Distribution of protein kinase C isoforms after infection of macrophages with Leishmania major

We characterized the effects of Leishmania infection on activation-induced translocation of protein kinase C (PKC) isoforms in murine bone marrow-derived macrophages. Although PKC-dependent gene expression was attenuated by infection, the distribution and translocation of PKC isoforms were unaffected. However, subsequent dissociation from membranes was substantially delayed for some isoforms, particularly PKCbeta.     

Experimental infection of dogs with the nasal mite Pneumonyssoides caninum

Cisplatin (CP) has been reported to activate murine macrophages to tumoricidal state, however, its mechanism of action is not known. In the present study it is reported that the production of: (a) interleukin-1 (IL-1); (b) tumor necrosis factor (TNF); (c) nitric oxide (NO); and (d) macrophage-mediated cytotoxicity by cisplatin-treated bone marrow-derived macrophages were inhibited by PKC inhibitors H-7 and chelerythrine chloride. Also, it was observed that treatment of macrophages with CP resulted in the translocation of PKC from the cytosol to the membrane fraction. These findings suggest the involvement of PKC in the activation of bone marrow-derived macrophages with cisplatin.     

Selective role for beta-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells

A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete beta-PKC, both betaI- and betaII-PKC, but not alpha-PKC, delta-PKC, or zeta-PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells). Depletion of beta-PKC in dHL60 cells elicited selective inhibition of O-2 generation triggered by fMet-Leu-Phe, immune complexes, or phorbol myristate acetate, an activator of PKC. In contrast, neither ligand-elicited beta-glucuronidase (azurophil granule) release nor adherence to fibronectin was inhibited by beta-PKC depletion. Ligand-induced phosphorylation of a subset of proteins was reduced in beta-PKC-depleted dHL60 cells. Phosphorylation of p47(phox) and translocation of p47(phox) to the membrane are essential for activation of the NADPH oxidase and generation of O-2. beta-PKC depletion had no effect on the level of p47(phox) in dHL60 cells but did significantly decrease ligand-induced phosphorylation of this protein. Furthermore, translocation of p47(phox) to the membrane in response to phorbol myristate acetate or fMet-Leu-Phe was reduced in beta-PKC-depleted cells. These results indicate that beta-PKC is essential for signaling for O-2 generation but not cell adherence or azurophil degranulation. Depletion of beta-PKC inhibited ligand-induced phosphorylation of p47(phox), translocation of p47(phox) to the membrane, and activation of O-2 generation.     

Decreased protein kinase C activation mediates inhibitory effect of norathyriol on serotonin-mediated endothelial permeability

We examined the mechanisms of norathyriol on the serotonin-induced increased permeability of rat heart endothelial cell monolayers. The present study showed that the activation of rat heart endothelial cell protein kinase C by phorbol myristate acetate led to the dose-dependent increase in endothelial permeability to albumin, an effect that was inhibited by staurosporine (a protein kinase inhibitor). Staurosporine also attenuated the serotonin-induced increase in permeability. Norathyriol abolished both serotonin- and phorbol myristate acetate-induced permeability. We investigated whether norathyriol, by inhibiting protein kinase C activation, attenuated the serotonin-induced permeability. Immunofluorescence studies demonstrated that norathyriol prevented the redistribution of protein kinase C isozymes following stimulation with serotonin. Western blot analysis showed that norathyriol significantly inhibited the serotonin-induced translocation of the alpha protein kinase C isozyme from the cytosolic to the particulate fraction. In conclusion, norathyriol attenuates the serotonin-induced permeability of rat heart endothelial cells to macromolecules in association with inhibition of protein kinase C activation. This decrease in endothelial cell permeability may be one of the mechanisms for the protective effects of norathyriol against edema formation in response to inflammatory agonists in vivo.     

pH dependence of the formation of an M-type intermediate in the photocycle of 13-cis-bacteriorhodopsin

Phosphatidic acid (PA) dose-dependently induced superoxide (O2-) production of electropermeabilized human neutrophils but not of intact neutrophils, indicating that PA induces the activation of NADPH oxidase by acting on an intracellular target. The O2- production by PA was not inhibited by protein kinase C (PKC) inhibitors, such as staurosporine and calphostin C, and an inhibitor of PA phosphohydrolase, propranolol. These observations suggest that the activation of the oxidase by PA is independent of the activity of PKC and may dominate the activation by diacylglycerol which is formed from PA via the action of PA phosphohydrolase. Furthermore, the production by PA, as well as that by phorbol myristate acetate, was inhibited by cyclic AMP and GDP beta S. Therefore, PA seems to act at a site downstream of PKC.     

Cisplatin-induced activation of murine bone marrow-derived macrophages require protein tyrosine phosphorylation

The aim of the present study is to evaluate the involvement of tyrosine phosphorylation in the signal transduction mechanism of cisplatin-induced macrophage activation in vitro. Stimulation of bone marrow-derived macrophages (BMDM) with cisplatin (CP) resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 18, 20, 21, 30, 33, 35, 39, 41, 44, 58 and 123 kD, detected by immunoblot using anti-phosphotyrosine antibody. CP-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor genistein. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of CP on murine bone marrow-derived macrophages (BMDM). Treatment of macrophages with genistein before incubation with CP completely inhibited the CP-induced tumoricidal activation of macrophages as well as production of TNF and NO, whereas pre-treatment of macrophages with phosphatase inhibitor sodium vanadate upregulated macrophage activation in addition to enhanced protein tyrosine phosphorylation. Taken together, these data suggest that tyrosine phosphorylation play a critical regulatory role in the activation of macrophages with CP.     

Binding profile of the novel 5-HT1B/1D receptor antagonist, [3H]GR 125,743, in guinea-pig brain: a comparison with [3H]5-carboxamidotryptamine

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M3 subtype. Acute exposure to phorbol esters (1 microM phorbol 12,13-dibutyrate, PDB, or 0.1 microM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 microM) and H7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 microM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [3H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.     

Responses of neurons of the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic tract in the awake monkey

The in vitro effect of recombinant human Granulocyte Macrophage Colony Stimulating Factor (rh-GMCSF) on the leishmanicidal activity and superoxide anion productivity of macrophages derived from human blood monocytes (MOs) were investigated. MOs treated with 25, 125, or 250 U/mL of rh-GMCSF for 72 h prior to infection with leishmania parasites, manifested significant dose-dependent increase in its leishmanicidal activities against Leishmania major and Leishmania donovani parasites. The percentage of increase in leishmanicidal activity of L. major-infected MOs were 22.71, 64.34 and 81.34, respectively while in L. donovani-infected MOs, it reached 3.01, 32.28 and 74.38, respectively. Treatment of leishmania-infected MOs with rh-GMCSF (250 U/mL) for different periods of time up to 96 hours, induced a significant time-dependent reduction in the percentage of infected cells and the parasitic load (No. of amastigotes/100 MOs). After 96 h of treatment with rh-GMCSF, the percentages of reduction in the infection rates were 82.45 in L.major-infected MOs (p < 0.001) and 39.65 in L. donovani-infected cells (p < 0.01). The percentage of reduction in the parasitic load reached 90.82 (p < 0.001) and 36.6 (p < 0.05) in MOs infected with L. major and L. donovani, respectively. The priming effect of rh-GMCSF on superoxide anion production by human MOs stimulated with phorbol myristate acetate (PMA) was both dose-dependent and time-dependent. In 72 hour-old human MOs, the maximum superoxide anion release was generated by MOs primed for 45 min with 500 U/mL of rh-GMCSF. These cells produced 8.960 +/- 2.075 nmol/5 x 10(4) MOs/ 180 min as compared to 4.563 +/- 1.773 nmol/5 x 10(4) unprimed cell control/180 min (p < 0.001).     

A heritable component for external apical root resorption in patients treated orthodontically

Double-stimulation was used to demonstrate that, in a T lymphocytic cell line (CEM), phorbol myristate acetate (PMA) rapidly induced NF-kappa B through a signaling pathway which did not involve reactive oxygen species (ROS) and was different from the activation triggered by either H2O2 or tumor necrosis factor-alpha (TNF-alpha). Since these latter compounds were known to activate NF-kappa B translocation in a redox-sensitive way, we have demonstrated that NF-kappa B activation by PMA was resistant to antioxidant N-acetyl-L-cysteine (NAC) and sensitive to kinase inhibitors staurosporine and H7 while activation by H2O2 or TNF-alpha were not.     

Leishmania major infection in C57BL/10 mice differing at the Lps locus: a new non-healing phenotype

The course of cutaneous leishmaniasis was examined in mice from two genetically closely related strains, C57BL/10ScCr (Cr) and C57BL/10ScSn (Sn). Sn mice are able to heal Leishmania major infections, while Cr mice are unable to heal. The cutaneous lesions of the Cr mice progressed continuously and the increase in lesion size was paralleled by an unrestricted growth of the parasites in vivo. Cr mice, in contrast to their Sn counterparts, are highly resistant to all effects of lipopolysaccharide (LPS). The nonhealing L. major infection in Cr mice is in sharp contrast to the course of infection in another endotoxin-nonresponder mouse strain, C3H/HeJ, which heal infections with L. major. Cr mice exhibit, in addition to the defective LPS responsiveness, an impaired interferon-gamma (IFN-gamma) response after infection with a variety of microorganisms. The insufficient activation of parasitized macrophages to kill intracellular L. major could be due to the inability of splenocytes from infected Cr mice to secrete IFN-gamma upon restimulation with L. major. IFN-gamma is essential for the efficient activation of parasitized macrophages to kill intracellular L. major by producing nitric oxide (NO). Although bone marrow-derived Cr macrophage do not produce NO in response to LPS, both Sn and Cr macrophages release NO upon stimulation with IFN-gamma and tumor necrosis factor, indicating that they are responsive to activation by these cytokines.     

Influence of twinline, an elemental diet, on the generation of nitric oxide and reactive-oxygen intermediates from mouse peritoneal macrophages and polymorphonuclear leukocytes

The influence of Twinline (SNN-6010), an elemental diet containing medium-chain triglycerides, on the generation of nitric oxide (NO) and superoxide (O2.-) has been examined in mouse peritoneal macrophages and polymorphonuclear leukocytes (PMN). When PMN and peritoneal macrophages obtained from untreated mice were cultured in medium containing 0.1% and 1% (v/v) Twinline for 48h and stimulated with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine, their chemiluminescence and O2.- generation were strongly suppressed, as was NO generation from peritoneal macrophages. PMN and peritoneal macrophages obtained from mice fed Twinline for 30 days generated much smaller amounts of 02.- and NO compared with PMN and peritoneal macrophages from control mice. In conjunction with this suppressed NO generation, inducible NO synthase and its mRNA expression in peritoneal macrophages were suppressed by Twinline both in-vivo and ex-vivo. Although phagocytosis of PMN and peritoneal macrophages was not suppressed by Twinline; their candida-killing activity was markedly suppressed. These results indicate that Twinline suppresses the host-defence function of PMN and peritoneal macrophages by down-regulating their generation of reactive-oxygen intermediates and NO.     

Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actin-crosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin.     

The influence of various factors on breast-feeding in Slovenia

During natural infections, Leishmania is in contact with a variety of mononuclear phagocytic cells in different tissues, including resident macrophages and monocytes mobilized to the site of infection from the bone marrow and blood circulation. Because the functional capabilities of fully differentiated macrophages and blood monocytes differ, the outcome of infection by Leishmania may depend upon the stage of differentiation of the host cells. To address this question, we evaluated Leishmania panamensis infection of (1) the human promonocytic/histiocytic cell line U-937 before and after induction of differentiation by phorbol myristate acetate; (2) fresh human peripheral blood monocytes; and (3) macrophages derived from monocytes by differentiation in vitro. Based on the percentage of cells infected and the number of parasites per cell, macrophages derived from monocytes or by induction of differentiation of U-937 cells were significantly more permissive to infection by stationary-phase L. (Viannia) panamensis promastigotes than monocytes. Increasing time and maturation in culture prior to exposure to infective promastigotes was associated with the increased permissiveness of differentiated macrophages to infection (P<0.05). The percentage of cells infected and number of amastigotes per cell increased with time postinfection for both monocytes and macrophages but remained significantly greater for macrophages. The increased expression of CD68, CD16, and lysozyme, and decreased expression of peroxidase by macrophages cultured for 5 days in vitro compared with fresh monocytes, whether adherent or in suspension, supported the distinct maturation status of these cells.     

Reduced expression of lipophosphoglycan (LPG) and kinetoplastid membrane protein (KMP)-11 in Leishmania donovani promastigotes in axenic culture

The expression of surface lipophosphoglycan (LPG) and the lipophosphoglycan-associated kinetoplastid membrane protein (KMP)-11 was studied in the strain AG83 of Leishmania donovani in axenic culture. The expression of LPG and KMP-11 decreased along with parasite virulence as a function of the time of the subculture.     

2-Hydroxypropyl-beta-cyclodextrin enhances phorbol ester effects on glucose transport and/or protein kinase C-beta translocation to the plasma membrane in rat adipocytes and soleus muscles

In rat adipocytes and soleus muscles, 2-hydroxypropyl-beta-cyclodextrin (CD) was found to have a relatively small or no effect on basal or insulin-stimulated hexose uptake, but markedly enhanced hexose uptake effects of phorbol esters and/or diacylglycerol. In rat adipocytes, the CD-induced enhancement of hexose uptake during concurrent phorbol ester treatment was not associated with an increase in GLUT4 glucose transporter translocation to the plasma membrane, which was stimulated comparably by insulin and phorbol esters. Moreover, CD appeared to activate or facilitate the activation of glucose transporters subsequent to their translocation to the plasma membrane during ongoing phorbol ester treatment. In rat adipocytes, CD also enhanced the translocation of protein kinase C (PKC)-beta to the plasma membrane during the action of phorbol esters, which alone had little or no effect on this specific PKC translocation. Although it is uncertain how CD alters the function of plasma membranes to enhance the translocation of PKC-beta to, and the activation of glucose transporters within, this subcellular fraction during phorbol ester treatment, our findings provide direct support for a two-step model in the activation of glucose transport. In addition, it seems clear that, at least in some cell types, simple phorbol ester treatment does not necessarily serve as a ubiquitous activator of all activable PKC pools and all potential PKC-mediated responses.     

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.     

Modulation of activator protein-1 (AP-1) transcription factor and protein kinase C by hydrogen peroxide and D-alpha-tocopherol in vascular smooth muscle cells

The effects of hydrogen peroxide D-alpha-tocopherol and of D-beta-tocopherol on proliferation, protein kinase C and activator protein-1 (AP-1) activation have been studied in vascular smooth muscle cells. Cell proliferation, when activated by foetal calf serum, was inhibited by D-alpha-tocopherol. Protein kinase C activity was stimulated by hydrogen peroxide in a manner similar to phorbol myristate acetate; in the latter case, but not in the former, D-alpha-tocopherol inhibited the reaction. Hydrogen peroxide prevented phorbol-myristate-acetate-stimulated AP-1 binding to DNA but stimulated it if protein kinase C was down-regulated or inhibited. D-alpha-Tocopherol promoted AP-1 activation in quiescent cells but prevented its activation by phorbol myristate acetate. None of the described effects of D-alpha-tocopherol were shared by D-beta-tocopherol, suggesting a non-antioxidant mechanism as the basis of its action. The data show that hydrogen peroxide and D-alpha-tocopherol affect more than one element in the cell signal-transduction cascade.