Biomedical subjects

J T O'Flaherty

Publications and source records attributed to J T O'Flaherty.

At least 19 recordsLinked to original sources

PubMed1996

5-Oxo-eicosanoids and hematopoietic cytokines cooperate in stimulating neutrophil function and the mitogen-activated protein kinase pathway.

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.

Arachidonic Acid↗

PubMed1995

5-Lipoxygenase products modulate the activity of the 85-kDa phospholipase A2 in human neutrophils.

Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1994

Chemical and biological characterization of oxo-eicosatetraenoic acids.

Eicosatetraenoates (ETEs) with 5-oxo residues are known to induce human neutrophil (PMN) Ca2+ transients and chemotaxis. We find that 5-oxoETE, 5-oxo-8-trans-ETE, 5-oxo-15-hydroxy-ETE, 5-hydroxy-ETE, 5-hydroxy-15-oxoETE, 5,15-dioxoETE, and 5,15-dihydroxy-ETE have respective relative potencies of 10, 5, 3, 1, 0.2, 0.1, and 0.02 in: a) causing PMN to mobilize Ca2+, aggregate, and release small amounts of granule enzymes and b) promoting large degranulation and oxidative burst responses in PMN co-challenged with platelet-activating factor, tumor necrosis factor-alpha, or ATP. Contrastingly, 12(R)-hydroxy-ETE, 12(S)-hydroxy-ETE, and 12-oxoETE induced PMN Ca2+ transients and aggregation [respective potencies (5-hydroxy-ETE = 1) of 0.1, 0.01, and 0.003] but did not effect degranulation, and 15-hydroxy-ETE, 15-oxoETE, and 15-oxo-11-trans-ETE were inactive in all assays. Finally, 5-oxo/hydroxy-ETEs desensitized PMN to themselves but not to 12-oxo/hydroxy-ETEs or leukotriene (LT)B4; 12-oxo/hydroxy-ETEs and LTB4 desensitized PMN to themselves and each other but not to 5-oxo/hydroxy-ETEs; 15-oxo/hydroxy-ETEs did not desensitize PMN; and a LTB4 receptor antagonist blocked responses to LTB4 and 12-oxo/hydroxy-ETEs but not to 5-oxo/hydroxy-ETEs. Thus, 5-oxo/hydroxy-ETEs act by a common, LTB4 receptor-independent mechanism that recognizes 5- but not 12- or 15-oxo/hydroxy-ETEs and prefers oxo over hydroxy residues at C5 whereas 12-oxo/hydroxy-ETEs act via a LTB4 receptor mechanism that recognizes 12- but not 5- or 15-oxo/hydroxy-ETEs and prefers hydroxy over oxo residues at C12.

Arachidonic Acids↗

PubMed1994

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family.

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca2+, degranulate, and produce superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [3H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B4 and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyeicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.

Animals↗

PubMed1994

Human neutrophil degranulation responses to nucleotides.

BACKGROUND: Nucleotides have polymorphonuclear neutrophil (PMN)-stimulating actions resembling those of 5-hydroxyicosatetraenoate and its oxo analog, 5-oxoETE. Their effects on degranulation, however, are disputed even though this response may underlie their in vivo toxicity and is well-suited for comparing their mechanism of action with e.g., 5-oxoETE. EXPERIMENTAL DESIGN: We measured the direct, synergistic, and cross-desensitizing actions of nine nucleotides and six other stimuli in degranulating unprimed and tumor necrosis factor (TNF)-alpha-primed human PMN. RESULTS: Nucleotides weakly degranulated unprimed PMN but caused far larger responses in TNF-alpha-primed cells. Their actions, while differing from those of N-formyl-MET-LEU-PHE, platelet-activating factor, leukotriene B4, ionomycin, or dioctanoylglycerol, resembled those of 5-oxoETE. Nucleotides also enhanced PMN degranulation responses to the latter stimuli, particularly 5-oxoETE. Nucleotide degranulating and enhancing potencies were: UTP > or = ATP > or = ATP gamma S > ITP > ADP > 2-MeSATP, nonphosphohydrolyzable analogs lacked activity, and adenosine and AMP blocked PMN degranulation. Finally, nucleotides desensitized degranulation responses to each other but not to 5-oxoETE or other agonists, and 5-oxoETE desensitized to itself but not to nucleotides. CONCLUSIONS: Nucleotides have intrinsic and synergistic degranulating actions that under appropriate conditions (i.e., in concert with TNF-alpha or 5-oxoETE) are exceedingly prominent. Recognition systems mediating their effects differ from those for various stimuli including 5-oxoETE. These systems likely involve a common "nucleotide" receptor, but studies do not exclude possibilities that other purinergic receptors contribute to their actions.

Adenine Nucleotides↗

PubMed1993

5-hydroxyicosatetraenoate stimulates neutrophils by a stereospecific, G protein-linked mechanism.

We examined how 5-hydroxyicosatetraenoate (5-HETE) activates human neutrophils (PMN). 5-HETE stimulates PMN to mobilize Ca2+ but has little effect on degranulation or superoxide anion production. It nonetheless stereospecifically induced these responses in cells primed with tumor necrosis factor-alpha and likewise induced PMN plasma membranes to bind 35S-labeled guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and phosphohydrolyze [gamma-32P]GTP. Pertussis toxin blocked GTP gamma S binding responses. Scatchard analyses of GTP gamma S binding data indicated that 5-HETE raised the Ka of high affinity GTP gamma S binding sites without altering these sites' numbers or the parameters of low affinity GTP gamma S binding. Since N-formyl-Met-Leu-Phe, platelet-activating factor, and leukotriene (LT) B4 have these same bioactions, receptors for the latter agents might mediate responses to 5-HETE. However, 5-HETE desensitized degranulation responses to itself but not to the receptor agonists, the receptor agonists desensitized to themselves but not 5-HETE, and a LTB4 antagonist inhibited LTB4 but not 5-HETE in all assays. Finally, PMN and their membranes took up [3H] 5-HETE at 4 or 37 degrees C but, at both temperatures, also acylated the radiolabel into glycerolipids. Acylation nullified assessment of 5-HETE binding and questions reports that measure the cell binding, but not metabolism, of various HETEs. Our studies thus indicate 5-HETE acts by a down-regulatable, G protein-linked mechanism and represent the best available evidence that 5-HETE does not operate through, for example, LTB4 receptors.

Calcium↗

PubMed1993

5-Oxo-eicosatetraenoate, a potent human neutrophil stimulus.

5-Oxo-eicosatetraenoate (5-oxoETE), a newly defined arachidonate metabolite, resembled 5-hydroxyeicosatetraenoate (5-HETE) in stimulating neutrophils to mobilize Ca2+ an in promoting PMN degranulation responses to other agents. It was, however, 10-fold stronger than 5-HETE and, like leukotriene (LT) B4, had intrinsic PMN degranulating effects. Nonetheless, 5-oxoETE and 5-HETE desensitized PMN to themselves or each other but not to LTB4; LTB4 desensitized to itself but not to 5-oxoETE or 5-HETE; and an antagonist blocked LTB4 but not 5-oxoETE or 5-HETE. 5-OxoETE and 5-HETE thus induce diverse PMN responses using a shared, down-regulatable, and receptor-like mechanism that does not involve LTB4 receptors; 5-oxoETE is the preferred natural agonist for this mechanism.

Arachidonic Acids↗

PubMed1992

Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils.

Platelet-activating factor (PAF) desensitizes as well as stimulates its various target cells, We find that human polymorphonuclear neutrophils (PMN) exposed to PAF became maximally unresponsive to a second PAF challenge within 15-90 s in assays of Ca2+ mobilization and degranulation. The cells regained full PAF-sensitivity over the ensuing 20-40 min. These effects correlated with changes in PAF receptor availability. PMN treated with PAF, washed in regular buffer and assayed for PAF binding exhibited falls (maximal in 15 s), followed by rises (reaching control levels by 60 min), in the number of high-affinity PAF receptors. However, tracking studies showed that [3H]PAF accumulated on the cell surface for approximately 2 min before being internalized. Regular-buffer washes did not remove this superficial PAF, whereas a washing regimen using excess albumin to adsorb PAF removed 99% of the surface compound. PMN washed by the latter regimen after PAF exposure lost PAF receptors relatively slowly (maximal at approximately 5 min), but the ultimate extent of this loss and the rate at which receptor expression normalized were similar to those of cells washed in regular buffer. Neither cycloheximide nor actinomycin D influenced the course of the receptor changes, but two protein kinase C (PKC) blockers, staurosporine and 1-(5-isoquinolinesulphonyl)piperazine, inhibited the receptor-receptor-depleting actions of PAF. Indeed, a phorbol diester activator of PKC also caused PMN to decrease high-affinity PAF receptor numbers, and the two PKC blockers antagonized this action at concentrations that inhibited PAF-induced PAF receptor losses. We conclude that: (a) PAF induces PMN to down-regulate and then to re-express PAF receptors independently of protein synthesis; (b) these changes are likely to underlie the later stages and reversal of desensitization; (c) the onset (t < or = 2 min) of desensitization, however, precedes receptor down-regulation and must be due to receptor uncoupling from transductional elements; and (d) down-regulation of receptors for PAF appears to be mediated by PKC and/or elements inhibited by PKC blockers.

Calcium↗

PubMed1992

Propranolol, a phosphatidate phosphohydrolase inhibitor, also inhibits protein kinase C.

Propranolol, a beta-adrenergic receptor antagonist, also inhibits phosphatidate phosphohydrolase, the enzyme that converts phosphatidic acid into diacylglycerol. This latter effect has prompted recent use of propranolol in studies examining the importance of diacylglycerol and phosphatidic acid in cellular signalling events. Here, we show that propranolol is also an inhibitor of protein kinase C. At concentrations greater than or equal to 20 microM, propranolol reduced [3H]phorbol dibutyrate binding (IC50 = 200 microM) and phorbol myristate acetate-stimulated superoxide anion release (IC50 = 130 microM) in human neutrophils. Scatchard analysis showed that propranolol lowers the number of phorbol diester binding sites without significantly affecting their affinity. In vitro kinetic analysis, performed in a mixed micellar assay with protein kinase C purified from human neutrophils, suggested a competitive inhibition of propranolol with the cofactor phosphatidylserine. Complex kinetic patterns were observed with respect to diacylglycerol and ATP, approximating competitive and noncompetitive inhibition, respectively. Taken together, these results suggest that the drug interacts at the level of the regulatory domain of the enzyme. Fifty % inhibition occurred at approximately 150 microM propranolol. Similar levels of inhibition were obtained using exogenous (histone) and endogenous (p47-phox, a NADPH oxidase component) substrates. Protein kinase C-alpha and protein kinase C-beta, two protein kinase C isozymes present in human neutrophils, were inhibited by propranolol in a comparable manner. In the range of concentrations tested (30-1000 microM), neither cAMP-dependent protein kinase nor neutrophil protein tyrosine kinases were affected. The racemic form of propranolol and the (+) and the (-) stereoisomers were equally active, and other beta-adrenergic receptor antagonists (pindolol) and agonists (isoproterenol) were inactive. This suggests that the inhibitory action of propranolol on protein kinase C is related to the amphipathic nature of the drug rather than to its beta-adrenergic receptor blocking ability. Analogs of propranolol were synthesized and found to be more potent protein kinase C inhibitors, with IC50 values in the 10-20 microM range. We conclude that the ability of propranolol to inhibit both protein kinase C and PA phosphohydrolase complicates interpretation of results when this drug is used in signal transduction studies. In addition, propranolol may be a useful prototype for the synthesis of new protein kinase C inhibitors.

Cells, Cultured↗

PubMed1991

Tumor necrosis factor-alpha regulates expression of receptors for formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and platelet-activating factor. Dissociation from priming in human polymorphonuclear neutrophils.

TNF-alpha enhances polymorphonuclear responses to many stimuli, including chemotactic peptide FMLP. It also promotes expression of FMLP receptors and thus may prime polymorphonuclear neutrophils to this and other agonists by up-regulating signal recognition molecules. However, we find that the cytokine's actions on FMLP receptors lagged priming of FMLP-induced degranulation. Moreover, TNF-alpha enhanced degranulation responses to leukotriene B4 and platelet-activating factor but paradoxically down-regulated leukotriene B4 receptors and only transiently up-regulated platelet-activating factor receptors. Hence, TNF-alpha has pleiotropic effects on receptor expression; these effects diverge from priming; and a large part of the primed state must reflect enhancement of post-receptor events.

Cell Degranulation↗

PubMed1991

Roles of Ca2+ in human neutrophil responses to receptor agonists.

Previous studies have concluded that cytosolic Ca2+ [( Ca2+]i) transients are essential for neutrophils (PMN) to degranulate and make superoxide anion when challenged with the receptor agonists N-formyl-methionyl-leucyl-phenylalanine, platelet-activating factor and leukotriene B4. This view is based on the profound unresponsiveness of PMN that have their [Ca2+]i fixed at resting levels by removing storage Ca2+ and loading the cells with greater than or equal to 20 microM of a Ca2+ chelator, quin2 AM. We too observed this unresponsive state in PMN loaded with 10-32 microM-quin2 AM, fura-2 AM or 1,2-bis-(2-aminophenoxy) ethane-NNN'N'-tetra-acetic acid (BAPTA). When loaded with less than or equal to 1 microM fura-2 AM, however, Ca(2+)-depleted PMN failed to alter [Ca2+]i appreciably, yet still had substantial degranulation and superoxide-anion-generating responses to the receptor agonists. Function thus did not require [Ca2+]i transients. Moreover, Ca(2+)-depleted PMN had 20-35% decreases in receptor numbers for each of the three agonists, and chelator loading of these cells decreased receptor availability by 30-50%. All receptor losses were reversed by incubating PMN with Ca2+ at 37 degrees C, but not at 4 degrees C, and agonist binding at 4 degrees C was not influenced by the presence or absence of extracellular Ca2+. Ca2+ thus caused PMN to up-regulate their agonist receptors at 37 degrees C, and the effect persisted at 4 degrees C regardless of ambient Ca2+. We conclude that Ca2+ acts in at least three ways to regulate responses to receptor agonists. First, some pool of (probably cellular) Ca2+ maintains receptor expression. Second, [Ca2+]i transients potentiate, but are not required for, function. The [Ca2+]i pool may or may not be the same as that influencing receptors. Finally, another pool(s) of Ca2+ signals or permits responses. This last pool, rather than [Ca2+]i transients, appears essential for the bioactions of standard Ca(2+)-mobilizing stimuli.

Calcium↗

PubMed1991

Bioactions of 5-hydroxyicosatetraenoate and its interaction with platelet-activating factor.

In a variety of stimulated cells, platelet-activating factor (PAF) and numerous arachidonate derivatives are co-products that form as a consequence of receptor-mediated phospholipid mobilization. These lipid co-products produce a plethora of biological effects in a wide variety of cell systems. Furthermore, they often have a fascinating although less widely appreciated, interaction. 5-HETE, at submicromolar concentrations, exerts relatively few direct bioactions. It does, however, potently (16-160 nM) raise cytosolic free calcium [Ca2+]i and augment PAF-induced responses in human polymorphonuclear neutrophils (PMN) by as much as 100- to 1000-fold. 5-HETE acts on PMN by a structurally specific, stereospecific and pertussis toxin-inhibitable mechanism. In addition, PMN exposed to 5-HETE exhibit homologous but not heterologous desensitization. These findings suggest that 5-HETE, like PAF, may bind to its own specific plasmalemmal receptors to exert its unique set of bioactions. However, further investigation is required to demonstrate any putative 5-HETE receptors. Other potential mechanisms of 5-HETE-induced bioactions together with the possible effects of 5-HETE on PAF transduction mechanisms are also discussed.

Animals↗

PubMed1990

Stimulation and priming of protein kinase C translocation by a Ca2+ transient-independent mechanism. Studies in human neutrophils challenged with platelet-activating factor and other receptor agonists.

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 stimulate human polymorphonuclear neutrophils (PMN) to translocate protein kinase C from the cytosol to plasmalemma as judged by their abilities to increase PMN binding of and receptor numbers for [3H]phorbol dibutyrate [( 3H]PDB) (O'Flaherty, J.T., Jacobson, D.P., Redman, J.F., and Rossi, A.G. (1990) J. Biol. Chem. 265, 9146-9152). Platelet-activating factor (PAF) had these same effects. Moreover, two potent PAF analogs (but not an inactive analog) increased [3H]PDB binding; a PAF antagonist blocked responses to PAF without altering those to fMLP; and PMN treated with PAF became desensitized to PAF while retaining sensitivity to fMLP. Indeed, PMN incubated with 1-100 nM PAF for 5-40 min had markedly enhanced [3H]PDB binding responses to fMLP. PAF thus acted through its receptors to stimulate and prime protein kinase C translocation. Its effects, however, did not necessarily proceed by a standard mechanism: Ca2(+)-depleted PMN failed to raise Fura-2-monitored cytosolic Ca2+ concentrations [( Ca2+]i), yet increased [3H]PDB binding and receptor numbers almost normally after PAF challenge. PAF also primed Ca2(+)-depleted PMN to fMLP. Nevertheless, [3H]PDB binding responses to PAF were blocked in PMN loaded with Ca2+ chelators, viz. Quin 2, Fura-2, or 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Exogenous Ca2+ reversed Quin 2 inhibition, and a weak chelator 4,4'-difluoro-BAPTA, lacked inhibitory actions. The chelators similarly influenced fMLP and leukotriene B4. Thus, PMN can by-pass [Ca2+]i to translocate protein kinase C. They may achieve this using a regulatable pool of Ca2+ that evades conventional [Ca2+]i monitors or a signal that needs cell Ca2+ to form and/or act. This signal may mediate function in Ca2(+)-depleted cells, the actions of [Ca2+]i-independent stimuli, cell priming, and protein kinase C movements that otherwise seem [Ca2+]i-induced.

Calcium↗

PubMed1990

Translocation of protein kinase C in human polymorphonuclear neutrophils. Regulation by cytosolic Ca2(+)-independent and Ca2(+)-dependent mechanisms.

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector enzyme.

Binding, Competitive↗

PubMed1990

Mechanisms involved in the bidirectional effects of protein kinase C activators on neutrophil responses to leukotriene B4.

Three protein kinase C (PKC) activators (PMA, mezerein, and a diacylglycerol) had bidirectional effects on human polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4. Lower concentrations of the three agents enhanced, whereas higher concentrations inhibited, release of lysozyme and beta-glucuronidase stimulated by the arachidonic acid metabolite. Contrastingly, the activators inhibited but never enhanced LTB4-induced Ca2+ transients. We examined the causes for these varying effects. Each PKC activator reduced PMN specific binding of [3H]LTB4. Scatchard analyses revealed that PMA (greater than or equal to 0.16 nM) decreased the number of high affinity LTB4 receptors. The receptor losses correlated closely with inhibition of Ca2+ transients. PMN pretreated with 0.5 nM PMA for 5 min retained approximately 50% of their high affinity LTB4 receptors. These cells responded to 10 nM LTB4 with reduced but still substantial rises in cytosolic Ca2+, enhanced PKC mobilization, and increased granule enzyme release. The latter two effects appeared calcium-dependent because sequential exposure to PMA and LTB4 did not synergistically stimulate PKC mobilization or degranulation in PMN that were: 1) Ca2(+)-depleted; 2) challenged with 5 nM PMA; or 3) treated with LTB4 for 5 min before PMA. Each of the latter treatments completely interfered with the extent or timing of LTB4-induced Ca2+ transients. Accordingly, we suggest that the response-specific, bidirectional effects of PKC activators on LTB4 result from two opposing mechanisms. First, PKC activators down-regulate LTB4 high affinity receptors and thereby reduce those PMN responses that are not elicited by activated PKC (i.e., Ca2+ transients). Second, LTB4, by elevating cytosolic Ca2+, increases the amount of PKC mobilized by PKC activators and thereby promotes PKC-dependent responses (e.g., degranulation). The two mechanisms may be pertinent to the bidirectional effects of PKC activators on various other agonists. Furthermore, PKC, by down-regulating receptors, may serve as a physiologic stop signal for terminating function and producing a poststimulatory state of desensitization.

Calcium↗

PubMed1990

Cyclical binding, processing, and functional interactions of neutrophils with leukotriene B4.

Leukotriene (LT) B4 activates human polymorphonuclear neutrophils (PMN) by binding to plasmalemmal receptors. It stimulates PMN to raise cytosolic calcium and degranulate. Both responses end within 15-30 sec. However, in less than 15 sec, LTB4-treated PMN lose the ability to respond further to LTB4; decrease the affinity and number of high affinity receptors available for binding LTB4; sequester LTB4 in plasmalemma-associated sites that are inaccessible to a releasing buffer regimen; and begin internalizing LTB4. Over the next 90 min, the cells increasingly internalize LTB4 and convert it to less potent metabolites; release the metabolites; recover LTB4 binding sites; and become fully sensitive to LTB4. Contrastingly, during the entire 90 min incubation with LTB4. PMN retained the capacity to bind and respond normally to a second stimulus, platelet-activating factor. We therefore suggest the following model. LTB4 receptors, when ligand-bound, initiate function but rapidly lose this capacity as they lower their ligand binding affinity and sequester, internalize, or otherwise uncouple from transducing elements. These LTB4 receptor changes contribute to terminating PMN responses and producing a stimulus-selective state of desensitization. During the desensitization period, PMN progressively process and metabolize LTB4. This removes LTB4 from the environment, thereby allowing PMN to recover functional receptors for and sensitivity to the ligand.

Calcium↗

PubMed1989

Protein kinase C blockers and neutrophil receptors for leukotriene B4.

Three protein kinase C blockers (staurosporin, Cl, and sphinganine) acted temperature- and time-dependently on human neutrophils to lower the affinity and number of high affinity plasmalemma receptors available to leukotriene B4. The drugs did not alter the ligand's binding to isolated plasma membranes or reduce intact cell binding of platelet-activating factor. Thus, protein kinase C may regulate the expression of certain receptors in resting cells and blockers of this enzyme, by interfering with receptor expression, have secondary effects that complicate their use as pharmacological probes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Receptor-independent metabolism of platelet-activating factor by myelogenous cells.

Human neutrophils incorporate and metabolize platelet-activating factor (PAF). We dissociated these events from PAF binding to its receptors. Cells were pretreated with either pronase, a PAF antagonist (L652731), or excess PAF. This reduced PAF receptor numbers by 70 to almost 100% but had no comparable effect upon the neutrophil's ability to metabolize PAF. Furthermore, HL-60 cells efficiently metabolized, but did not specifically bind, PAF. Thus, PAF receptor availability did not correlate with PAF metabolic capacity and we conclude that myelogenous tissues can process this bioactive ligand by a receptor-independent pathway.

Humans↗