Staurosporine, a protein kinase inhibitor, up-regulates the stimulation of human neutrophil respiratory burst by N-formyl peptides and platelet activating factor

Staurosporine (STAR), a potent protein kinase C (PKC) antagonist, was found to modulate the chemoattractant-induced respiratory burst of human polymorphonuclear leukocytes (PMNs) according to drug concentration. Low STAR concentrations from 10 to 200 nM potentiated the N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet activating factor (Paf)-induced respiratory burst, affecting both the initial rate and the total amount of superoxide anion generated. The maximal increase occurred in the presence of 100 nM STAR and optimal fMLP concentration and reached 60-100% of control values. Above 250 nM, STAR inhibited the respiratory burst with an IC50 of 360 and 320 nM for fMLP and Paf, respectively. The respiratory burst induced by PKC activators such as phorbol myristate acetate or phorbol 12, 13 dibutyrate was inhibited effectively by STAR, with a low IC50 (25 nM) for both stimuli. Thus, the use of low STAR concentrations points to two possible roles of PKC in the regulation of NADPH oxidase activity, i.e. a positive regulation in phorbol ester-treated cells and a negative regulation in chemoattractant-stimulated PMNs.     

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.     

Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry.

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type- and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLP/cytochalasin B-induced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMA-induced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst.     

A novel type of cytoplasmic granule in bovine neutrophils

We obtained cell preparations containing greater than 95% neutrophils from freshly drawn bovine blood. The cells were suspended in sucrose and disrupted in a Dounce homogenizer, and the postnuclear supernate was fractionated by zonal differential sedimentation and by isopycnic equilibration. The subcellular fractions were characterized biochemically by testing for marker enzymes and other constituents known to occur in azurophil and specific granules of other species, and by electrophoretic analysis of extracts of the particulate material. In addition, each fraction was examined by random-sampling electron microscopy. We found that bovine neutrophils contain in addition to azurophil and specific granules a third type of granule, not known to occur in neutrophils of other species. These novel granules are larger, denser, and considerably more numerous than the two other types. Except for lactoferrin, they lack the characteristic constituents of azurophil granules (peroxidase, acid hydrolases, and neutral proteinases) and of specific granules (vitamin B12-binding protein). Instead, they contain a group of highly cationic proteins not found in the other granules, and they are the exclusive stores of powerful oxygen-independent bactericidal agents. We studied the fate of the large granules in bovine neutrophils exposed to opsonized particles, the ionophore A 23187, or phorbol myristate acetate. The appearance in the cell-free media of antibacterial activity and of the characteristic highly cationic proteins as revealed by electrophoresis was monitored and compared with the release of azurophil and specific granule markers. In addition, changes of the relative size of the large granule compartment induced by phagocytosis were assessed by morphometry. The results show that exocytosis of the large granules occurs following both phagocytosis and exposure to soluble stimuli. Like the specific granules, the large granules appear to be discharged by true secretion under conditions where the azurophil granules are fully retained.     

Effects of inhibitors on aldolase breakdown after its microinjection into HeLa cells.

The tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces insulin secretion from isolated pancreatic islets, and this suggests a potential role for protein kinase C in the regulation of stimulus-secretion coupling in islets. In the present study, the hypothesis that the insulinotropic effect of TPA is mediated by activation of protein kinase C in pancreatic islets has been examined. TPA induced a gradual translocation of protein kinase C from the cytosol to a membrane-associated state which correlated with the gradual onset of insulin secretion. The pharmacologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not mimic this effect. TPA also induced a rapid time-dependent decline of total protein kinase C activity in islets and the appearance of a Ca2+- and phospholipid-independent protein kinase activity. Insulin secretion induced by TPA was completely suppressed (IC50 approximately 10 nM) by staurosporine, a potent protein kinase C inhibitor. Staurosporine also inhibited islet cytosolic protein kinase C activity at similar concentrations (IC50 approximately 2 nM). In addition, staurosporine partially (approximately 60%) inhibited glucose-induced insulin secretion at concentrations (IC50 approximately 10 nM) similar to those required to inhibit TPA-induced insulin secretion, suggesting that staurosporine may act at a step common to both mechanisms, possibly the activation of protein kinase C. However, stimulatory concentrations of glucose did not induce down-regulation of translocation of protein kinase C, and the inhibition of glucose-induced insulin release by staurosporine was incomplete. Significant questions therefore remain unresolved as to the possible involvement of protein kinase C in glucose-induced insulin secretion.     

Biochemical responses in activated human neutrophils mediated by protein kinase C and a Ca2+-requiring proteinase

Low concentrations of phorbol 12-myristate 13-acetate (PMA) elicit a specific response in human neutrophils, characterized by the production of oxygen radicals and the release into the medium of a membrane-bound serine proteinase (Pontremoli, S., Melloni, E., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Damiani, G. and Horecker, B. L. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 1685-1689). The following evidence indicates that this response is mediated by membrane-bound protein kinase C: 1) it is blocked by inhibitors of protein kinase C; and 2) it is enhanced in cells preloaded with leupeptin which prevents proteolysis of protein kinase C and its subsequent dissociation from the cell membrane. This response is not accompanied by significant exocytosis of granule enzymes. With higher concentrations of PMA, and more particularly on stimulation with formylmethionyl-leucyl-phenylalanine (fMLP) plus cytochalasin B, a substantial exocytosis of constituents of both specific and azurophil granules is observed. With fMLP, exocytosis of granule enzymes is the predominant event, with little production of H2O2 and negligible release of membrane-bound serine proteinase. Exocytosis promoted either by a high concentration of PMA or by fMLP is inhibited by leupeptin, indicating that it is due to the action of an intracellular Ca2+-dependent thiol proteinase (calpain), either directly or by conversion by calpain of membrane-bound protein kinase C to the soluble Ca2+/phospholipid-independent form. Intracellular mobilization of Ca2+ is also observed following stimulation with either PMA or fMLP, but only the latter results in a net increase in the intracellular concentration of free Ca2+; under these conditions maximum exocytosis of granule contents is observed.     

Calcium stimulates luteinizing-hormone (lutropin) exocytosis by a mechanism independent of protein kinase C.

Using permeabilized gonadotropes, we examined whether Ca2(+)-stimulated luteinizing-hormone (LH) exocytosis is mediated by the Ca2(+)-activated phospholipid-dependent protein kinase (protein kinase C). In the presence of high [Ca2+]free (pCa 5), alpha-toxin-permeabilized sheep gonadotropes secrete a burst of LH and then become refractory to maintained high [Ca2+]free. The protein kinase C activator phorbol myristate acetate (PMA) is able to stimulate further LH release from cells made refractory to high [Ca2+]free, suggesting that Ca2+ does not stimulate LH release by activating protein kinase C. Staurosporine, a protein kinase C inhibitor, inhibited PMA-stimulated (50% inhibition at 20 nM), but not Ca2(+)-stimulated, LH exocytosis. In cells desensitized to PMA by prolonged exposure to a high PMA concentration, Ca2(+)-stimulated LH exocytosis (when corrected for depletion of total cellular LH) was not inhibited. Ba2+ was able to stimulate LH exocytosis to a maximal extent similar to Ca2+, although higher Ba2+ concentrations were necessary. Ba2+ and Ca2+ stimulated LH exocytosis with a similar time course, and both were inhibitory at high concentrations. Furthermore, cells made refractory to Ca2+ were also refractory to Ba2+. These data strongly suggest that Ba2+ and Ca2+ act through the same mechanism. Since Ba2+ is a poor activator of protein kinase C, these findings are additional evidence against a major role for protein kinase C in mediating Ca2(+)-stimulated LH exocytosis.     

Human immunoglobulin G potentiates superoxide production induced by chemotactic peptides and causes degranulation in isolated human neutrophils

Neutrophils are major cellular mediators of host defense and inflammation. They can be activated to produce superoxide and to release the contents of their granules to the extracellular space. We observed that monomeric human immunoglobulin G (IgG) sensitizes these cells to the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLP). In cells submaximally stimulated by fMLP this enhancement was especially prominent. With saturating fMLP concentrations, the rate of O2- production was still about twice that in the control. No synergy with other activators (phorbol myristate acetate, concanavalin A) was observed. Binding of fMLP to the cells was decreased by IgG, resembling the effect of cytochalasin B. IgG did not induce O2- production on its own, but it stimulated degranulation of the neutrophils.     

Contribution of mitogen-activated protein kinase to stimulation of phospholipase D by the chemotactic peptide fMet-Leu-Phe in human neutrophils.

Phospholipase D (PLD) plays an important role in signaling through phosphatidylcholine (PC) and in the production of superoxide (respiratory burst) by polymorphonuclear leukocytes (PMN) stimulated by the chemoattractant fMet-Leu-Phe (fMLP). However, the regulation of PLD activity by protein kinases is not fully understood. In the present study, we have used a mitogen-activated protein (MAP) kinase inhibitor (PD 98059) to investigate a possible connection between extracellular signal-regulated kinase (ERK) and PLD activity and respiratory burst. Using a range of concentrations (3-20 microM) which inhibit ERK activity, PD 98059 inhibited PLD activity induced by fMLP in cytochalasin B-primed PMN, as assessed by production-tritiated phosphatidylethanol (PEt), phosphatidic acid (PA), and hydrolysis of PC. However, the inhibition was partial (approximately 50%), while inhibition of PC hydrolysis was almost complete, suggesting a concomitant inhibition of PLA2 activity. In addition, PD 98059 reduced fMLP-induced respiratory burst by 50%, an effect which was correlated with PLD inhibition of PLD (r = 0.981, P < 0.01), and neither did PD 98059 inhibit the PLD activity and respiratory burst induced by PKC upon its direct activation by phorbol myristate acetate. These data provide the first evidence for implication of the ERK cascade in the stimulation of PLD through Gi signaling. They further indicate that PLD stimulation by fMLP receptors occurs through two pathways, dependent and independent on MAP kinase, the former pathway being linked to superoxide production.     

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.     

The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranes

Staurosporine induced the association of purified protein kinase C (PKC) with inside-out vesicles from erythrocyte membranes. This effect was Ca2+ and concentration dependent, and maximum PKC translocation was observed at 50 nM staurosporine and 0.5 microM Ca2+, or higher. A significant effect of staurosporine was already obtained at free Ca2+ concentrations in the range found in resting cells. Under these conditions, the PKC activator 4-phorbol 12,13-dibutyrate was by itself inactive, but enhanced translocation by staurosporine. Protein phosphorylation by staurosporine-translocated PKC was inhibited in the presence or absence of phorbol esters. Translocation and inhibition of PKC occurred in the same staurosporine concentration range.     

The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis

Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (<2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t_1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 μM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.     

N-formyl-Met-Leu-Phe-induced oxidative burst in DMSO-differentiated HL-60 cells requires active Hsp90, but not intact microtubules

In this study we examined whether microtubules and heat shock protein 90 (Hsp90) are involved in phorbol myristate acetate (PMA) and N-formyl-Met-Leu-Phe (fMLP)-induced oxidative burst in DMSO-differentiated HL-60 cells. Our results showed that microtubule interfering agents, paclitaxel (1-5 microM), colchicine (1-100 microM), nocodazole (1-20 microM), and vincristine (1-50 microM), did not affect either PMA or fMLP-induced oxidative burst. In contrast, radicicol, an inhibitor of Hsp90, inhibited fMLP-induced oxidative burst in time and concentration-dependent manner where IC50 value for 30 min pre-incubation was 16.5 +/- 3.5 microM radicicol. We conclude that both PMA and fMLP-induced oxidative burst in DMSO-differentiated HL-60 cells is microtubule-independent while the latter requires Hsp90 activity.     

Two transduction sequences are necessary for neutrophil activation by receptor agonists

The effects of 17-hydroxywortmannin (HWT), a powerful inhibitor of the respiratory burst associated with phagocytosis (Baggiolini, M., Dewald, B., Schnyder, J., Ruch, W., Cooper, P. H., and Payne, T. G. (1987) Exp. Cell Res. 169, 408-418), were studied in human neutrophils stimulated with chemotactic agonists or phorbol myristate acetate. At nanomolar concentrations HWT inhibited superoxide production and the release of granule contents induced by N-formyl-Met-Leu-Phe, C5a, platelet-activating factor, and leukotriene B4, but not by phorbol myristate acetate, indicating that it interferes with receptor-mediated activation of the neutrophils, without directly affecting protein kinase C (Ca2+/phospholipid-dependent enzyme), the NADPH-oxidase, or the process of granule exocytosis. Moreover, HWT did not influence agonist-induced [Ca2+]i changes, indicating that it does not interfere with the function of agonist receptors, G-proteins or the phosphatidylinositol-specific phospholipase C. By studying the effect of HWT on the respiratory burst elicited in normal and Ca2+-depleted cells by combined stimulation with N-formyl-Met-Leu-Phe and phorbol myristate acetate, evidence was obtained that two transduction sequences, both of which are G-protein-dependent, are necessary for the induction of the response by receptor agonists. One sequence is Ca2+-dependent, HWT-insensitive, and leads to activation of protein kinase C, the other is Ca2+-independent and HWT-sensitive. Ca2+ depletion, which blocks the first, and HWT, which blocks the second, can be used to show that both processes must be functional for the transduction of agonist signals into a respiratory burst response.     

Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation

We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.     

Chemistry and biological activities of constituents from Morus australis.

A novel constituent named australone B (1) was further isolated from the cortex of Morus australis (Moraceae). The structure of 1 has been elucidated by one- and two-dimension spectra. In human citrated platelet-rich plasma, 1 showed strong inhibition of aggregation induced by adrenaline in a concentration-dependent manner with an IC(50) value of about 33.3 microM. Compound 1 (30 microM) also showed inhibitory effects on superoxide anion formation from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP)/cytochalasin B (CB). Morusin (2) inhibited superoxide anion formation from rat neutrophils stimulated with phorbol myristate acetate (PMA) in a concentration-dependent manner with an IC(50) value of 66.9+/-2.5 microM.     

Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.     

Differential regulation of neutrophil phospholipase d activity and degranulation

One of the proposed functions of phosphatidic acid (PA) formation from phospholipase D (PLD) activation in neutrophils is to promote degranulation induced by receptor agonists. The present study shows that the time course and dose response of PA formation and degranulation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) differed. PLD activation and degranulation also exhibited different dose response to genistein and epigallocatechin gallate (EGCG), inhibitors of protein tyrosine kinases. Genistein inhibited PLD activity with an IC(50) value of 12.2 microM in fMLP- and 107 microM in phorbol myristate acetate (PMA)-stimulated cells. It required higher concentrations of genistein to inhibit degranulation than to inhibit PLD activity induced by fMLP. EGCG in the range of 40-400 microM had no effect on PLD activity but it inhibited the release of beta-glucuronidase and elastase by fMLP-stimulated cells. These results demonstrate differential regulation of PLD activity and degranulation of primary granules by genistein and EGCG in fMLP-stimulated neutrophils.     

The ability of staurosporine to modulate pancreatic acinar cell desensitization by TPA, carbamylcholine and caerulein.

The implication of protein kinase C in the phenomenon of pancreatic acinar cell desensitization to carbamylcholine, caerulein and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated using a potent PKC inhibitor, staurosporine. At a concentration of 1 microM, staurosporine caused a maximum 64% inhibition of amylase release from rat pancreatic acini stimulated by 100 nM TPA. At 100 nM, staurosporine reduced by 50 to 55% amylase secretion elicited by maximal concentrations of carbamylcholine or caerulein without affecting their potency. Staurosporine was also able to prevent completely desensitization by TPA of the subsequent secretory response to carbamylcholine and caerulein. Furthermore, staurosporine also totally prevented desensitization by caerulein of the subsequent secretory response to caerulein. In contrast, staurosporine only partially prevented desensitization by carbamylcholine of the subsequent secretory response to carbamylcholine. These results indicate that staurosporine is a potent inhibitor of protein kinase C as it inhibited the secretory response to carbamylcholine, caerulein and TPA. They also suggest that desensitization of the secretory response induced by TPA and caerulein used a common pathway involving protein kinase C activation. Finally, desensitization by carbamylcholine is more complex as it is only partially prevented at staurosporine; therefore, protein kinase C activation seems to be one of the factors involved.     

Inhibition of stimulus-dependent epidermal growth factor receptor and transforming growth factor-alpha mRNA accumulation by the protein kinase C inhibitor staurosporine

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10(-9) M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF-alpha mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF-stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti-phosphotyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF-alpha expression.