses were normal in all patients. No correlation was found between low or high responses and clinical manifestations of disease.

Animals↗
PubMed1981

Antitumor effects of hydrogen peroxide in vivo.

Glucose oxidase, covalently coupled to polystyrene microspheres (GOL), produced H(2)0(2) at an average rate of 3.6 nmol/min per 10(9) beads under standard assay conditions. Injection of 1.3 x 10(10) to 1.1 x 10(11) GOL i.p. prolonged the survival of mice by 27 percent after injection of 10(6) P388 lymphoma cells in the same site, consistent with destruction of 97.6 percent of the tumor cells. Placing mice for several hours in 100 percent O(2), the probable rate-limiting substrate for GOL, afforded a 42 percent prolongation of survival from P388 lymphoma, consistent with destruction of 99.6 percent of the tumor cells. When the P388 inoculum was 10(5), 10(4), or 10(3) cells, GOL led to long-term survival (presumed cure) of 23 percent, 77 percent, and 92 percent of the mice, respectively, consistent with reduction of the injected tumor dose to less than 10 cells. Subcutaneous growth of 10(5) P388 cells (approximately 300 lethal dose to 50 percent of mice) was suppressed in 83 percent of mice by admixture of GOL with the tumor cell inoculum. GOL alone had no effect against a more peroxide-resistant tumor, P815 mastocytoma. However, P815 cell glutathione reductase could be inhibited in vivo by well-tolerated doses of the antitumor agent, 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU). BCNU alone cured few mice with P815. Together, BCNU and GOL apparently cured 86 percent of mice injected with 10(6) P815 cells i.p. The protective effect of GOL was abolished by boiling it to inactivate the enzyme, by co-injection of catalase coupled to latex beads, or by delaying the injection of tumor cells for 3 h, by which time the beads had formed aggregates. Soluble glucose oxidase, in doses threefold higher than that bound to GOL, had no detectable antitumor effect. A single injection of preformed H(2)0(2) readily killed P388 cells in the peritoneal cavity, but only at doses nearly lethal to the mice. In contrast, GOL had very little toxicity, as judged by the normal appearance of the mice for over 400 d, gross and microscopic findings at autopsy, and various blood tests. GOL injected i.p. remained in the peritoneal cavity, where it was gradually organized into granulomata by macrophages, without generalized inflammation. Thus, an H(2)0(2)-generating system confined to the tumor bed exerted clear- cut antitumor effects with little toxicity to the host.

Animals↗
PubMed1981

Phorbol myristate acetate stimulates phagosome-lysosome fusion in mouse macrophages.

The effect of the tumor promoter phorbol myristate acetate (PMA) on phagosome-lysosome (P-L) fusion in mouse macrophages has been studied using a previously described (10) fluorescence assay. Treatment with 0.1--1.0 microgram PMA/ml caused a striking increase in the rate and extent of P-L fusion. Exposure of cells to phorbol, free myristate, or the monoesters of PMA did not reproduce this effect. Macrophages required from 2 to 3 h of pretreatment to express maximal P-L fusion, and this was maintained for at least 20 h when cells were returned to PMA-free medium. Catalase, superoxide dismutase, indomethacin, and hydrocortisone, agents that are known to block the effect of PMA on H2O2, O2-, prostaglandins, or plasminogen activator, did not affect the stimulation of P-L fusion by PMA. The protein-synthesis inhibitors puromycin and cycloheximide did block the PMA effect under conditions in which the high fusion rate of 4-d cells was not affected. Labeled PMA was rapidly taken up by macrophages, with a plateau of uptake at approximately 3 h. When cells were returned to PMA-free medium, cel-associated label was rapidly released, returning to background level within 1 h. The released label was found to be a metabolite of PMA by thin-layer chromatography. This product migrated between the monoester phorbol-12-myristate and free phorbol. Rapid metabolism of PMA was also observed by a macrophage cell line, J774, and, to a lesser extent, by primary rat embryo fibroblasts.

Animals↗