Hydrogen peroxide-induced phospholipase D activation in rat pheochromocytoma PC12 cells: possible involvement of Ca2+-dependent protein tyrosine kinase

The mechanism for hydrogen peroxide (H2O2)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H2O2-induced PLD activity. Thus, H2O2-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H2O2-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca2+-dependent protein tyrosine kinase(s) somehow participates in H2O2-induced PLD activation in PC12 cells.     

Possible involvement of mitogen-activated protein kinase in phospholipase D activation induced by H2O2, but not by carbachol, in rat pheochromocytoma PC12 cells

We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H2O2-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+-independent cytosolic PLA2-type enzyme was suggested. In contrast to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2-induced PLD activation may be mediated by MAP kinase and also that H2O2-mediated OA release, which would be catalyzed by a Ca2+-independent cytosolic PLA2-like enzyme, is not linked to the PLD activation in PC12 cells.     

Depolarization-induced tyrosine phosphorylation in PC12h cells

Depolarization induced by KCl was found to induce tyrosine phosphorylation of cellular proteins in PC12h cells. By Western blotting with anti-phosphotyrosine antibody, we detected tyrosine phosphorylation of proteins with molecular weights of 120, 110, 105, 95, 75, 70, 66, 44, and 42 kDa in response to KCl. The immunoprecipitates from KCl-treated cells with the antibody contained large amounts of tyrosine-phosphorylated proteins and increased activity of tyrosine kinase. Incubation of the immunoprecipitates with [gamma-32P]ATP resulted in tyrosine phosphorylation of two proteins with the molecular weights of 120 and 140 kDa. These effects were completely abolished by the addition of EGTA before KCl treatment, suggesting that the depolarization-induced tyrosine phosphorylation may require calcium entry into the cells from the medium. Increased activity of tyrosine kinase phosphorylating the 120 and 140 kDa proteins was also recovered from cells stimulated with nerve growth factor, basic fibroblast growth factor, epidermal growth factor, and vasoactive intestinal peptide. Among them, depolarization by KCl elicited the strongest effect. These results indicate that a protein tyrosine kinase that phosphorylate the 120 and 140 kDa proteins is phosphorylated or activated in response to calcium ion, cAMP, and growth factors acting through tyrosine kinase receptors.     

Depolarization-induced tyrosine phosphorylation of paxillin in PC12h cells

Treatment of PC12h cells with a high concentration of KC1 induces depolarization of the plasma membrane and Ca2+ influx into the cells. We have previously shown that KC1 induced tyrosine phosphorylation of cellular proteins of 120, 110, 68, 44 and 42 kDa. In the present study, we found that the 68-kDa protein is paxillin, a tyrosine kinase substrate associated with the actin cytoskeleton. A calcium ionophore, A23187, also induced tyrosine phosphorylation of the 68-kDa protein, while KC1 did not in the presence of EGTA or nifedipine, indicating that the effect of KC1 was due to the Ca2+ influx into the cells. Tyrosine phosphorylation of paxillin was also induced by nerve growth factor and epidermal growth factor, but its migration patterns on an SDS/polyacrylamide gel were different, that is, nerve growth factor and epidermal growth factor caused upward shifts of the bands, while KC1 did not. However, both forms could associate with Csk and Crk. The effect of KC1 was blocked by cytochalasin D, indicating that tyrosine phosphorylation required the integrity of actin filaments. These results suggest that tyrosine phosphorylation of paxillin may be involved in Ca2+ -dependent events in neuronal and neuroendocrine cells.     

Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells

Rat basophilic RBL-2H3 cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen, contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C. These isoforms associated, to variable extents (i.e. delta the most and zeta the least), with the membrane fraction upon antigen stimulation but without external Ca2+; only the Ca(2+)-independent isoforms did so. Both types of isozymes were probably necessary for optimal responses to antigen as indicated by the following observations. All Ca(2+)-dependent isozymes were degraded in cells treated with 20 nM phorbol 12-myristate 13-acetate for 6 h, whereas the Ca(2+)-independent isozymes were not degraded and were retained when the cells were subsequently permeabilized and washed. Cells so treated still exhibited antigen-induced secretion (25-33% of normal) which was suppressed by selective inhibitors of protein kinase C (Ro31-7549 and calphostin C) thereby indicating a possible contribution of the Ca(2+)-independent isozymes in secretion. Normally, washed permeabilized cells lost all isozymes of protein kinase C and failed to secrete in response to antigen. A full secretory response to antigen could be reconstituted by the subsequent addition of nanomolar concentrations of either beta or delta isozymes of protein kinase C (other isozymes were much less effective) but only in the presence of 1 microM free Ca2+ to indicate distinct roles for Ca2+ and protein kinase C in exocytosis.     

Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Calpha (PKCalpha), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCalpha. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2 levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.     

Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells

The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca2+ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

CDK4, a possible critical regulator of apoptosis in rat pheochromocytoma PC12 cells

Rats infused subcutaneously with 9 mg/kg/day nicotine tartrate for 7 days exhibit behavioral abstinence signs following either termination of nicotine infusion or injection of the noncompetitive nicotinic antagonists mecamylamine (s.c.) or hexamethonium (ic.c.v.). This study examined the abstinence precipitating effects of dihydro-beta-erythroidine (DHbetaE), a competitive nicotinic antagonist. Twenty-four nicotine-dependent rats were injected in the third ventricle with 10, 18, or 25 microg DHbetaE in 20 microl saline or with saline alone and observed for abstinence signs over a 20-min period. There was a significant positive linear trend of overall abstinence signs as a function of dose, p < 0.01. In 12 nondependent rats, the high dose of DHbetaE did not induce more abstinence-like signs than saline alone. In a second experiment, 18 nicotine-dependent rats were injected s.c. with 1 or 6 mg/kg of the muscarinic antagonist scopolamine or with saline alone. Few abstinence signs were observed in any group: there was no significant drug effect. The results suggest that nicotine abstinence signs observed in the rat are specific to reduced stimulation of previously overstimulated nicotinic receptors.     

Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line

The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes transient activation of phospholipase C and an enhancement of antigen-induced secretion in a rat mast cell (RBL-2H3) line via adenosine A3-receptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethasone (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NECA synergizes the secretory response to Ca(2+)-ionophore as well as to antigen. The ability of NECA to synergize the secretory responses persisted for 10 to 20 min, long after the early phospholipase C-mediated reactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [3H]phosphatidic acid, or in the presence of 0.3% ethanol, [3H]phosphatidylethanol. This activation was associated with a sustained increase in diglycerides, in protein kinase C activity and in the phosphorylation of myosin light chains by protein kinase C. The generation of diglycerides was enhanced in dexamethasone-treated cells and suppressed in cells that had been treated with cholera toxin or pertussis toxin. Collectively, the studies suggested that the generation of diglycerides via phospholipase D and the associated activation of protein kinase C were, by themselves, insufficient signals for secretion in RBL-2H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]i.     

Characterization of Ca(2+) flows essential in ornithine decarboxylase induction by L-asparagine in rat hepatoma cells using Ca(2+) flow inhibitors

L-Asparagine stimulates bi-directional Ca(2+) flows and induces ornithine decarboxylase in Reuber H-35 hepatoma cells. Previously it has been shown that these effects are completely, but reversibly inhibited by lanthanum chloride. In this study we examined the role(s) of Ca(2+) flows using more specific Ca(2+) flow inhibitors. It was shown that ornithine decarboxylase induction was inhibited by CdCl(2) and verapamil at concentrations above 1 mu M and 100 mu M respectively, but was unaffected by as much as 300 mu M NiCl(2), 1 mM nifedipine, or 10 mu M omega-conotoxin. Enzyme induction was blocked by the Ca(2+)-ATPase pump antagonists vanadate and Compound 48/80 in a dose-dependent manner. These results, taken together with the observations that extracellular Ca(2+) is essential for enzyme induction but a substantial elevation of cytoplasmic [Ca(2+)] is not, suggest that Ca(2+) inflow independent of the receptor-activated Ca(2+) channels, and the Ca(2+)-ATPase mediated Ca(2+) out-flow, are both important factors in the action of L-asparagine.     

Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.     

Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells

Although knowledge of IgA Fc receptor (Fc(alpha)R) structure and gene organization has progressed in the past few years, signal transduction pathways elicited by its activation have hardly been studied. Previously, we have demonstrated that mesangial cells (MC) possess Fc(alpha)R stimulation triggers several biologic responses. In this work, we studied the early biochemical signals triggered by Fc(alpha)R stimulation in MC. MC incubation with aggregated IgA (AIgA) elicited a dose-dependent increase in cytosolic Ca2+ ([Ca2+]i). The response was rapid and transient, and slowly fell to the original baseline. Ca2+ mobilization was dependent on the Fc region of the IgA, because Fc, but neither Fab fragment nor carbohydrates, inhibited the [Ca2+] rise. The initial induction of [Ca2+]i rise was due to Ca2+ mobilization from inositol trisphosphate (IP3)-sensitive intracellular stores, while sustained levels were maintained through extracellular Ca2+ influx. Stimulation of Fc(alpha)R also resulted in production of IP3, temporally correlated with Ca2+ mobilization. Protein tyrosine kinase inhibitors abolished [Ca2+]i rise, indicating that tyrosine phosphorylation of some substrates is required for Ca2+ mobilization. Stimulation through Fc(alpha)R gave rise to a marked increase in tyrosine phosphorylation of several proteins, including the 147-kDa band, similar in size to phospholipase C-gamma(1) (PLC-gamma(1)). Tyrosine phosphorylation of PLC-gamma(1) reached a maximum 30 s after stimulation, as determined by immunoprecipitation and Western blot. Collectively, these results indicate that the Fc(alpha)R signaling pathway in MC involves PLC-(gamma(1) activation, IP3 formation, and Ca2+ mobilization, and is linked to activation of tyrosine kinases.     

Immunocytochemical localization of Ca(2+)-dependent protease from Allomyces arbuscula

The Ca(2+)-dependent protease antisera and the purified specific antibodies from Allomyces arbuscula have shown very specific recognition when blotted against the total protein extract or the purified 43-40 kDa Ca(2+)-dependent protease from this aquatic fungus. By immunoblotting and immunofluorescence techniques using specific antibodies, we have shown that the enzyme activity is developmentally regulated and is related to the presence of antigen and not to any specific inhibitor. The immunofluorescence was absent in zoospores but appeared in polarized forms in germinating spores. In elongating hyphae the protease was mainly localized along the cytoplasmic membrane and in the cytoplasm, with predominance at the apex.     

Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells

The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.     

Ca2+/Mg(2+)-dependent endonuclease in marrow CD34 positive and erythroid cells in myelodysplasia

Endonucleases capable of producing internucleosomal DNA cleavage are one of the key enzymes in apoptosis. We examined endonuclease activities contained in nuclei of CD34+ and erythroid cells in the bone marrow (BM) from 12 patients with the myelodysplastic syndromes. The levels of Mg(2+)-dependent and acidic endonucleases showed little changes as compared with those from normal BM. By contrast, the level of Ca2+/Mg(2+)-dependent endonuclease was appreciably higher in MDS erythroid cells than normal counterparts, although the activity varied markedly in CD34+ and erythroid cells. Our results suggested that Ca2+/Mg(2+)-dependent endonuclease is related to ineffective erythropoiesis in MDS.     

Beta 1 integrin ligation stimulates tyrosine phosphorylation of phospholipase C gamma 1 and elevates intracellular Ca2+ in pancreatic acinar cells

We have recently reported increased tyrosine (TYR) phosphorylation of a number of pancreatic acinar cell proteins following antibody ligation of beta 1 integrins (Wrenn and Herman, Biochem, Biophys. Res. Commun. 208, 1995, 978-984). Concurrent with this TYR phosphorylation was a marked activation of protein kinase C (PKC). This led us to investigate phospholipase C gamma 1 (PLC gamma 1), a key enzyme responsible for diacylglycerol generation, as a target for integrin-mediated TYR phosphorylation. Staining with antiphosphotyrosine antibodies revealed increased TYR phosphorylation of immunoprecipitated PLC gamma 1 prepared from beta 1 integrin-ligated acinar cells. Subsequent stripping and reprobing of Western blots with polyclonal anti-PLC gamma 1 was confirmatory. Over this same time period, intracellular [Ca2+] increased from < 100 nM to 600 nM, further suggesting a functional relevance of integrin-linked phosphorylation as a regulatory mechanism in exocrine pancreas.     

A non-radioactive, sensitive method for the detection of DNA fragmentation in apoptotic cells (rat pheochromocytoma PC12 and rat cortical cells)

Cartilage diseases include a wide variety of clinical phenotypes from common osteoarthrosis to several different types of chondrodysplasias, i.e. 'disorders of cartilage', of which more than 100 different have been described. Patients frequently suffer from various symptoms affecting their joints and/or the growth of their long bones. The amount of hyaline cartilage at articular surfaces is often diminished and structurally abnormal. The surface of the cartilage may have an irregular appearance with defects extending into the subchondral bone. The major constituents of this hyaline cartilage are collagens and proteoglycans, the most abundant protein being type II collagen. It is a homotrimer of three identical alpha-chains, which are encoded by a single gene on human chromosome 12. The gene for type II collagen therefore became a likely candidate for some forms of chondrodysplasias and cartilage degeneration. Recently, both linkages and exclusions between this gene and various cartilage diseases have been reported and a growing number of mutations within the gene have also been identified.     

Characterization of acidic Ca(2+)-independent phospholipase A2 of bovine lung

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.