Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.     

Activation- and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3-dependent mast cells and serosal mast cells

As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.     

Beta2 integrins target Rap GTPases to the plasma membrane by means of degranulation

We report the novel observation that engagement of β2 integrins on human neutrophils is accompanied by increased levels of the small GTPases Rap1 and Rap2 in a membrane-enriched fraction and a concomitant decrease of these proteins in a granule-enriched fraction. In parallel, we observed a similar time-dependent decrease of gelatinase B (a marker of specific and gelatinase B-containing granules) but not myeloperoxidase (a marker of azurophil granules) in the granule fraction, and release of lactoferrin (a marker of specific granules) in the extracellular medium. Furthermore, inhibition of Src tyrosine kinases, or phosphoinositide 3-kinase with PP1 or LY294002, respectively, blocked β2 integrin-induced degranulation and the redistribution of Rap1 and Rap2 to a membrane-enriched fraction. Consequently, the β2 integrin-dependent exocytosis of specific and gelatinase B-containing granules occurs via a Src tyrosine kinase/phosphoinositide 3-kinase signaling pathway and is responsible for the translocation of Rap1 and Rap2 to the plasma membrane in human neutrophils.     

Correlation between a specific molecular weight form of plasminogen activator and metabolic activity of 3T3 cells

In quiescent cultures of 3T3 cells, plasminogen activator (PA) is found predominantly as a 75,000 dalton species. When quiescent cells are exposed to mitogenic agents such as phorbol myristate acetate, Ca++, or 25% serum, the absolute levels of PA in cell lysates may either increase or decrease. However, a consistent observation is that in the stimulated cultures PA is found predominantly as a 49,000 dalton species. This also is the predominant form of PA in growing and transformed cells. Concomitant with the mitogen-induced stimulation of the 49,000 dalton PA in quiescent cultures is a change in morphology to one that is characteristic of growing and transformed cells. The data suggest that PA is not operative in causing the morphological change that occurs with activation; however, the 49,000 dalton PA in particular is closely related to the pleiotypic response accompanying growth stimulation and transformation.     

Reactive oxygen species‐induced activation of ERK and p38 MAPK mediates PMA‐induced NETs release from human neutrophils

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013. © 2012 Wiley Periodicals, Inc.     

Protein kinase C-stimulated formation of ethanolamine from phosphatidylethanolamine involves a protein phosphorylation mechanism: negative regulation by p21 Ras protein

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alpha (PKC-alpha)-activated, presently unknown, mechanism. Now we report that addition of a PKC-alpha-enriched purified PKC preparation or recombinant PKC-alpha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly ( approximately 6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-alpha, ATP, and PMA on [(14)C]PtdEtn hydrolysis were inhibited by GF 109203X (10 microM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller ( approximately 3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-alpha is inhibited by p21 Ras.     

Phorbol myristate acetate-induced release of granule enzymes from human neutrophils: inhibition by the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride

Phorbol myristate acetate (PMA) stimulated the extracellular release of the granule-associated enzyme lysozyme from human neutrophils. The extrusion of lysozyme was not accompanied by the release of β-glucuronidase or the cytosol enzyme lactate dehydrogenase. A time dependent PMA-induced release of lysozyme occurred in the absence of extracellular calcium and when neutrophils were preincubated with EGTA. 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of intracellular calcium, caused a dose-dependent inhibition of lysozyme release from neutrophils exposed to PMA in a calcium-free medium. This effect of TMB-8 could be reversed by the addition of calcium to the extracellular medium. These studies indicate that TMB-8 represents a valuable pharmacologic tool used to define the dependence of a secretagogue such as PMA on intracellular as opposed to extracellular calcium.     

PMA-sensitive protein kinase C is not necessary in TRH-stimulated prolactin release from female rat primary pituitary cells.

In GH3 cells and other clonal rat pituitary tumor cells, TRH has been shown to mediate its effects on prolactin release via a rise of cytosolic Ca2+ and activation of protein kinase C. In this study, we examined the role of protein kinase C in TRH-stimulated prolactin release from female rat primary pituitary cell culture. Both TRH and PMA stimulated prolactin release in a dose-dependent manner. When present together at maximal concentrations, TRH and PMA produced an effect which was slightly less than additive. Pretreatment of rat pituitary cells with 10(-6) M PMA for 24 hrs completely down-regulated protein kinase C, since such PMA-pretreated cells did not release prolactin in response to a second dose of PMA. Interestingly, protein kinase C down-regulation had no effect on TRH-induced prolactin release from rat pituitary cells. In contrast, PMA-pretreated GH3 cells did not respond to a subsequent stimulation by either PMA or TRH. Pretreatment of rat pituitary cells with TRH (10(-7) M, 24 hrs) inhibited the subsequent response to TRH, but not PMA. Forskolin, an adenylate cyclase activator, stimulated prolactin release by itself and in a synergistic manner when incubated together with TRH or PMA. The synergistic effects of forskolin on prolactin release was greater in the presence of PMA than TRH. Down-regulation of protein kinase C by PMA pretreatment abolished the synergistic effect produced by PMA and forskolin but had no effect on those generated by TRH and forskolin. sn-1,2-Dioctanylglycerol (DOG) pretreatment attenuated the subsequent response to DOG and PMA but not TRH. The effect of TRH, but not PMA, on prolactin release required the presence of extracellular Ca2+. In conclusion, the mechanism by which TRH causes prolactin release from rat primary pituitary cells is different from that of GH3 cells; the former is a protein kinase C-independent process whereas the latter is at least partially dependent upon the activation of protein kinase C.     

Characterization of the formyl peptide chemotactic receptor appearing at the phagocytic cell surface after exposure to phorbol myristate acetate

We examined the biochemistry and subcellular source of new formyl peptide chemotactic receptor appearing at the human neutrophil and differentiated HL-60 (d-HL-60) cell surface after stimulation with phorbol myristate acetate (PMA). Formyl peptide receptor was analyzed by affinity labeling with formyl-norleu-leu-phe-norleu-[125I]iodotyr-lys and ethylene glycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis of autoradiographs. PMA, a specific granule secretagogue, increases affinity labeling of formyl peptide receptors on the neutrophil surface by 100%, and on d-HL-60, which lack specific granule markers, by 20%. Papain treatment markedly reduces surface labeling of formyl peptide receptor in both neutrophils and d-HL-60, and results in the appearance of a lower m.w. membrane-bound receptor fragment. PMA stimulation of papain-treated cells increases uncleaved surface receptor on neutrophils by 400%, and on d-HL-60 by only 45%. This newly appearing receptor is the same apparent m.w. (55,000 to 75,000 for neutrophils; 62,000 to 80,000 for d-HL-60) and yields the same papain cleavage product (Mr, 31,000 for neutrophils; Mr, 29,000 for d-HL-60) as receptor on the surface of unstimulated cells. Formyl peptide receptor detected by affinity labeling in neutrophil specific granule-enriched subcellular fractions is identical to receptor found on the surface of unstimulated cells appearing as equal amounts of two isoelectric forms (isoelectric points, 5.8 and 6.2) at Mr 55,000 to 70,000. There is twice as much receptor present in the specific granule-enriched fraction per cell equivalent compared with plasma membrane. Azurophil granules contain trace amounts of receptor. Similar analysis of neutrophils treated with papain before subcellular fractionation shows that papain cleaved receptor fragment is detectable almost exclusively in the plasma membrane-enriched fraction. Most of the affinity-labeled formyl peptide receptor present in specific granule enriched fraction is present in membranes other than plasma membrane or Golgi membrane, because specific granule-enriched fraction contains only a small amount of plasma membrane marker and an amount of Golgi membrane marker equal to that found in plasma membrane-enriched fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular reactions in single human granulocytes upon phorbol myristate acetate activation using confocal Raman microspectroscopy

We have obtained new evidence for the occurrence of intracellular NADPH-oxidase activity in neutrophilic and eosinophilic granulocytes upon stimulation with phorbol myristate acetate (PMA). PMA activation leads to a partial translocation of cytochrome b(558) from the membranes of the specific granules to the plasma membrane. It was suggested that NADPH-oxidase activity only takes place in the plasma membrane, leading to an extracellular release of oxygen metabolites because cellular self-destruction can be avoided in this way. The effects of PMA activation were indirectly studied in recent experiments employing scavengers of extracellular superoxide anion and hydrogen peroxide, and support for intracellular NADPH-oxidase activity was obtained. In this paper we use Raman microspectroscopy as a direct method to study intracellular molecular reactions that result from cellular triggering by PMA. The molecular specificity of this microscopic method enables us to show that intracellular reduction of both myeloperoxidase (MPO) and cytochrome b(558) occurs in neutrophilic granulocytes. Control measurements with cytochrome b(558)-deficient neutrophilic granulocytes did not show a reduction of intracellular MPO. This is direct support for the occurrence of intracellular NADPH-oxidase activity in organelles that must be in close contact with the azurophilic granules that contain MPO. Furthermore, a comparison was made with chemical reactions occurring in eosinophilic granulocytes after activation with PMA. Moreover, in these cells an intracellular reduction of eosinophil peroxidase was observed.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Fas/FasL-independent activation-induced cell death of T lymphocytes from HIV-infected individuals occurs without DNA fragmentation

We assessed the effects of activation with phorbol myrystic acetate (PMA) and ionomycin on peripheral blood mononuclear cells (PBMC) from HIV-infected individuals by (51)Cr release, propidium iodide (PI) uptake, electron microscopy, and DNA analysis. Up to 70% (51)Cr release was induced from PBMC of HIV-infected individuals, versus up to 26% (51)Cr release from PBMC of non-HIV-infected volunteers. Flow cytometry identified mostly T cells undergoing activation-induced cell death (AICD). The kinetics of (51)Cr release and the effects of cold target inhibitors were consistent with cell-mediated cytotoxicity. Certain anti-CD3 antibodies or extracellular Ca(2+) chelation prevented AICD, but antagonistic anti-Fas antibodies, caspase inhibitors, and cycloheximide had no effect. The antioxidants thiourea and N-acetylcysteine reduced AICD, indicating a role for oxidative stress. Electron microscopy revealed plasma membrane disruption with nuclear integrity, while DNA analysis showed intact chromosomal DNA. This form of T cell AICD triggered by PMA and ionomycin differs from classical apoptosis in the absence of either caspase involvement or DNA fragmentation.     

Partial characterization of a growth-inhibiting protein in 3T3 cell plasma membranes

A plasma membrane-enriched fraction from 3T3 cells has been detergent-solubilized, and the supernatant of this solubilization was reconstituted into liposomes using soybean lecithin. When these vesicles were added to actively growing cells, cell growth rates were inhibited to levels that were comparable to those observed with the original plasma membranes (at least 50% of maximum growth rates). Liposomes without proteins, or liposomes containing proteins from SV3T3 plasma membranes did not significantly inhibit growth of 3T3 cells. Treatment of the reconstituted vesicles with urea or high concentrations of salt did not eliminate the growth-inhibiting properties of these reconstituted membranes. These results indicate that the specific growth-inhibiting factor in 3T3 cell plasma membranes is a membrane protein that has significant non-polar interactions with the membrane bilayer.     

Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.     

Partial characterization of fusicoccin binding to receptor sites on oat root membranes

The possibility that fusicoccin (FC) binds to plasma membrane-associated ATPases of oat (cv. Victory) roots has been examined. Specific FC-binding in vitro is localized primarily on plasma membrane-enriched fractions. This FC-binding is greatly reduced by pretreatment of the membrane vesicles at temperatures above 45 C or with trypsin, and the same treatments cause the release of already bound FC. These results support the idea that the FC receptor is a protein located on the plasma membrane.     

Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 microM) but is unaffected by the nitric oxide synthase inhibitor N omega-nitro-l-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22 phox, a NOX4 isotype; p47 phox; and p67 phox (real-time PCR). Exposure to the Ca2+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22 phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca2+.     

Alterations in binding of inositol 1,4,5-trisphosphate to subcellular structures of rat liver during chronic endotoxemia

Inositol 1,4,5-trisphosphate (IP3) binding to, and Ca2+ uptake and release by plasma membrane- and endoplasmic reticulum-enriched fractions of rat liver were measured after continuous Escherichia coli endotoxin (ET) administration in vivo. IP3 binding to both fractions was significantly reduced by ET treatment. This was associated with decreased Ca2+ uptake and impaired IP3-dependent Ca2+ release. A decrease of 5'-nucleotidase specific activity of plasma membrane-enriched fraction was also observed in ET treated rats. The results suggest that previously observed impairments in the ability of hepatocytes to mobilize Ca2+, to activate glycogen phosphorylase and to respond--when saponin permeabilized--by Ca2+ release upon IP3 addition during chronic endotoxemia are due to alterations in both IP3 binding to the subcellular fractions that are imputed to be targets of IP3, and a decrease in the size of IP3-sensitive pool of releasable Ca2+.     

Individual C1 domains of PKD3 in phorbol ester-induced plasma membrane translocation of PKD3 in intact cells

Protein kinase D3 is a novel member of the serine/threonine kinase family PKD. The regulatory region of PKD contains a tandem repeat of C1 domains designated C1a and C1b that bind diacylglycerol and phorbol esters, and are important membrane targeting modules. Here, we investigate the activities of individual C1 domains of PKD3 and their roles in phorbol ester-induced plasma membrane translocation of PKD3. Truncated C1a of PKD3 binds [(3)H]phorbol 12, 13-dibutyrate with high affinity, but no binding activity is detected for C1b. Meanwhile, mutations in C1a of truncated C1ab of PKD3 lead to the loss of binding affinity, while these mutations in C1b have little impact, indicating that C1a is responsible for most of the phorbol ester-binding activities of PKD3. C1a and C1b of the GFP-tagged full length PKD3 are then mutated to assess their roles in phorbol ester-induced plasma membrane translocation in intact cells. At low concentration of phorbol 12-myristate 13-acetate (PMA), the plasma membrane translocations of the C1a and C1ab mutants are significantly impaired, reflecting an important role of C1a in this process. However, at higher PMA concentrations, all C1 mutants exhibit increased rates of translocation as compared to that of wild-type PKD3, which parallel their enhanced activation by PMA, implying that PKD3 kinase activity affects membrane targeting. In line with this, a constitutive active PKD3-GFP translocates similarly as wild-type PKD3, while a kinase-inactive PKD3 shows little translocation up to 2 muM PMA. In addition, RO 31-8220, a potent PKC inhibitor that blocks PMA-induced PKD3 activation in vivo, significantly attenuates the plasma membrane translocation of wild-type PKD3 at different doses of PMA. Taken together, our results indicate that both C1a and the kinase activity of PKD3 are necessary for the phorbol ester-induced plasma membrane translocation of PKD3. PKC, by directly activating PKD3, regulates its plasma membrane localization in intact cells.     

Diminished protein kinase C-activated arachidonate metabolism accompanies rat macrophage differentiation in the lung

Alveolar macrophages (AM) differ from other macrophage (m phi) populations in their profile of eicosanoids synthesized from arachidonic acid (AA)3. Little information is available regarding possible differences in the regulation of AA metabolism among various m phi populations. In our study, we compared the ability of cultured resident rat AM and peritoneal m phi (PM) to release and metabolize AA in response to exogenous activators of protein kinase C (PKC). When stimulated with PMA, prelabeled PM released free [3H]AA in a dose-dependent manner over the concentration range 1 to 100 nM. As assessed by HPLC, PMA-stimulated PM metabolized AA to a variety of predominantly cyclooxygenase products. The dose-dependent synthesis of PGE2 by unlabeled PM stimulated with PMA was confirmed using RIA. The ability of PMA to trigger AA release and metabolism in PM was a function of its capacity to activate PKC, as indicated by the following: 1) an additional activator of PKC, oleoyl acetylglycerol, also triggered PM AA metabolism, whereas phorbol didecanoate, which lacks the ability to activate PKC, did not; 2) two structurally unrelated inhibitors of PKC activation (staurosporine and sphinganine) both abrogated PMA induced AA release in PM; and 3) pretreatment for 18 h with high dose PMA (used to deplete cellular PKC), but not phorbol didecanoate, rendered PM refractory to subsequent PMA stimulation of AA release. In contrast to PM, AM cultured in identical fashion failed to release or metabolize AA in response to either PMA or oleoyl acetylglycerol. PM and AM were also compared for their ability to release extracellular superoxide anion in response to PMA; once again, PM exhibited significantly greater release than did AM. Inasmuch as this unresponsiveness to activation of PKC distinguishes AM from other m phi populations, we conclude that it is a unique consequence of m phi differentiation in the lung. Moreover, because both AA metabolism and the respiratory burst are affected, this refractoriness appears to reflect a defect at some proximal level in PKC-mediated signaling.     

Effect of phorbol myristate acetate on release of arachidonic acid and its metabolites in the osteoblastic MOB 3-4 cell line and its subclone, MOB 3-4-F2.

We examined the effect of phorbol 12-myristate 13-acetate (PMA) on release of arachidonic acid (AA) and its metabolites in osteoblastic cells in an attempt to study mechanism of the regulation of phospholipase A2 (PLA2) activity. In the MOB 3-4-F2 cell line, a subclone of the clonal osteoblastic MOB 3-4 cell line, PMA (0.1-100 nM) changed its appearance and increased AA release in a dose- and time-dependent manner, whereas 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) did not show a significant affect on the release. The addition of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, greater than or equal to 1.5 mM), a Ca2+ chelator, almost completely inhibited the PMA-induced AA release without affecting the intrinsic AA release. Preincubation with staurosporine (5-20 nM), an inhibitor of protein kinase C (PKC), partially (approximately 60%) blocked the AA release. However, 30-min preincubation with H-7 (50-200 microM), an inhibitor of PKC, failed to block the AA release. PMA, thus, appeared to stimulate AA release partially by a staurosporine-sensitive mechanism, probably an activation of PKC, in an external Ca(2+)-dependent manner. On the other hand, MOB 3-4 cells responded to PMA with an increased AA release but not with a drastic change of its shape. Both staurosporine and BAPTA exerted similar inhibitory effects. Prolonged exposure (48 h) to PMA (0.1-10 nM) enhanced DNA synthesis of MOB 3-4-F2 cells, but not MOB 3-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)