calcimycin and 4-bromophenacyl-bromide

Topics: 5,8,11,14-Eicosatetraynoic Acid; Acetophenones; Acetylcholine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Catechols; Endothelium; Histamine; Hydroquinones; Hypoxia; Masoprocol; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; Prostaglandins; Quinacrine; Serotonin; Substance P; Thrombin

Activation of cytosolic phospholipase A2 (cPLA2) by TNF has been shown to be an important component of the signaling pathway leading to cell death. The role of cPLA2 in the cytotoxic action of TNF was investigated in a panel of human leukemic cell lines. TNF could activate cPLA2 only in U937 and HL60 TNF-sensitive leukemic cells, but not in KG1a, CEM, and CEM/VLB100 cells that are relatively resistant to TNF. Pretreatment with 4-bromophenacyl bromide, a cPLA2 inhibitor, rendered U937 and HL60 cell lines resistant to the cytotoxic effect of TNF. Immunoblot and reverse-transcriptase PCR demonstrated that cPLA2 expression was detectable at both transcriptional and translational levels in all leukemic cell lines studied, although CEM and CEM/VLB100 cells expressed cPLA2 mRNA and protein at lower levels. The protein synthesis inhibitor, cycloheximide, increased TNF-induced cPLA2 activity and cytotoxicity in both CEM and CEM/VLB100 cell lines. Low levels of cPLA2 activity in the KG1a cell line could be activated by the cPLA2 activator mellitin, or the calcium ionophore A23187. The data suggest that cPLA2 activity is involved in TNF-induced cytotoxicity in leukemic cells. Resistance to TNF-induced cytotoxicity may involve either protein inhibitors that act upstream of cPLA2 in the TNF-signaling pathway or constitutive defects of cPLA2 itself, possibly involving calcium utilization.

Topics: Acetophenones; Antigens, CD; Apoptosis; Arachidonic Acid; Calcimycin; Cytosol; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; HL-60 Cells; Humans; Leukemia; Melitten; Phospholipases A; Phospholipases A2; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Tumor Necrosis Factor-alpha

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN(-)-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca2+]i (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN(-)-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN(-)-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A2, induced the envelope formation, acid production, augmentation of CN(-)-insensitive and sensitive respiration, but did not cause any increase in [Ca2+]i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A2 induced by Ca2+ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN(-)-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca(2+)-triggered sequential reactions other than Ca(2+)-caused activation of phospholipase A2.

Topics: Acetophenones; Animals; Calcimycin; Calcium; Cyanides; Enzyme Activation; Enzyme Inhibitors; Female; Fertilization; Ionophores; Male; Melitten; Ovum; Oxygen Consumption; Phospholipases A; Phospholipases A2; Sea Urchins

Serum-cultured rat W256 carcinosarcoma cells of the monocytoid origin undergo rapid apoptosis in response to the lipoxygenase inhibitor NDGA (nordihydroguaiaretic acid). Exogenous arachidonic acid (AA), in a time- and dose-dependent fashion, suppressed NDGA-induced W256 cell apoptosis as well as DNA fragmentation, with the maximal effect observed at approximately 25 microM. Mobilization of endogenous AA by calcium ionophore A23187 provided an even stronger and longer-lasting protection against NDGA-caused cell death. The A23187 effect on AA release as well as W256 cell death can be blocked by bromophenacyl bromide, thus suggesting involvement of phospholipase A2 activation. Serum withdrawal similarly caused W256 cells to undergo typical apoptosis, which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation-induced W256 cell apoptosis and significantly extended cell survival in a dose-dependent manner. Lipoxygenase products, 12(S)- and 15(S)-, but not 5(S)-hydroxyeicosatetraenoic acid (HETE), in a dose-dependent fashion, also prevented both NDGA- and serum-starvation-induced W256 cell apoptosis. AA appears to suppress W256 cell apoptosis via distinct signaling pathway(s) since it does not prevent cell death triggered by several other inducers. Examination of a panel of polyunsaturated fatty acids revealed that alpha-linolenic and linoleic acid can also suppress NDGA-induced W256 cell apoptosis. Our data suggest that AA and other polyunsaturated fatty acids and/or their metabolites may enhance tumor growth not only by promoting cell proliferation but also by suppressing apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acetophenones; Animals; Apoptosis; Arachidonic Acid; Calcimycin; Carcinosarcoma; Cell Line; Cell Survival; Cytokines; DNA Fragmentation; Fatty Acids, Nonesterified; Growth Substances; Hydroxyeicosatetraenoic Acids; Kinetics; Masoprocol; Rats; Tumor Cells, Cultured

The roles of phospholipase A2, 5-lipoxygenase and cyclooxygenase in activation of "oxidative burst" in human neutrophils have been studied under isoosmotic (320 mOsM), hypoosmotic (200 mOsM) and hyperosmotic (420 mOsM) conditions. The oxidative burst induced by phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 and opsonized zymosan was followed by luminol-dependent chemiluminescence (CL). There were no differences in the concentration dependences of CL inhibition at 320 mOsM and 200 mOsM in the presence of the phospholipase A2 inhibitor, p-bromphenacylbromide. Contrariwise, at 420 mOsM the sensitivity to inhibition was decreased by PMA and A23187 but increased by opsonized zymosan. A change in the medium osmolality in the presence of the 5-lipoxygenase inhibitor quercetin did not result in the modification of the concentration dependence of CL inhibition induced by any of the activators. In the presence of the cyclooxygenase inhibitor, meclofenamic acid, the "oxidative burst" induced by PMA and A23187 at 200 mOsM was manifested as a decrease of the sensitivity to inhibition, while at 420 mOsM this sensitivity was increased in comparison with that observed under isoosmotic conditions. The data obtained suggest that phospholipase A2 and cyclooxygenase play a role in the mechanism of the modulating effect of the medium osmolality on the "oxidative burst" in neutrophils, while 5-lipoxygenase is unimportant for this process.

Topics: Acetophenones; Arachidonate 5-Lipoxygenase; Calcimycin; Humans; Lipoxygenase Inhibitors; Meclofenamic Acid; Neutrophils; Osmolar Concentration; Oxygen; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Quercetin; Respiratory Burst; Tetradecanoylphorbol Acetate; Zymosan

Both IFN-alpha/beta and IFN-gamma have recently been demonstrated to induce a rapid but transient activation of phospholipase A2 (PLA2) in BALB/c 3T3 fibroblasts and a human neuroblastoma cell line. We report that IFN-gamma induces the synthesis and prolonged activation of cytosolic phospholipase A2 (cPLA2) in a human bronchial epithelial cell line (BEAS 2B). Treatment of the cells with IFN-gamma (300 U/ml) increased the release of [3H]arachidonic acid (AA) from prelabeled cells with a maximal effect at 12 h after stimulation. The increased [3H]AA release was inhibited by the PLA2 inhibitor p-bromophenacyl bromide (10(-5) M). Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the IFN-gamma-treated cells. Subcellular enzyme activity assay revealed that IFN-gamma increased PLA2 activity in both the cytosol and membrane fractions with a translocation of the cPLA2 to cell membranes in a Ca(2+)-free cell lysing buffer. Treatment with IFN-gamma also induced the release of 15-HETE, an arachidonic acid metabolite. Immunoblot showed that IFN-gamma induced the synthesis of cPLA2 protein. Nuclear run-on assay demonstrated that IFN-gamma initiated cPLA2 gene transcription within 15 min, and this effect was sustained at 4 h and returned to near control level at 12 h. The cPLA2 mRNA level was assayed by reverse transcription and PCR. IFN-gamma was found to increase the cPLA2 mRNA after 2-24 h treatment. Furthermore, the IFN-gamma induced cPLA2 mRNA increase was blocked by inhibitors of protein kinase C and calcium/calmodulin-dependent protein kinases, suggesting the involvement of these protein kinases in IFN-gamma-induced gene expression of cPLA2. This study shows that IFN-gamma induces the synthesis and prolonged activation of cPLA2.

Topics: Acetophenones; Arachidonic Acid; Base Sequence; Bronchi; Calcimycin; Cell Line; Cell Membrane; Cell Nucleus; Cytosol; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epithelium; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Interferon-gamma; Kinetics; Molecular Sequence Data; Phospholipases A; Phospholipases A2; Polymerase Chain Reaction; Protein Processing, Post-Translational; RNA, Messenger; Transcription, Genetic

1. The patch-clamp technique of whole-cell current recording was applied to single, enzymatically isolated, rat pancreatic acinar cells to investigate the current responses evoked by internal perfusion of inositol polyphosphates (InsPx). The InsPx were included in the solution filling the recording pipette and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3; 10 microM) evoked transient current responses generally of less than 1 min duration, inositol 2,4,5-trisphosphate (Ins(2,4,5)P3; 10 microM) evoked smaller current transients while inositol 1,3,4,5-tetrakisphosphate (InsP4; 10 microM) evoked no detectable current response. However, in the presence (in external bathing solution) of the phospholipase A2 inhibitor 4-bromophenacyl bromide (4-BPB; 8 microM) all three of the InsPx now evoked prolonged current responses lasting for several minutes. The current responses to all three InsPx were abolished by inclusion of the Ca2+ chelator EGTA (5 mM) in the internal, pipette-filling solution indicating that the responses are calcium dependent and reflect the effect of the InsPx in increasing intracellular Ca2+. Inositol 1,3,4,5,6-pentophosphate (InsP5) induced no current response when tested up to 20 microM in the presence or absence of 4-BPB. 2. The potentiating effect of 4-BPB on the InsPx-induced current responses was not mimicked by application of arachidonic acid (AA) oxidation inhibitors; indomethacin (20 microM), nordihydroguaiaretic acid (20 microM) or proadifen (SKF525A, 100 microM). The effects of 4-BPB were countered however, by the inclusion of 2 microM AA in the external solution. The results suggest that the 4-BPB potentiates the response by inhibiting the activity of phospholipase A2, thereby reducing the formation of AA. 3. In the presence of 4-BPB (8 microM) the InsPx-evoked responses were dose dependent with an increase in both the amplitude and speed of onset with increasing concentrations. In the presence of 4-BPB InsP4 was as efficient as Ins(1,4,5)P3 both in terms of speed of onset and amplitude of responses; the efficacy and dissociation constant (Kd) for both of these InsPx were the same at 1 microM and 45 nM respectively. Ins(2,4,5)P3 was always less effective, with an efficacy and Kd of 10 microM and 750 nM respectively. 4. If 4-BPB was applied after the current responses evoked by the InsPx were over, or if guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was included in the recording pipette then the phospholipase inhibitor gave rise to

Topics: Acetophenones; Animals; Arachidonic Acid; Calcimycin; Calcium; Calcium Channels; Cytosol; Electrophysiology; Guanosine 5'-O-(3-Thiotriphosphate); In Vitro Techniques; Inositol Phosphates; Kinetics; Male; Pancreas; Phospholipases A; Phospholipases A2; Rats; Rats, Wistar

A mechanism by which vasopressin enhances phospholipase A2 activation in rabbit platelets was investigated. Stimulation of the platelets with vasopressin enhanced arachidonic acid liberation, as well as aggregation and ATP secretion in the presence of submaximal concentration of A23187, although vasopressin alone had no effect. The vasopressin-enhanced liberation was inhibited by p-bromophenacyl bromide, a phospholipase A2 inhibitor, and by genistein, a tyrosine kinase inhibitor. Though epinephrine also caused a similar enhancement of the liberation, this effect of epinephrine was insensitive to genistein. Staurosporine, a protein kinase C inhibitor, completely suppressed phorbol 12-myristate 13-acetate-enhanced arachidonic acid liberation, but suppressed the vasopressin-induced enhancement only slightly. These results suggest that vasopressin-enhanced phospholipase A2 activation may be regulated by a genistein-sensitive mechanism, most likely by a protein tyrosine kinase-mediated pathway, but not by guanine nucleotide-binding protein- or protein kinase C-mediated pathway.

Topics: Acetophenones; Adenosine Triphosphate; Alkaloids; Animals; Arachidonic Acid; Blood Platelets; Calcimycin; Drug Synergism; Epinephrine; Genistein; Isoflavones; Phospholipases A; Phospholipases A2; Platelet Aggregation; Protein Kinase C; Protein-Tyrosine Kinases; Rabbits; Staurosporine; Tetradecanoylphorbol Acetate; Vasopressins

Typhus rickettsiae were incubated with mouse exudative polymorphonuclear leukocytes (PMN), and supernatants were examined for leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) secretion by radioimmunoassay. PMN incubated with native rickettsiae secreted significantly more LTB4 and PGE2 than did those incubated with buffer alone. Autacoid secretion was dependent on both the time of PMN incubation with rickettsiae and the number of rickettsiae present in the incubation suspension. Rickettsial stimulation of LTB4 secretion was associated with rickettsial hemolytic activity; treatments which inactivated the rickettsial hemolysin abolished the ability of rickettsiae to stimulate PMN LTB4 secretion. Trifluoperazine, which did not alter the rate of phagocytosis of rickettsiae by PMN, stimulated rickettsial effects on secretion of both LTB4 and PGE2 but inhibited the PMN LTB4 response to A23187. This suggested that the PMN response to rickettsiae and to the calcium ionophore involved differing mechanisms of activation. Finally, rabbit antirickettsial antiserum, which inhibited rickettsial hemolysis and was opsonic, did not block the effects of rickettsiae on PMN LTB4 secretion.

Topics: Acetophenones; Animals; Antibodies, Bacterial; Calcimycin; Dinoprostone; Leukotriene B4; Mice; Mice, Inbred BALB C; Neutrophils; Rickettsia; Trifluoperazine

a simple gas chromatographic method for the assay of phospholipase A2 (PLA2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA2 activity in cell-free preparations derived from physically disrupted human neutrophils.

Topics: Acetophenones; Arachidonic Acids; Blood Platelets; Calcimycin; Chromatography, Gas; Fatty Acids, Unsaturated; Humans; Kinetics; Neutrophils; Phospholipases A; Phospholipases A2; Serum Albumin, Bovine; Tritium

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 15S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca2+ concentration [( Ca2+]i) induced by the Ca2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca2+]i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca2+]i rise triggered by the Ca2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca2+]i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 microM whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca2+]i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pHi): the former elevated the pHi while the latter induced a pronounced acidification of the cytoplasm. This difference in the pHi responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca2+]i signal to LO inhibitors.

Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Cyclooxygenase Inhibitors; Hydroxyeicosatetraenoic Acids; Indomethacin; Ion Channel Gating; Ionomycin; Masoprocol; Melitten; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; T-Lymphocytes

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostagl

Topics: Acetophenones; Calcimycin; Cell Line; Dinoprostone; Fatty Acids, Unsaturated; Kinetics; Macrophages; Melitten; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Substrate Specificity; Terpenes

Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Clone Cells; Complement Membrane Attack Complex; Complement System Proteins; Glioma; Humans; Hybrid Cells; Isoquinolines; Neuroglia; Oligodendroglia; Phosphatidylinositols; Piperazines; Protein Kinase C; Quinacrine; Rats

The present study investigates the effect of described nonsteroidal phospholipase inhibitors [mepacrine, papaverine, trifluoperazine (TFP), p-bromophenacyl bromide (pBPB), compound CB 874] and of glucocorticoids on endothelium-dependent and -independent relaxations of rabbit aorta. Endothelium-dependent relaxations were elicited by acetylcholine, Ca2+ ionophore A23187, melittin, and thimerosal. These four agents also stimulated vascular prostacyclin formation. Prostacyclin does not relax rabbit aorta; it was measured as an indicator of phospholipid hydrolysis. All putative nonsteroidal phospholipase inhibitors blocked acetylcholine-induced relaxations. Since papaverine did not inhibit prostacyclin production, it cannot be considered a phospholipase inhibitor in this tissue. Relaxations in response to A23187 were blocked only by pBPB and CB 874, which can interact with phospholipases directly. Melittin-induced relaxations were suppressed by mepacrine, TFP, pBPB, and CB 874. Relaxations elicited by thimerosal were not affected by mepacrine, but were abolished by the other three inhibitors. All inhibitors were ineffective against endothelium-independent relaxations induced by glyceryl trinitrate. Effective blockade of endothelial relaxations correlated with inhibition of prostacyclin formation. In the presence of glucocorticoids, no inhibition of endothelium-mediated relaxations and no inhibition of prostacyclin formation occurred, indicating unimpaired phospholipase activity. These findings suggest that cleavage of phospholipids may be important in the triggering of the production of endothelium-derived relaxing factor.

Topics: Acetophenones; Acetylcholine; Animals; Aorta; Biological Products; Calcimycin; Epoprostenol; Female; Glucocorticoids; In Vitro Techniques; Male; Nitric Oxide; Nitroglycerin; Phospholipases; Phospholipids; Rabbits; Trifluoperazine; Vasoconstriction; Vasodilation

Prostaglandin synthesis by isolated rat renal cortical tubular cells was studied in vitro with a superfusion system. The cells were introduced in Teflon chambers and intermittently stimulated. The PGE2 production was measured in the effluent. ANG II (10(-10)-10(-6)M) induced a dose-dependent increase in PGE2 synthesis. Saralasin antagonized the response to ANG II. Hyperosmolar mannitol or NaCl and Ca2+-ionophore A23187 also stimulated PGE2 synthesis. The PGE2 response to all stimuli was blocked in Ca2+-free media containing EGTA. The Ca2+-channel blocker nifedipine (10(-10)-10(-6)M) did not significantly inhibit the PGE2 response to ANG II, hyperosmolar mannitol or NaCl, and A23187, whereas the phospholipase-inhibitors p-bromophenacyl bromide (10(-4)M) and chloroquine (10(-4)M) inhibited the response. Thus, PGE2 synthesis in response to these stimuli in rat renal cortical tubular cell is a Ca2+-dependent process, acting via phospholipases by a mechanism which does not appear to involve voltage-dependent Ca2+-channels.

Topics: Acetophenones; Angiotensin II; Animals; Calcimycin; Chloroquine; Dinoprostone; Female; Hypertonic Solutions; Kidney Tubules, Proximal; Mannitol; Phospholipases A; Prostaglandins E; Rats; Rats, Inbred Strains; Saline Solution, Hypertonic

The effect of 5-lipoxygenase products of arachidonic acid on 14-C-alkyl-acetyl-glycero-phosphocholine (14C-alkyl-acetyl GPC) production in rat peritoneal macrophages was investigated, using macrophages prelabeled with N-methyl-14C-alkyl-lyso-glycero-phosphocholine (14C-alkyl-lyso GPC) (prelabeled macrophages). Bromophenacyl bromide (BPB: phospholipase A2 inhibitor), and AA861 (5-lipoxygenase inhibitor) suppressed the production of 14C-alkyl-acetyl GPC in the A23187-stimulated prelabeled macrophages in a dose-dependent manner. A23187-induced hydrolysis of 14C-alkyl-acyl-glycero-phosphocholine (14C-alkyl-acyl GPC) and formation of 14C-alkyl-lyso GPC were also reduced by BPB and AA861. However, indomethacin (IND: cyclo-oxygenase inhibitor) had no significant effect on 14C-alkyl-acetyl GPC production in the A23187-stimulated prelabeled macrophages. Exogenously supplied 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) reversed the inhibitory effect of AA861 on 14C-alkyl-acetyl GPC production in A23187-stimulated prelabeled macrophages. Reduced hydrolysis of 14C-alkyl-acyl GPC and formation of 14C-alkyl-lyso GPC in A23187-stimulated prelabeled macrophages, which were pretreated with AA861, were also reversed by the addition of 5-HPETE and 5-HETE. However, LTB4 had no such effects. 5-HPETE and 5-HETE augmented the stimulatory effect of A23187 on 14C-alkyl-acetyl GPC production in prelabeled macrophages, while they could not stimulate alkyl-acetyl GPC production in the absence of A23187. These results suggest that 5-lipoxygenase products, especially 5-HPETE and 5-HETE, may play an important role in alkyl-acetyl GPC production in rat peritoneal macrophages.

Topics: Acetophenones; Animals; Arachidonic Acids; Benzoquinones; Calcimycin; Carbon Radioisotopes; Dose-Response Relationship, Drug; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Macrophages; Male; Platelet Activating Factor; Quinones; Rats; Rats, Inbred Strains

The activity of cyclic AMP-dependent protein kinase (cyclic AMP-PK) was significantly higher (P less than 0.001) in thioglycollate-elicited than in resident rat peritoneal macrophages. The activity ratio of the enzyme (its activity in the absence of added cyclic AMP divided by that in the presence of 5 microM cyclic AMP) was similar in the two cell types. The divalent ion ionophore A23187 induced a rapid increase in the activity ratio of cyclic AMP-PK in both macrophage types. This effect was blocked by pretreating the cells with indomethacin or aspirin (inhibitors of cyclo-oxygenase) and bromo-phenacyl bromide (an inhibitor of phospholipase A2), implicating the synthesis of a prostanoid as an intermediary step. Prostaglandin (PG) E2, 8-bromo cyclic AMP and cholera toxin, all of which inhibit chemiluminescence and/or PG formation in macrophages, increased the activity ratio of cyclic AMP-PK in these cells. We propose that the activation of cyclic AMP-PK plays a central role in the response of macrophages to both endogenously-generated and exogenously added PGE.

Topics: Acetophenones; Animals; Aspirin; Calcimycin; Enzyme Activation; Indomethacin; Kinetics; Macrophages; Protein Kinases; Rats; Thioglycolates

The present experiments were undertaken to investigate the role of phospholipase A2 (PLA2) activation in histamine release from human basophils. A PLA2 inhibitor, P-bromophenacyl bromide (BPB), inhibited IgE-mediated anti-IgE-induced histamine release from human basophils with a concentration of drug required to produce 50% inhibition (IC50) of 1.5 x 10(-6) M when leukocytes were preincubated with this agent for 15 min. Histamine release induced by calcium ionophore A23187 and formyl-L-methionyl-L-leucyl-L-phenylalanine was also blocked by BPB with IC50 of 4.1 x 10(-6) M, and 3.5 x 10(-6) M, respectively. A PLA2 activator, 12-0-tetradecanoylphorbol-13-acetate (TPA) caused basophil histamine release with a dose-dependent fashion. BPB inhibited TPA-induced histamine release (IC50: 2.5 x 10(-6) M). However, another PLA2 activator, melittin, and PLA2 did not release histamine through non-cytotoxic mechanisms. Collectively, these results suggest that PLA2 activation plays a central role in histamine release from human basophils via generation of lysophosphatidylcholine or products of the lipoxygenase pathway of arachidonic acid metabolism.

Topics: Acetophenones; Basophils; Calcimycin; Histamine Release; Humans; Melitten; N-Formylmethionine Leucyl-Phenylalanine; Phospholipases; Phospholipases A; Phospholipases A2; Tetradecanoylphorbol Acetate

Horse eosinophils stimulated with the calcium ionophore A23187 were examined by transmission and scanning electron microscopy. Secretion was characterized by granule movement to the cell periphery and fusion of adjacent granules. The granules became swollen and less electron-dense as their contents were released into large intracellular vacuoles, which opened to the outside of the cell through surface pores. A23187-induced eosinophil peroxidase (EPO) release, as measured by guaiacol oxidation, was blocked by eicosa-5,8,11,14-tetraynoic acid (ETYA) (which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism) but not by indomethacin (which inhibits only the cyclooxygenase pathway). Highly purified porcine phospholipase A2 induced noncytotoxic eosinophil degranulation (as measured by the release of EPO without the concomitant release of the cytoplasmic marker lactate dehydrogenase), which was blocked by pretreatment of the enzyme with the phospholipase A2 inhibitor 4-bromophenacyl bromide. These results suggest that calcium-dependent activation of phospholipase A2 and generation of lipoxygenase products of arachidonic acid metabolism are important in the initiation of eosinophil degranulation.

Topics: Acetophenones; Animals; Anti-Bacterial Agents; Calcimycin; Cytoplasmic Granules; Enzyme Inhibitors; Eosinophils; Horses; L-Lactate Dehydrogenase; Microscopy, Electron; Microscopy, Electron, Scanning; Peroxidases; Phospholipases; Phospholipases A; Phospholipases A2

The role of phospholipase A2 (PLA2) in the formation of platelet-activating factor (PAF-acether) by rabbit platelets is supported by several pieces of evidence. First, the release of PAF-acether was accompanied by that of its deacetylated analog, lyso-PAF-acether. Second, EDTA, EGTA, db-AMPc, p'-bromophenacylbromide and 874 CB, which, in spite of their structural diversity, are all PLA2 blockers, inhibited the release of both PAF-acether and of the lyso-compound. Third, addition of hog pancreas PLA2 to platelets as well as platelet lysis resulted in the release of lyso-PAF-acether, thus mimicking the metabolic events initiating formation of PAF-acether. These results indicate that PLA2 activation triggers both the second and the third pathway of platelet activation.

Topics: Acetophenones; Animals; Blood Platelets; Calcimycin; Edetic Acid; Egtazic Acid; Kinetics; Lysophosphatidylcholines; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Rabbits; Thrombin