Arachidonate metabolites, platelet-activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils

In contrast to our previous report (Biochem. Biophys. Res. Comm. 134:587, 1986), we now find that protein kinase C (PKC) is mobilized in human polymorphonuclear neutrophils (PMN) stimulated with platelet-activating factor (PAF) or leukotriene (LT)B4. Thus nanomolar concentrations of each compound caused PMN to lose cytosolic, PKC-specific protein phosphorylating activity, as well as receptors for phorbol myristate acetate (PMA). Smaller gains in membrane-associated PMA receptors accompanied these changes. Diacylglycerol and PMA had very similar effects on PKC. However, unlike these direct PKC activators, PAF and LTB4 induced only moderate decreases in cytosolic PKC; acted only on PMN pretreated with cytochalasin B; did not mobilize PKC in disrupted PMN or activate PKC in a cell-free system; and with respect to PAF, induced responses that partially reversed within 30 min. Furthermore, PAF, LTB4, and several of their structural analogues mobilized PKC at concentrations correlating closely with their respective affinities for cellular LTB4 or PAF receptors. Thus PAF and LTB4 acted by indirect and apparently receptor-mediated mechanisms. Four observations indicated that the cytochalasin B-dependent degranulating actions of PAF and LTB4 involved PKC. First, PKC mobilization and degranulation occurred at the same stimulus concentrations. Second, 5-hydroxyicosatetraenoate dramatically enhanced both PKC mobilization and degranulation when elicited by PAF; it had relatively little influence on LTB4-induced responses. Third, PAF-induced mobilization (t1/2 less than 7 sec) preceded degranulation (t1/2 approximately 20 sec). Finally, a PKC blocker, polymyxin B, was similarly effective in inhibiting degranulation responses to PAF, LTB4, and PMA. Because stimulated PMN may produce and use PAF, LTB4, and 5-hydroxyicosatetraenoate as secondary intracellular mediators, our results implicate PKC as a central and potentially critical regulator of function.     

Inhibitors of protein kinase C selectively enhanced leukotriene D4-induced calcium mobilization in differentiated U-937 cells

U-937 cells differentiated with dimethylsulphoxide for 3-4 days express receptors for leukotriene D4 (LTD4), which are coupled to Ca2+ mobilization and phosphatidylinositol (PI) metabolism. Treatment of U-937 cells with an inhibitor of protein kinase C (PKC) [staurosporine (100 nM)] augmented the Ca2+ mobilized by LTD4. The peak concentration of the LTD4-induced increase in [Ca2+]i was 1500 nM in untreated cells and 3000 nM in cells treated with staurosporine for 30 s. Maximal mobilization responses were observed at 1-10 microM LTD4 in both control and staurosporine-treated cells. The increased Ca2+ response to LTD4 after staurosporine treatment occurred within 30 s and was attributable to both intracellular and extracellular stores. Additionally, a second phase of Ca2+ mobilization occurred after stimulation with LTD4, which was elevated by pretreatment with staurosporine--this effect was maximal after 5-10 min of treatment. Staurosporine either had no effect or decreased the Ca2+ mobilization response of differentiated U-937 cells to other agonists, such as LTB4, platelet activating factor, ATP or the chemotactic peptide f-Met-Leu-Phe. Although staurosporine alone had no effect on basal PI metabolism it increased LTD4-induced PI metabolism. Staurosporine did not prevent the tachyphylaxis observed upon second challenge with LTD4, nor did it prevent LTD4-induced homologous densensitization. Other compounds which inhibit PKC (sphingosine and 1-O-hexadecyl-2-O-methylglycerol), also enhanced the Ca2+ response of U-937 cells to LTD4, but not to other agonists. These data show that inhibition of PKC enhanced responses of LTD4, suggesting that PKC plays a role in determining the responsiveness of LTD4 receptors.     

Identification of glycation at the N-terminus of albumin by gas chromatography-mass spectrometry.

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neutrophil degranulation: evidence pertaining to its mediation by the combined effects of leukotriene B4, platelet-activating factor, and 5-HETE

Stimulus-activated polymorphonuclear neutrophils (PMN) produce leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoate (5-HETE), and platelet-activating factor (PAF). Each of these lipids promotes PMN degranulation; in combination they have additive and potentiating effects that result in prominent degranulation responses at relatively low concentrations. Thus, the combined interactions of LTB4, 5-HETE, and PAF may mediate responses in PMN activated by other stimuli. This possibility was examined by measuring the responses of PMN made insensitive to one or more of these lipids. Cells were pretreated with LTB4, 5-HETE, and/or PAF for 8 min; exposed for 2 min to cytochalasin B (which is required for lipid-induced degranulation); and then challenged. PMN challenged with only buffer released minimal amounts of granule-bound enzymes. Furthermore, the lipid-pretreated cells were hyporesponsive to challenge with 1) various combinations of these same lipids or 2) ionophore A23187. The relative potencies of the lipids in producing hyporesponsiveness to themselves or A23187 were: 5-HETE less than PAF less than or equal to LTB4 less than PAF + LTB4 less than PAF + LTB4 + 5-HETE. For both types of challenge, reduced responsiveness occurred in cells pretreated with greater than 0.1 nM LTB4 and/or greater than 0.2 nM PAF, persisted in cells washed after lipid pretreatment, and did not develop in cells pretreated with various combinations of bioinactive structural analogues of the lipids. Thus, PAF, LTB4, and 5-HETE interacted to desensitize PMN, and the degranulating actions of A23187 required cells that were fully responsive to each of the three lipids. This supports the concept that the lipids act together in mediating certain of the ionophore's effects. However, lipid-desensitized PMN degranulated fully when challenged with C5a, a formylated oligopeptide, or phorbol myristate acetate. Degranulation responses, therefore, may proceed through various pathways, only some of which involve the lipid products studied here.     

Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors

LTB4-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB4 to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [3H]-LTB4 binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20 degrees C, binding of [3H]-LTB4 to the membrane receptor was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [3H]-LTB4 bound to high affinity (dissociation constant, Kd = 1.5 nM), and low capacity (density, Bmax = 40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB4, LTB4-epimers, 20-OH-LTB4, 2-nor-LTB4, 6-trans-epi-LTB4 and 6-trans-LTB4, in decreasing order of affinity, bound to the [3H]-LTB4 receptors. The mean binding affinities (Ki) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PMN chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB4 in human PMN plasma membrane.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Prostaglandin binding sites in human polymorphonuclear neutrophils

Prostaglandin (PG) E2 (greater than or equal to 1.6 nM) and PGD2 (greater than or equal to 16 nM) inhibited polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4 and platelet-activating factor (PAF) whereas PGF2 alpha was bioinactive. [3H]PGE2 and [3H]PGD2 bound to PMN and isolated, plasmalemma-enriched PMN membranes. Binding was time-dependent, specific, saturable, and reversible. Competitive studies indicated that the two PGs bound to distinctly different sites. PMN had high (Kd = 1 nM; Rt = 150/cell) and low (Kd = 100 nM; Rt = 5800/cell) affinity PGE2 binding sites. Only a single type of PGD2 binding site (Kd = 13 nM; Rt = 5100/cell) was detected. We conclude that PGE2 and PGD2 bind to their respective, plasmalemmal receptors to attenuate PMN function. The PGs may act as endogenous stop signals to limit the action of concurrently formed excitatory signals, eg., LTB4 and PAF.     

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.     

Selective modulation by guanine nucleotides of the high affinity subset of plasma membrane receptors for leukotriene B4 on human polymorphonuclear leukocytes

Isolated human polymorphonuclear (PMN) leukocyte plasma membranes express high affinity (mean Kd = 0.12 nM) and low affinity (mean Kd = 50 nM) receptors for the chemotactic factor leukotriene B4 (5(S),12(R)-dihydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid; LTB4) that are similar to those on intact PMN leukocytes. A portion of high affinity LTB4-R on PMN leukocyte membranes were converted to the low affinity state by GTP (mean +/- SE = 28.6 +/- 14.0%) and nonhydrolyzable GTP analogues, such as 5'-guanylylimidodiphosphate (GMP-PNP), in a concentration-dependent, nucleotide-specific, and reversible manner, without altering the intrinsic binding affinities of either class. [3H]GMP-PNP bound specifically to one class of receptors (mean Kd = 13 nM) on PMN leukocyte membranes. The interdependence of the LTB4-binding membrane protein and guanine nucleotide-binding protein was suggested by the capacity of LTB4 to enhance by a maximum of 150% the binding of [3H]GMP-PNP to PMN leukocyte membranes by increasing the number, but not altering the affinity, of receptors for GMP-PNP. Pertussis toxin, but not cholera toxin, reversed the enhancement of binding of [3H]GMP-PNP produced by LTB4. Guanine nucleotide-binding proteins and high affinity LTB4-R thus exhibit a mutual regulation that differs mechanistically from that of peptide chemotactic factor receptors on PMN leukocytes.     

Inflammatory chemoreceptor cross-talk suppresses leukotriene B4 receptor 1-mediated neutrophil calcium mobilization and chemotaxis after trauma

G protein-coupled chemoattractants recruit neutrophils (PMN) to sites of injury and infection. The leukotrienes (LT) and CXC chemokines (CXC) and their receptors (BLT1/BLT2 and CXCR1/CXCR2) are all known to play roles in these responses. Each system has been studied separately in vitro, but in vivo they act concurrently, and the clinical interactions between the two systems are unstudied. We prospectively studied calcium mobilization and chemotactic responses to LTB(4) in PMN from major trauma patients. The responses of the high affinity BLT1 receptor were suppressed at the 3-day postinjury time point, but recovered by 1 wk. Trauma patients had transient elevations of plasma LT and CXC levels. Functional deficits identical with those in trauma PMN were reproduced in vitro by exposing healthy PMN to CXCs at the elevated plasma concentrations found. Functional responses to LTB(4) were suppressed by cross-talk with CXC and BLT2 receptors that desensitize BLT1. Since the suppression of intracellular calcium mobilization was prominent, we also studied the role of suppressed cell calcium mobilization in the defective chemotactic responses to LTB(4). We noted that PMN chemotaxis to LTB(4) showed far more dependence on store-operated calcium entry than on the release of cellular calcium stores, and that store-operated calcium responses to BLT1 activation were markedly inhibited during the same time period as was chemotaxis. The intermittent release of inflammatory mediators after injury can blunt PMN responses to LTs by suppressing BLT1 as well as downstream calcium entry. Diminished LT receptor activity due to cross-talk with CXC receptors can inhibit PMN recruitment to infective sites. This may predispose injured patients to septic complications.     

Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release.

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.     

Desensitization by protein kinase C activation differentially uncouples formyl peptide receptors from effector enzymes in HL-60 granulocytes

The hypothesis that protein kinase C (PKC) participates in agonist-mediated desensitization of formyl peptide receptors in HL-60 granulocytes was tested. fMet-Leu-Phe and leukotriene B₄(LTB₄) produced homologous desensitization of agonist-stimulated intracellular calcium transients. Pre-treatment with the PKC activator, phorbol myristate acetate (PMA; 10 nM), abolished both fMet-Leu-Phe and LTB₄-stimulated calcium transients. Membranes prepared from control HL-60 granulocytes (NM) or cells treated with 10 nM PMA (PMA-M) demonstrated increased formyl peptide receptor and G protein density, as determined by radioligand binding and pertussis toxin- and cholera toxin-catalysed ADP ribosylation. fMet-Leu-Phe stimulation of guanosine 5′-[γ-thio]-triphosphate (GTPγS) binding and GTP hydrolysis and GDP inhibition of fMet-Leu-Phe binding were not different between NM and PMA-M. Pre-treatment with 10 nM PMA did not inhibit subsequent fMet-Leu-Phe-stimulated superoxide generation or phospholidase D activation. We conclude that PKC desensitizes fMet-Leu-Phe-stimulated phospholipase C, but not phospholipase D, responses and that PKC activation does not mediate agonist-induced desensitization of formyl peptide receptors.     

Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes

The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.     

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.     

Intracellular Ca2+ mobilization in immature and more mature U937 induced to differentiate by dimethyl sulfoxide or phorbol myristate acetate

Intracellular Ca2+ mobilization in U937 cells was studied. Stimulation of immature U937 cells with leukotriene B4 (LTB4) increased intracellular Ca2+ levels, whereas stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) failed to increase intracellular Ca2+ levels. U937 cells cultured with 1.5% dimethyl sulfoxide (DMSO) for 4 days (DMSO-U937 cells) responded to LTB4 and possessed the ability to respond to fMLP. U937 cells cultured with 1 ng/ml phorbol myristate acetate (PMA) for 4 days (PMA-U937 cells) lost the ability to respond to LTB4, although they responded to fMLP. Treatment of DMSO-U937 cells with 100 ng/ml PMA for 3 min suppressed intracellular Ca2+ increase induced by LTB4 and fMLP. The fMLP-induced Ca2+ rise in PMA-U937 cells was not suppressed by a further treatment with 100 ng/ml PMA. DMSO-U937 cells responded to inositol 1,4,5-trisphosphate (IP3), indicating that IP3 functions as a messenger of intracellular Ca2+ mobilization from endoplasmic reticulum in U937. The magnitude and duration of the rise in Ca2+ induced by IP3 in DMSO-U937 cells treated with 100 ng/ml PMA for 3 min were similar to those of the controls. When DMSO-U937 cells were Ca2+-depleted, addition of Ca2+ resulted in a transient overshoot of Ca2+ influx. However, the transient overshoot was not observed, when PMA-U937 cells were tested. These results indicate that Ca2+ efflux in PMA-U937 cells is increased by an activated exit pump, which may be directly or indirectly related to the functional state of PMA-U937 cells.     

Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C.

The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.     

Activation of oxidative burst and degranulation of porcine neutrophils by a homologous spleen galectin-1 compared to N-formyl-L-methionyl-L-leucyl-L-phenylalanine and phorbol 12-myristate 13-acetate

Galectins are a family of animal lectins defined by their beta-galactoside-binding activities and a consensus sequence in their carbohydrate-recognizing domain (CRD). Relevant roles of galectins are described in adaptive immune response, innate immunity and modulation of the acute inflammatory response. We have extended our previous studies on a porcine spleen galectin-1 in relation to its functional roles such as polymorphonuclear neutrophils (PMNs) stimulation compared to well known PMN activators e.g. N-formyl-L-methionyl-L leucyl-L-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Relative to activation of NADPH-oxidase fMLP and PMA are stronger than galectin-1 plus cytochalasin B (CB) when the lectin is employed at low concentrations (gal-1 1 microM, 3.6+0.8 nm O(2)(-)/min/10(7) PMN). Higher doses of galectin-1 (10 microM) plus CB produced a significant activation of NADPH-oxidase (27.9+14.8 nm O(2)(-)/min/10(7) PMN) and stimulated PMN degranulation up to 50%. We propose that local galectin-1 concentrations under physiological conditions might reach suitable levels for pig PMN stimulation, and might be a natural inducer of O(2)(-) formation or degranulation. Porcine galectins might produce enhanced responses in vivo when they stimulate neutrophils in combination with some other stimuli.     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.