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Neutrophil extracellular traps (NETs) formation is a key process in inflammatory diseases like gout, but the underlying molecular mechanisms remain incompletely understood. This study aimed to establish a model to examine the formation of NETs induced by monosodium urate (MSU) and phorbol 12-myristate 13-acetate (PMA) and to elucidate their molecular pathways. Laser confocal microscopy was used to visualize NET formation, while flow cytometry was employed to detect reactive oxygen species (ROS) production. The microstructure of neutrophils was observed by transmission electron microscopy, and the expression of key proteins was determined by Western blotting. Additionally, the effect of various inhibitors targeting the MAPK signaling pathway on NET formation was evaluated. They include the Ras inhibitor Salirasib, Raf inhibitor Vemurafenib, ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, as well as NADPH oxidase inhibitor DPI and neutrophil elastase inhibitor Alvelestat. The results showed that MSU and PMA triggered significant NET formation, which was accompanied by increased ROS levels, lactate dehydrogenase release, dsDNA, and IL-8. Notably, selective MAPK pathway inhibitors and DPI and Alvelestat, except for SB203580, effectively down-regulated these indicators. These data indicated that the activation of a signaling pathway involving Ras-Raf-ERK, which is dependent on ROS, is crucial for the induction of NET formation by MSU and PMA. Given the involvement of NETs in multiple pathologies, our findings could potentially serve as molecular targets for the intervention and treatment of crystal-related diseases, especially for gout. [ABSTRACT FROM AUTHOR]

People's health is adversely impacted by diseases caused by abnormal inflammation; therefore, more effective treatments as well as research into the underlying mechanisms are urgently required. According to our preliminary research, geniposidic acid exhibits anti-inflammatory properties. Even though geniposidic acid has been linked to inflammation and shows promise in the treatment of aberrant inflammatory illnesses, its exact antiinflammatory mechanism is unknown. To create in vitro cellular inflammatory models for 48 h, we employed THP- 1 cells treated with PMA (phorbol 12-myristate 13-acetate) and LPS (lipopolysaccharide), which were then treated with geniposidic acid. These models were further validated by ELISA-based detection of the expression of the interleukins IL-1, IL-6, IL-8, and tumor necrosis factor TNF-κ. The anti-inflammatory properties of geniposidic acid were investigated using proteomics based on TMT-LC-MS/MS analysis and bioinformatic analysis. Geniposidic acid can act as an anti-inflammatory agent in inflammatory cell models, according to the successfully established THP-1 cell inflammatory models. GO and KEGG analyses further established numerous bioprocesses, molecular activities, and signaling pathways related to the regulation of geniposidic acid. Additionally, western blot analysis confirmed that geniposidic acid can suppress inflammation by lowering the expression of proteins linked to inflammation such as CXCL10, PPM1H, and TLR5. After more thorough mechanistic, animal, and clinical research were included, the current study revealed that anti-inflammatory efficacy of geniposidic acid from the standpoint of proteomics made it a promising medicine to cure and prevent diseases caused by aberrant inflammation. [ABSTRACT FROM AUTHOR]

Background: Neutrophil extracellular traps (NETs) consist of chromatin DNA networks that are studded with cytosolic and granular antimicrobial proteins to trap or kill an infected microorganism. A lipid emulsion, the solvent of pure propofol for intravenous application, is given to clinical patients who require intravenous feeding of fatty acids and fat for energy. Intravenous propofol is widely used to sedate critically ill patients. Both intravenous propofol and its lipid emulsion have immunomodulatory activity. However, the role of lipid emulsion of intravenous propofol on NET induction remains unclear. Methods: In this study, neutrophils were stimulated with phorbol myristate acetate (PMA) or Escherichia coli (E. coli) in the absence or presence of intravenous propofol (Propofol-Lipuro®), its solvent lipid emulsion (Lipofundin) or pure propofol, and NETs were stained with SYTOX Green for visualization and quantification. Total HOCl was determined by measuring the taurine-chloramine complex, and intracellular HOCl was evaluated with BioTracker™ TP-HOCl 1 dye. Results: PMA-induced NETs were not efficiently inhibited when Propofol-Lipuro® was added after PMA stimulation. Clinically relevant concentrations of Lipofundin exerted a significant reduction in PMA-induced NETs and total reactive oxidative species (ROS), which was comparable to that observed for Propofol-Lipuro®. Lipofundin transiently reduced intracellular HOCl production and the phosphorylation level of extracellular regulated kinase (p-ERK) but did not scavenge HOCl. Moreover, Lipofundin decreased E. coli-induced NETs in a ROS-independent pathway, similar to Propofol-Lipuro®. Conclusions: All data agree that Lipofundin, the major component of Propofol-Lipuro®, inhibits intracellular HOCl and p-ERK to suppress PMA-induced NET formation but reduces E.coli-induced NETs in a ROS-independent pathway. [ABSTRACT FROM AUTHOR]

Background: Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells., Methods: Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE 2 ) levels were measured using enzyme-linked immunosorbent assay., Results: Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE 2 , and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered., Conclusions: PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE 2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented., Competing Interests: CONFLICT OF INTEREST: No potential conflict of interest relevant to this article was reported., (Copyright © 2024 by The Korean Orthopaedic Association.)

Introduction: The formation of neutrophil extracellular traps (NETs) is a process in which several kinds of enzymes participate generating posttranslational modifications of proteins. NETs have been associated with infectious, autoimmune, and inflammatory diseases. Inhibition of several proteases reduces the formation of NETs. In the present work, we analyzed the role of several broad-acting and specific inhibitors of proteases in the formation of NETs. Methods: Neutrophils were isolated from peripheral blood of healthy individuals by density gradient. The neutrophils were quantified and seeded into cell culture plates. Phorbol myristate acetate and A23187 were used as NETs inducers, and several specific inhibitors of proteases were used. The cells were stained for cytoskeleton or DNA. The cell-free supernatants were used to assess DNA release. Statistical analysis was carried out by a Kruskal-Wallis or ANOVA test. Results: We observed marked changes in actin organization after the induction of NETs, suggesting that the cytoskeleton is being actively regulated. When we used protease inhibitors, the release of DNA was reduced, suggesting the participation of actin remodeling in the process. Further characterization of the specific proteases revealed that calpain modulates the reorganization of actin cytoskeleton and DNA release. Preservation of part of the actin cytoskeleton suggests that DNA release is not only a mechanic process associated to the chromatin decondensation; rather the process is highly regulated by active proteases that promote cytoskeleton reorganization and chromatin decondensation that culminates in DNA release. Conclusion: Calpain mediates the DNA release in the NET formation process by the modification of cortical actin cytoskeleton in a calcium-dependent manner. [ABSTRACT FROM AUTHOR]

THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Here we elucidated whether NO relates to PMA-induced monocyte-to-macrophage differentiation of THP-1 cells. The mutual regulation of calcium and NO was also investigated. THP-1 cells were incubated with PMA for 24 h, followed by assay of adherence, morphological change, migration or IL-1β release. L-NG-Nitroarginine methyl ester (l -NAME, a nitric oxide synthase inhibitor) or BAPTA-AM (a calcium chelator) was added before PMA stimulation, and levels of calcium and NO were measured. Furthermore, a selective inhibitor of inducible nitric oxide synthase (iNOS) activity was employed to study the role of iNOS. Effects of PMA on upregulation of adherence, lipopolysaccharide-triggered IL-1β, and migration ability of THP-1 cells were consistent with NO concentrations. Both l -NAME and BAPTA-AM mitigated effects of PMA on THP-1 cells differentiation. BAPTA-AM decreased levels of NO, while l -NAME had no effect on calcium levels. Of note, inhibition of iNOS activity decreased PMA-triggered upregulation of NO. PMA induced differentiation of THP-1 cells partially in a NO-dependent manner. The calcium signaling may mediate PMA-triggered upregulation of NO. • Phorbol myristate acetate induces monocyte-to-macrophage differentiation ofTHP-1 cells. • Differentiation of THP-1 cell partially depends on inducible nitric oxide synthase-triggered nitric oxide. • Calcium signaling may mediate upregulation of nitric oxide by phorbol myristate acetate. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1β transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals. [ABSTRACT FROM AUTHOR]

Phorbol 12-myristate 13-acetate (PMA) promotes skin cancer in rodents. The mutations found in murine tumors are similar to those found in human skin cancers, and PMA promotes proliferation of human skin cells. PMA treatment of human keratinocytes increases the synthesis of APOBEC3A, an enzyme that converts cytosines in single-stranded DNA to uracil, and mutations in a variety of human cancers are attributed to APOBEC3A or APOBEC3B expression. We tested here the possibility that induction of APOBEC3A by PMA causes genomic accumulation of uracils that may lead to such mutations. When a human keratinocyte cell line was treated with PMA, both APOBEC3A and APOBEC3B gene expression increased, anti-APOBEC3A/APOBEC3B antibody bound a protein(s) in the nucleus, and nuclear extracts displayed cytosine deamination activity. Surprisingly, there was little increase in genomic uracils in PMA-treated wild-type or uracil repair-defective cells. In contrast, cells transfected with a plasmid expressing APOBEC3A acquired more genomic uracils. Unexpectedly, PMA treatment, but not APOBEC3A plasmid transfection, caused a cessation in cell growth. Hence, a reduction in single-stranded DNA at replication forks may explain the inability of PMA-induced APOBEC3A/APOBEC3B to increase genomic uracils. These results suggest that the proinflammatory PMA is unlikely to promote extensive APOBEC3A/APOBEC3B-mediated cytosine deaminations in human keratinocytes. [ABSTRACT FROM AUTHOR]

LPA 1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA 1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA 1 -Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA 1 -Rab5 interaction and receptor internalization. LPA-induced LPA 1 -Rab9 interaction was only observed at 60 min, and LPA 1 -Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA 1 -Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA 1 -Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA 1 receptor internalization varies with the stimuli. [Display omitted] • LPA and PMA induce LPA 1 internalization with different time-courses. • LPA 1 -Rab protein FRET was employed to analyze receptor trafficking. • LPA- and PMA-induced differential LPA 1 early endosome association and recycling. • LPA and PMA induce LPA 1 internalization through distinct endocytic routes. [ABSTRACT FROM AUTHOR]

ObjectivesDoxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated.MethodsIn human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis.ResultsPMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9.ConclusionThe results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.

Moxifloxacin is useful in the treatment of respiratory infections, including community-acquired pneumonia, and also shows promise in the treatment of tuberculosis, a clinical setting which necessitates extended administration of this agent. Relatively little is known, however, about the effects of this agent on the antimicrobial and proliferative activities of human neutrophils and T-lymphocytes, respectively. In the current study, we have investigated the effects of moxifloxacin at therapeutic concentrations and greater (1–20 µg/mL) on cytosolic Ca2+ fluxes, generation of antimicrobial reactive oxygen species (ROS), and release of the primary granule protease, elastase, following activation of the cells with the chemoattractant, fMLP (1 µM), or the phorbol ester, PMA (25 ng/mL), using radiometric, chemiluminescence, and colourimetric procedures, respectively. The effects of moxifloxacin on mitogen-activated proliferation of T cells and expression of the interleukin-2 (IL-2) receptor (CD25) were measured using radiometric and flow cytometric procedures respectively. With the exception of elastase release, which was significantly increased (P < 0.05) by treatment of the cells with moxifloxacin at 10 and 20 µg/mL, none of the other neutrophil or lymphocyte functions was affected by moxifloxacin. These observations suggest that extended use of this agent is unlikely to compromise the protective functions of neutrophils and T-lymphocytes and may even potentiate neutrophil-mediated antimicrobial activity by increasing the release of elastase.

Objective: Oral lichen planus (OLP) is a T cell-mediated inflammatory condition in the oral cavity. Mucosal-associated invariant T (MAIT) cells are gaining more relevance in immune diseases because they can be activated by cytokines without T cell receptor stimulation. Herein, we tested the effect of interleukin-23 (IL-23) on the activation status of OLP MAIT cells., Methods: Peripheral blood mononuclear cells (PBMCs) isolated from OLP patients were stimulated by IL-23 in the absence or presence of phorbol myristate acetate (PMA) and ionomycin. The activation status of MAIT cells was analyzed by flow cytometry after staining for CD3, CD4, CD8, CD161, TCR Vα7.2, and CD69., Results: The fraction of MAIT cells in OLP peripheral blood was approximately 0.38% to 3.97%, and CD8 + subpopulations overwhelmed CD4 + cells. The mean percentages of OLP MAIT cells in PBMCs and CD8 + MAIT cells in MAIT cells were approximately 40%. PMA and ionomycin significantly increased CD69 expression on OLP T cells, MAIT cells, and CD8 + MAIT cells. Cells with enhanced activation had different responsiveness to exogenous IL-23, showing increased CD69 expression on OLP T cells, decreased CD69 on OLP CD8 + MAIT cells, and no significant change on OLP MAIT cells., Conclusions: IL-23 showed different effects on the activation status of OLP MAIT cells and CD8 + MAIT cells.

Background: We previously reported that phorbol 12-myristate 13-acetate (PMA) treatment can induce the cardiac differentiation of mesenchymal stem cells (MSCs). In the present study, we investigated how PMA induces cardiac differentiation of MSCs, focusing on its effect on the transcription factors responsible for increased cardiac marker gene expression. Methods: Human MSCs (hMSCs) were treated with 1 μM PMA for 9 days. The expression of MSC markers and cardiac markers in the PMA-treated hMSC, as well as the nuclear translocation of transcription factors, nuclear factor of activated T cells (NFAT), and myogenic differentiation 1 (MyoD), was examined. Transcriptional activity of NFAT was examined by utilizing a green fluorescent protein (GFP) vector containing NFAT motif of human interleukin-2 promoter. The effect of PMA on the expression of key cell cycle regulators was examined. Results: PMA induces the transcriptional activity of NFAT and MyoD, which have been associated with increased expression of cardiac troponin T (cTnT) and myosin heavy chain (MHC), respectively. Our data suggested that protein kinase C (PKC) mediates the effect of PMA on NFAT activation. Furthermore, PMA treatment increased cell-cycle regulator p27kip1 expression, suggesting that PMA triggers the cardiac differentiation program in MSCs by regulating key transcription factors and cell cycle regulators. Conclusions: The results of this study demonstrate the importance of NFAT activation during PMA-induced MSC differentiation and help us to better understand the underlying mechanisms of small molecule-mediated MSC differentiation so that we can develop a strategy for synthesizing novel and improved differentiation-inducing small molecules. [ABSTRACT FROM AUTHOR]

Phorbol myristate acetate (PMA), as a potent tumor promoter, may induce microglial senescence. The present study investigated the effect of PMA infection on microglial senescence. From 58 male Sprague-Dawley rats, 10 were randomly selected and divided into a PMA injection group, containing five rats (0.5 μg/μl PMA) and a control group, containing five rats (commensurable 0.9% saline). Immunofluorescent staining of Iba-1 and enzyme-linked immunosorbent assay analyses of the expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1 β were performed in these two groups. The remaining 48 rats were randomly divided into the following three groups, each containing 16 rats: Repeated injection control group (commensurable normal saline, once a week for 4 weeks), single PMA injection group (0.5 μg/μl PMA, once in the first week) and repeated injection PMA group (0.5 μg/μl PMA, once a week for 4 weeks). The expression levels of p21, detected using double immunofluorescence staining with Iba-1, and β-galactosidase, via double immunohistochemical staining of Iba-1, were examined in these three groups. The results indicated that a single injection of PMA did not change the microglial morphology and had no significant effects on the expression levels of TNF-α and IL-1β, compared with the control group (P>0.05). Following four repeated injections of PMA, the microglia in the substantia nigra presented with features of senescence, characterized by increased expression levels of β-galactosidase (P<0.001) and p21 (P<0.001), compared with the repeated injection control group. In conclusion, repeated intra-nigrostriatal treatment with PMA induced microglial senescence with increased expression levels of β-galactosidase and p21 in the substantia nigra of the rats. [ABSTRACT FROM AUTHOR]

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase ( AMPK), extracellular signal-regulated kinase ( ERK), and autophagy in phorbol myristate acetate ( PMA)-induced neuronal differentiation of SH- SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 ( LC3)-I to autophagosome-bound LC3- II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH- SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH- SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ ERK signaling are counteracted by the coinciding autophagic response. [ABSTRACT FROM AUTHOR]

The respiratory burst is a rapid cellular consumption of oxygen resulting in abundant production of reactive oxygen species (ROS), most often associated with primary mediators of innate immunity, neutrophils and macrophages. These myeloid cells convert ROS into potent antimicrobial oxidants that efficiently kill pathogens. The respiratory burst also can have destructive consequences, as ROS are well known to support chronic inflammation and aberrant autoimmune responses. Interestingly, ROS perform conflicting roles in the tumor microenvironment; ROS and derived cytotoxic products can destroy cancer cells but also suppress important tumor-fighting functions of T cells or natural killer cells, or yield mutagenized proteins that can promote tumorigenesis or support tumor cell growth. Moreover, high numbers of neutrophils or macrophages in tumors are associated with poor prognosis. Therefore, accurate and quantitative assays to assess the respiratory burst are an important tool for measuring ROS production by neutrophils or cells of the monocyte/macrophage system, each recently identified in the tumor microenvironment. Described are methods to derive mouse or human models of neutrophils or macrophages, which are then used in a detailed assay to quantitatively measure ROS produced by either cell type using luminescence-enhanced reagents and a multi-well platform along with different stimulants that cause rapid ROS production., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Genetic, epidemiological and clinical evidence has demonstrated the importance of the human apolipoproteinA5 (apoA5), apolipoproteinA4 (apoA4), apolipoproteinC3 (apoC3), and apolipoproteinA1 (apoA1) genes in the control of the triglyceride and cholesterol concentrations in the blood. However, little is known about the mechanism by which protein kinase C (PKC) regulates the expression of these genes in hepatic and intestinal cells. The aim of this study was to explore the regulatory role of PKC on the expression of apoA5, apoA4, apoC3 and apoA1.Methods: Hepatic HepG2 and intestinal Caco-2 cells were treated with a potent PKC activator, Phorbol myristate acetate (PMA). The real time quantitative RT-PCR (qRT-PCR) technique was used to evaluate the effects of PMA on the expression of apoA1, apoA4, apoA5 and apoC3 genes. Nuclear run on assay was used to determine whether the effect of PMA on apoA4 and apoC3 was due to its ability to regulate the transcription of these genes.Results: PMA specifically down-regulated the transcription of apoA4 and apoC3, but exhibited no effects on apoA1 or apoA5 in either HepG2 or Caco-2 cells. Further study by nuclear run on assay proved that the suppressive effect of PMA on apoA4 and apoC3 resulted from PMA's regulation of the transcription rate of the two genes.Conclusions: PMA down-regulated transcription of apoA4 and apoC3 possibly through the common regulatory element shared by these two genes, suggesting a suppressive role of PKC on the transcriptional regulation of specific apolipoproteins in hepatic and intestinal cells. [ABSTRACT FROM AUTHOR]

Background: Genetic, epidemiological and clinical evidence has demonstrated the importance of the human apolipoproteinA5 (apoA5), apolipoproteinA4 (apoA4), apolipoproteinC3 (apoC3), and apolipoproteinA1 (apoA1) genes in the control of the triglyceride and cholesterol concentrations in the blood. However, little is known about the mechanism by which protein kinase C (PKC) regulates the expression of these genes in hepatic and intestinal cells. The aim of this study was to explore the regulatory role of PKC on the expression of apoA5, apoA4, apoC3 and apoA1.Methods: Hepatic HepG2 and intestinal Caco-2 cells were treated with a potent PKC activator, Phorbol myristate acetate (PMA). The real time quantitative RT-PCR (qRT-PCR) technique was used to evaluate the effects of PMA on the expression of apoA1, apoA4, apoA5 and apoC3 genes. Nuclear run on assay was used to determine whether the effect of PMA on apoA4 and apoC3 was due to its ability to regulate the transcription of these genes.Results: PMA specifically down-regulated the transcription of apoA4 and apoC3, but exhibited no effects on apoA1 or apoA5 in either HepG2 or Caco-2 cells. Further study by nuclear run on assay proved that the suppressive effect of PMA on apoA4 and apoC3 resulted from PMA's regulation of the transcription rate of the two genes.Conclusions: PMA down-regulated transcription of apoA4 and apoC3 possibly through the common regulatory element shared by these two genes, suggesting a suppressive role of PKC on the transcriptional regulation of specific apolipoproteins in hepatic and intestinal cells. [ABSTRACT FROM AUTHOR]

Background: Fat embolism syndrome (FES) associated with acute lung injury (ALI) is a clinical condition following long bone fracture. We have reported 14 victims due to ALI with FES. Our laboratory has developed an animal model that produced fat emboli (FE). The major purpose of this study was to test whether neutrophil activation with phorbol myristate acetate (PMA) and inhibition with sivelestat (SVT) exert protection on the lung. Methods: The lungs of Sprague-Dawley rats were isolated and perfused. FE was produced by addition of corn oil micelles into the lung perfusate. PMA and SVT were given simultaneously with FE. Parameters such as lung weight/body weight ratio, LW gain, exhaled nitric oxide (NO), protein concentration in bronchoalveolar lavage relating to ALI were measured. The neutrophil elastase (NE), myeloperoxidase, malondialdehyde and phopholipase A2 activity were determined. We also measured the nitrate/nitrite, methyl guanidine (MG), and cytokines. Pulmonary arterial pressure and microvascular permeability were assessed. Lung pathology was examined and scored. The inducible and endothelial NO synthase (iNOS and eNOS) were detected. Results: FE caused ALI and increased biochemical factors. The challenge also resulted in pulmonary hypertension and increased microvascular permeability. The NE appeared to be the first to reach its peak at 1 hr, followed by other factors. Coadministration with PMA exacerbated the FE-induced changes, while SVT attenuated the effects of FE. Conclusions: The FE-induced lung changes were enhanced by PMA, while SVT had the opposite effect. Sivelestat, a neutrophil inhibitor may be a therapeutic choice for patients with acute respiratory distress syndrome (ARDS) following fat embolism. [ABSTRACT FROM AUTHOR]

Abstract: Injection of endothelial progenitor cells (EPCs) into arteries for cell therapy is a promising field in regenerative medicine. However, adhesion of EPCs during capillary passage is restricted, and non-adhering cells are lost into circulation. Here we demonstrate that it is possible to achieve a three- to sevenfold higher rate of EPC adhesion to endothelium and extracellular matrix molecules after short-term activation with phorbol myristate acetate (PMA). In addition, differentiation and toxicity analyses of PMA activated EPCs showed no impact on cell differentiation and negligible impact on cell survival. [ABSTRACT FROM AUTHOR]

Moxifloxacin is useful in the treatment of respiratory infections, including community-acquired pneumonia, and also shows promise in the treatment of tuberculosis, a clinical setting which necessitates extended administration of this agent. Relatively little is known, however, about the effects of this agent on the antimicrobial and proliferative activities of human neutrophils and T-lymphocytes, respectively. In the current study, we have investigated the effects of moxifloxacin at therapeutic concentrations and greater (1-20 μg/mL) on cytosolic Ca2+ fluxes, generation of antimicrobial reactive oxygen species (ROS), and release of the primary granule protease, elastase, following activation of the cells with the chemoattractant, fMLP (1 μM), or the phorbol ester, PMA (25 ng/mL), using radiometric, chemiluminescence, and colourimetric procedures, respectively. The effects of moxifloxacin on mitogen-activated proliferation of T cells and expression of the interleukin-2 (IL-2) receptor (CD25) were measured using radiometric and flow cytometric procedures respectively. With the exception of elastase release, which was significantly increased (P < 0.05) by treatment of the cells with moxifloxacin at 10 and 20 μg/mL, none of the other neutrophil or lymphocyte functions was affected by moxifloxacin. These observations suggest that extended use of this agent is unlikely to compromise the protective functions of neutrophils and T-lymphocytes and may even potentiate neutrophilmediated antimicrobial activity by increasing the release of elastase. [ABSTRACT FROM AUTHOR]

Abstract: How HIV-1 affects the monocyte proteome is incompletely understood. We posit that one functional consequence of virus-exposure to the monocyte is the facilitation of protein transformation from the cytosol to the plasma membrane (PM). To test this, cell surface labeling with CyDye fluorophores followed by 2 dimensional differential in-gel electrophoresis (2D DIGE) and liquid chromatography tandem mass spectrometry (LC–MS/MS) was performed. Fifty three percent of HIV-1 induced proteins were PM associated. These were linked, in large measure, to cellular activation and oxidative stress. They included, but not limited to, biliverdin reductase, leukotriene hydrolase A4, heat shock protein 70, and cystatin B. HIV-1 induced PM protein translocation was associated with cathepsin B- and caspase 9, 3-dependent apoptosis. In contrast, PMA-treated monocytes bypassed caspase 3, 9 pathways and lead to cathepsin B-dependent necrosis. These results demonstrate that HIV-1 uniquely affects monocyte activation and oxidative stress. These do not affect viral infection dynamics but are linked to stress-induced cell death. [Copyright &y& Elsevier]

It was previously found that re-seeding monocyte-derived macrophages (MDM) on polycarbonate-based polyurethanes (PCNUs) in the presence of the protein kinase C (PKC) activator phorbol myristate acetate (PMA) inhibited MDM-mediated degradation of PCNUs synthesized with 1,6-hexane diisocyanate (HDI), as well as esterase activity and monocyte-specific esterase (MSE) protein. However, no effect on the degradation of a 4,4′-methylene bisphenyl (MDI)-derived PCNU (MDI321) occurred. This finding suggested that oxidation, a process linked to the PKC pathway, was not activated in the same manner for all PCNUs. In the current study MDM were re-seeded onto the above PCNU surfaces with PMA, PKC-inactive 4αPMA and the PKC inhibitor bisindolylmaleimide I hydrochloride (BIM) for 48 h before assaying for PCNU degradation, esterase activity, MSE protein, DNA, cell viability and cell morphology. 4αPMA did not alter MDM-mediated HDI PCNU degradation but MDI321 degradation increased in this condition. BIM alone had no effect on any parameter; however, when BIM and PMA were added together, the PMA inhibition of biodegradation, esterase activity and MSE protein was partially reversed for MDM on HDI PCNUs only. Adding PMA to MDM on HDI PCNUs increased intercellular connections, whereas 4αPMA or BIM+PMA increased cell size. Although this study demonstrated a role for oxidation via a PKC-activated pathway in MDM-mediated PCNU degradation, phorbol esters appear to also activate non-PKC pathways that have roles in biodegradation. Moreover, the sensitivity to material surface chemistry in the MDM response to each PCNU dictates a multi-factorial degradative process involving alternate material specific oxidative and hydrolytic mechanisms. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to investigate the role of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the signalling pathway of the novel protein kinase C (PKC)- and reactive oxygen species (ROS)-dependent stimulation of intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) production in human eosinophils. Materials and Methods: Immunomagnetically purified human eosinophils were stimulated in vitro with a PKC activator, phorbol myristate acetate (PMA), and the cAMP response in the presence of a phosphodiesterase inhibitor, rolipram, was determined. The role of ERK1/2 phosphorylation was investigated using specific inhibitors and Western blot analysis. Results: The PMA-stimulated eosinophils responded with a profound increase in intracellular levels of cAMP that was dependent on both PKC and ROS, as confirmed by the use of specific inhibitors: Ro 31-8220 for PKC and diphenyleneiodonium (DPI) for the ROS-generating enzyme NADPH oxidase. Pre-treatment of cells with the ERK1/2 inhibitor PD 98059, but not the p38-MAPK inhibitor SB203580, nor the PI3 kinase inhibitor, wortmannin, abolished the response. PMA treatment induced the phosphorylation of ERK1/2 with a time course that is consistent with a role in the cAMP response. The ERK1/2 phosphorylation was abolished by the ERK1/2 inhibitor PD 98059 and the PKC inhibitor Ro 31-8220, but not the NADPH oxidase inhibitor DPI. Conclusion: These results reveal the involvement of ERK1/2 in the signalling mechanism of PMA-stimulated, PKC- and ROS-dependent stimulation of cAMP production in human eosinophils, and show that ERK1/2 phosphorylation is upstream of ROS production in the signalling pathway. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Phorbol myristate acetate (PMA) causes acute lung injury (ALI). The present study was designed to elucidate the role of nitric oxide (NO), inducible NO synthase (iNOS), neutrophil elastase (NE) and other mediators in the ALI caused by PMA. In isolated rat’s lungs, PMA at various doses (1, 2 and 4 μg/g lung weight) was added into the lung perfusate. Vehicle group received dimethyl sulfoxide (the solvent for PMA) 100 μg/g. We measured the lung weight changes, pulmonary arterial pressure, capillary filtration coefficient, exhaled NO, protein concentration in bronchoalveolar lavage (PCBAL) and Evan blue dye leakage. Nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and myeloperoxidase (MPO) in lung perfusate were determined. Histopathological examination was performed. We detected the iNOS mRNA expression in lung tissue. PMA caused dose-dependent increases in variables for lung changes, and nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and MPO in lung perfusate. The pathology was characterized by alveolar hemorrhagic edema with inflammatory cell infiltration. Scanning electron microscopy revealed endothelial damage. PMA upregulated the expression of iNOS mRNA. Our results suggest that neutrophil activation by PMA causes release of NE, upregulation of iNOS and a series of inflammatory responses leading to endothelial damage and ALI. [ABSTRACT FROM AUTHOR]

The article examines whether intravascular Fluorinert (FC-77) attenuates phorbol myristate acetate (PMA)-induced acute lung injury. The study reveals that intravascular FC-77 ameliorated acute lung injury induced by PMA in a dose-dependent manner. It also reveals that the protective mechanism may act by decreasing neutrophil infiltration, inhibiting proinflammatory cytokine production and by preventing the release of free radicals.

Abstract: Matrix metalloproteinase-9 (MMP-9) plays an important role in mediating the invasion and angiogenic process of malignant gliomas. This study was undertaken to determine if an isoflavone metabolite, irisolidone, inhibits MMP-9 expression in human astroglioma cells. Irisolidone was found to inhibit the secretion and protein expression of MMP-9 induced by PMA in U87 MG glioma cells, accompanied by the inhibition of MMP-9 mRNA expression and promoter activity. Further mechanistic studies revealed that irisolidone inhibits the binding of NF-κB and AP-1 to the MMP-9 promoter and suppresses the PMA-induced phosphorylation of ERK and JNK, which are upstream signaling molecules in MMP-9 expression. The Matrigel-invasion assay showed that irisolidone suppresses the in vitro invasiveness of glioma cells. Therefore, the strong inhibition of MMP-9 expression by irisolidone might be a potential therapeutic modality for controlling the growth and invasiveness of gliomas. [Copyright &y& Elsevier]

Abstract: Gene expression changes are used with increasing frequency to assess the effects of exposure to environmental agents. Housekeeping (Hk) genes are essential in these analyses as internal controls for normalizing expression levels evaluated with Real-Time PCR (RT-PCR). Ideal Hk genes are constitutively expressed, do not respond to external stimuli and exhibit little or no sample-to-sample or run-to-run variation. Previous studies indicate that some commonly used Hk genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin have differential expression in various cell lines. Here we examine the expression of 11Hk genes in four normal human lymphoblastoid cell lines and one T-cell leukemia (Jurkat) cell line following exposure to graded doses of ionizing radiation or to varying ratio concentrations of phytohemagglutinin (PHA) and phorbol myristate acetate (PMA). PHA and PMA are known to have synergistic effects on the expression of some genes and have very different effects from those of radiation. There has been no systematic study performed to ascertain the best control genes for radiation and/or PHA/PMA exposures in lymphoblastoid cells. Using a two-step reverse-transcriptase RT-PCR protocol we show that following radiation doses ranging from 0 to 400 cGy, 18S rRNA, acidic ribosomal protein, beta-actin, cyclophilin, GAPDH, phosphoglycerokinase, beta-2 microglobulin (B2M), beta-glucuronidase, hypoxanthine phosphoribosyltransferase and transferrin receptor showed no significant variation in expression in normal lymphoblastoid cells. In contrast, only 18S rRNA levels were unchanged in Jurkat cells. After PHA/PMA treatment of the same normal cell lines, B2M showed no significant variation and 18S rRNA, GAPDH and transcription binding protein (TBP) were minimally responsive, whereas in Jurkat cells all these genes were unresponsive. While our results suggest that the utility of a particular Hk gene should be determined for each experimental condition, 18S rRNA and B2M appear to be excellent candidates for use as internal controls in RT-PCR in human lymphoblastoid cells because they have the most constant levels of expression across cell lines following exposure to ionizing radiation as well as to PHA/PMA. [Copyright &y& Elsevier]

Abstract: Acute lung injury (ALI) caused by phorbol myristate acetate (PMA) is characterized by pulmonary edema and inflammatory cells infiltration. PMA-activated neutrophils in vivo and in vitro to release free radicals, pro-inflammatory cytokines, nitric oxide (NO) and other mediators. These mediators may be the causes of pulmonary hypertension and increased microvascular permeability. In the present study, we used isolated perfused rat lungs from Sprague–Dawley (SD) rats. The purpose was to evaluate the effects of pretreatment of N-acetylcysteine (NAC) on the PMA-induced ALI and associated changes. PMA (2μgkg−1) was introduced into the lung perfusate. NAC (150mgkg−1) was administered 10min before PMA. Thirty isolated lungs were randomly assigned to receive vehicle (dimethyl sulfoxide, DMSO, the solvent for PMA, 100μgg−1), PMA alone and PMA with NAC pretreatment. There were 10 lungs in each group. We measured the lung weight (LW) to body weight (BW) ratio (LW/BW), LW gain (LWG), exhaled nitric oxide (NO) and protein concentration in bronchoalveolar lavage (PCBAL). The pulmonary arterial pressure (PAP) and microvascular permeability (Kfc ) were assessed. The concentration of nitrate/nitrite, methyl guanidine (MG), tumor necrosis factor α (TNF α ) and interleukin-1 β (IL-1 β ) in lung perfusate were determined. In addition, we also evaluate the lung injury by histopathological examination and by grading system for the lung injury score (LIS). PMA caused severe ALI as evidenced by the marked increases in LW changes, exhaled NO, PCBAL, histopathological changes, and LIS. It also increased the nitrate/nitrite, MG, TNF α , and IL-1 β in lung perfusate. Pretreatment with NAC significantly attenuated these changes and abrogated the extent of ALI. Our results suggest that NAC exerts strong protective effects on the PMA-induced ALI and associated alterations. The mechanisms are possibly attributable to its antioxidant actions, inhibition of pro-inflammatory cytokines, and restoration of glutathione enzymes. [Copyright &y& Elsevier]

Background: Neutrophils (PMNs) are critical components of the innate immune system and help to maintain oral health in the face of a constant bacterial challenge. However, along with protecting the periodontium from microbial invasion, these cells release potent lysosomal enzymes and oxygen radicals that can be destructive to periodontal tissues and lead to tooth loss. We examined neutrophil function in a unique population of patients diagnosed with refractory aggressive periodontitis (RAP). Methods: Venous blood was obtained from 12 non-smoking patients who had been diagnosed with RAP, 10 patients with chronic periodontitis who had responded to periodontal therapy (CP), and 13 periodontally healthy controls (HCs). Peripheral blood PMNs were loaded with dihydrorhodamine 123 and stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the receptor independent respiratory burst of these key immune cells. Phagocytosis via the complement and Fc-gamma receptors was also assessed. Results: PMNs from patients with RAP displayed significantly increased PMA-induced oxygen radical production compared to those from the HC and CP patients. PMNs from RAP patients also displayed increased phagocytosis compared to those from the CP group. Conclusions: Our findings demonstrated a larger receptor-independent respiratory burst and higher phagocytotic activity in PMNs derived from patients with RAP compared to PMNs derived from CP patients and periodontally HCs. We speculate that the higher intrinsic intracellular activity of the nicotinamide adenine dinucleotide phosphate oxidase system may account for the continued periodontal breakdown, despite ongoing periodontal therapy in these challenging patients. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine the mechanism of cytosolic calcium ion concentration [Ca2+]i elevation in chicken and rat phagocytes stimulated with phorbol myristate acetate (PMA), leukotriene B4 (LTB4), formyl-methionyl-leucyl-phenylananine (fMLP) and Saccaromyces cerevisiae culture supernatant (SCS). Pretreatment with EGTA completely suppressed the PMA-induced [Ca2+]i elevation in rat and chicken phagocytes, suggesting that all the [Ca2+]i elevation induced in the PMA-stimulated rat and chicken phagocytes was attributable to the influx of extracellular Ca2+. On the other hand, the elevation of LTB4-, FMLP- and SCS-induced [Ca2+]i was only partially suppressed by ethyleneglycol-bis (β-aminoethyl)-N,N,N′,N′-tetraacetic acid ethylene (EGTA) pretreatment of phagocytes. The results indicated that two pathways of [Ca2+]i elevation, recruitment from the intracellular Ca2+ store and influx of extracellular Ca2+, are involved in the [Ca2+]i elevation of LTB4-, fMLP- and SCS-stimulated phagocytes. In fMLP-stimulated rat neutrophils, [Ca2+]i elevation showed a two-phase pattern in which the time lag between the first and second phase was approximately 1 min. The EGTA treatment of the fMLP-stimulated cells induced a reduction of the first phase level and a disappearance of the second phase. The reason for the special influence of EGTA observed in fMLP-stimulated cells is unknown, but the disappearance of the second phase of the [Ca2+]i may be elicited by the EGTA-induced decrease of the first phase [Ca2+]i elevation that depends on IP3 and diacylglycerol induced by fMLP. [ABSTRACT FROM AUTHOR]

Abstract: Recently, we showed that phorbol 12-myristate 13-acetate (PMA) can cause a direct, PKC-dependent, stimulation of intracellular cAMP in human eosinophils. Since PMA also stimulates the release of reactive oxygen species in these cells, we have investigated whether reactive oxygen species are involved in the cAMP response. Provided eosinophils were incubated for <20 min at 37 °C before stimulation, PMA potently stimulated cAMP generation that surpassed that of histamine. Pre-treatment of the cells with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI) and apocynin, strongly inhibited the cAMP production induced by PMA, but not that induced by histamine. This treatment also strongly inhibited the release of superoxide anions (O2 −). The cAMP response was also inhibited by pre-treatment with the specific peroxide scavenger, ebselen, but not superoxide dismutase, or NG-nitro-l-arginine methyl ester (L-NAME), thus, suggesting the possible involvement of a peroxide rather than O2 − or nitric oxide (NO). These results reveal a novel involvement of intracellular reactive oxygen species in protein kinase C (PKC)-dependent stimulation of cAMP production in human eosinophils. [Copyright &y& Elsevier]

Conventionally, a semi‐quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O 2 - ) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water‐soluble, yellow‐colored, nitroblue tetrazolium (Y‐NBT) by O 2 - . However, this assay is semi‐quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2 M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620 nm. The absorbance of dissolved NBT increased in proportion to cell number (r=0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O 2 - . The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB‐985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O 2 - produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O 2 - produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay. [ABSTRACT FROM AUTHOR]

Abstract: The in vivo contribution of neutrophil elastase (NE) in phorbol myristate acetate (PMA)-induced acute lung injury has so far been unclear. This study examined the role of NE in PMA-induced acute lung injury in conscious rabbits, using a specific NE inhibitor, sivelestat sodium hydrate (Sivelestat). A single bolus injection of PMA (40μg/kg) caused acute lung injury as indicated by an increase in protein concentration and hemorrhage in bronchoalveolar lavage fluid (BALF) 4h after PMA injection. These changes were associated with mild decrease in arterial oxygen pressure and peripheral white blood cell and platelet. When continuously infused starting 1h before and ending 4h post-PMA injection, Sivelestat at 3–30mg/kg/h that are able to inhibit rabbit NE activity by 60–90%, dose-dependently attenuated both PMA-induced hemorrhagic pneumonitis and the increase in protein concentration in BALF without affecting myeloperoxidase activity in the lung. Histopathological study indicated that sivelestat (30mg/kg/h) markedly attenuated lung histopathological changes, alveolar hemorrhage and white blood cells migration with evidence of inhibition of NE activity in BALF. These results suggest that NE plays a significant role in PMA-induced acute lung injury and further supports the importance of this enzyme in acute lung injury. [Copyright &y& Elsevier]

Results from earlier experiments in our laboratories revealed that both selective and nonselective inhibitors of cyclooxygenase-2 possess little potential for decreasing in vitro phagocytosis by rat macrophages or canine neutrophils and no potential for decreasing in vivo phagocytosis by the intact murine immune system. We now report the results of studies to assess in vitro and ex vivo effects of the drugs on 1) canine complement activation, 2) generation of superoxide anion and hydrogen peroxide (oxidative burst) by canine neutrophils, and 3) leukocytic chemotaxis and transmigration through endothelial cell monolayers. In vitro concentrations of naproxen sodium, SC-236, SC-245, and SC-791 ranging from 0.1 to 10 μM were tested for their abilities to inhibit canine complement-mediated hemolysis of opsonized sheep erythrocytes and to block phorbol myristate acetate-induced oxidative burst in canine neutrophils. Both models responded to known inhibitory agents, leupeptin in the complement activation test and staurosporine in the superoxide anion assay. In contrast, tested nonsteroidal anti-inflammatory drugs produced only trivial changes in complement activation and superoxide anion production. Experiments on plasma and neutrophils isolated from dogs administered an experimental selective COX-2 inhibitor during a 28-day toxicology study revealed no evidence of drug-associated changes in complement activation or formation of superoxide anion. SC-791 reduced chemotaxis of canine leukocytes toward zymosan-activated dog plasma, but not toward leukotriene B 4 . None of the other drugs tested significantly affected leukocytic chemotaxis. Ibuprofen, SC-245 and SC-791 but not SC-236, reduced transmigration of canine leukocytes through endothelial cell monolayers. Based on the results of these experiments and our earlier studies we have concluded that, although high (suprapharmacologic) concentrations of the drugs may induce in vitro evidence of apparent immunomodulation of the innate immune system, the findings are unlikely to represent a significant human health risk. [ABSTRACT FROM AUTHOR]

In mammals, neonatal positive calcium balance is required for adequate growth. Parathyroid hormone (PTH) plays a central role in this process mainly through its action on the distal nephron. We studied the effect of PTH on cytosolic calcium in distal segments from neonatal rat kidney. PTH elicited a concentration-dependent increase in cytosolic calcium in neonatal distal nephron (EC50=0.5 nM) but not in proximal tubules. At similar PTH concentrations the response was higher in the neonatal than in the adult tubules. The response was associated with protein kinase C (PKC), since phorbol myristate acetate (100 nM) increased [Ca2+]i, and staurosporin, an inhibitor of PKC, decreased (10 nM) or suppressed (100 nM) the PTH effect. cAMP analogues did not change [Ca2+]i. The response was diminished in low external calcium (0.1 mM) and absent at zero calcium, indicating dependency on external calcium. Resting calcium decreased from 80±10.8 to 28.6±2.6 nM at zero [Ca2+]e. PTH and nifedipine increased cytosolic calcium in an additive fashion. We show for the first time that PTH increased cytosolic calcium in the distal nephron of neonatal kidney, in a concentration-dependent pattern and in association with PKC activation. Higher sensitivity of the neonatal tubule might facilitate absorption of this cation during the neonatal period, when growth requires a positive balance of calcium. [ABSTRACT FROM AUTHOR]

1. The present study was designed to determine whether U74500A could ameliorate acute lung injury (ALI) induced by phorbol myristate acetate (PMA) in our rat isolated lung model compared with any amelioration induced by dimethylthiourea (DMTU), superoxide dismutase (SOD) and catalase. 2. Acute lung injury was induced successfully by PMA during 60 min of observation. At 2 µg/kg, PMA elicited a significant increase in microvascular permeability (measured using the capillary filtration coefficient Kfc), lung weight gain, the lung weight/bodyweight ratio, pulmonary arterial pressure and protein concentration of the bronchoalveolar lavage fluid. 3. Pretreatment with 1.5 mg/kg U74500A significantly attenuated ALI; there was no significant increase in any parameters measured, except for pulmonary arterial pressure. The protective effect of U74500A was approximately the same as that of 600 mg/kg DMTU. However, 6000 U/kg SOD, 50 000 U/kg catalase and 6000 U/kg SOD + 50 000 U/kg catalase had no protective effect. 4. These experimental data suggest that U74500A significantly ameliorates ALI induced by PMA in rats. [ABSTRACT FROM AUTHOR]

This study aimed to investigate further the mechanisms involved in the antinociception caused by dipyrone, given by intraperitoneal (i.p.) or intrathecal (i.t.) routes. The intraperitoneal administration of dipyrone to mice 30 min prior resulted in a significant and dose-related inhibition of the biting responses induced by i.t. injection of glutamate, trans-ACPD or substance P (SP). In addition, dipyrone given by i.t. route, 15 min before glutamate, trans-ACPD or SP, also produced a significant reduction in their nociceptive effects. In addition, dipyrone given by i.t. route, 15 min before glutamate, trans-ACPD or SP, also produced a significant reduction in their nociceptive effects. Dipyrone, given either systemically (i.p.) or by i.t. route also caused a dose-dependent inhibition of phorbol myristate acetate (PMA)-induced nociception. Given by systemic route, dipyrone inhibited PMA-induced paw oedema formation. Collectively, these results extend previous data from our group indicating that glutamatergic-mediated pain responses, specifically those mediated by metabotropic receptor subtype, together with inhibition of neurokinin NK1-mediated response, account for the antinociceptive action of dipyrone in mice. Furthermore, we have also produced experimental evidence indicating that the activation of the protein kinase C-dependent pathway plays a role in the dipyrone antinociceptive action. [Copyright &y& Elsevier]

Protein kinase C (PKC) has been implicated in the control of vascular tone and mitogenesis in the adult pulmonary vasculature, but little is known about the role of PKC in the neonatal pulmonary vasculature. In addition, the vasodilator nitric oxide (NO) is important in the transition of the pulmonary circulation from fetal to postnatal life, and it is thought that attenuated production of NO and therefore, cGMP may contribute to the pathophysiology of a variety of forms of neonatal pulmonary vascular disease states. Although evidence exists for an interaction between PKC and NO in the adult pulmonary vasculature, the identification of specific roles for PKC in neonatal pulmonary vascular smooth muscle (NPVSM) has not been determined, and no studies have been done on the modulation of cGMP by PKC in NPVSM. Accordingly, immunoblot analysis revealed the expression of the α, δ, ε, and ι PKC isozymes in NPVSM. Treatment of NPVSM with 10nM 4-β phorbol myristate acetate (PMA), a PKC activator, induced translocation of PKCα, and PKCδ from the soluble to the particulate fraction, while exposure to 10nM endothelin-1 (ET-1), a potent vasoconstrictor and mitogenic substance, caused translocation of PKCδ and PKCι from the soluble to the particulate fraction. Sodium nitroprusside (SNP) significantly increased intracellular cGMP levels, an effect attenuated by PMA but not by ET-1. In addition, pretreatment with the specific PKC isozyme antagonist Go 6983 blocked the effect of PMA on cGMP levels. Collectively, these data demonstrate the expression and activation of multiple PKC isozymes in NPVSM, and indicate that PKC inhibits SNP-stimulated cGMP production in NPVSM. These data also suggest that complex intracellular signaling pathways by specific PKC isozymes may be important in the development of neonatal pulmonary vascular function. [ABSTRACT FROM AUTHOR]

The induction of haemorrhagic necrosis by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in transplantable murine tumours depends on the in situ synthesis of cytokines, particularly tumour necrosis factor (TNF). Since the in vivo action of DMXAA would be greatly clarified by the development of an in vitro model, we investigated whether DMXAA could induce cytokines in cultured murine splenocytes. DMXAA alone induced low amounts of TNF with an optimal concentration of 10 μg/mL and an optimal time of 4 hr. When combined with low concentrations of lipopolysaccharide, deactivated-lipopolysaccharide (dLPS) or phorbol-12-myristate-13-acetate that did not elicit TNF production alone, synergistic TNF production was obtained. DMXAA also induced interferon-γ at an optimal dose of 300 μg/mL, but the addition of dLPS had no further effect. Decreasing culture pH, although not changing the optimal concentrations for stimulation, increased both TNF and interferon-γ production in response to DMXAA. The major DMXAA metabolites, DMXAA-glucuronide and 6-hydroxy-5-methylxanthenone-4-acetic acid, did not induce either cytokine alone, in combination with dLPS or at low pH. The results indicate that DMXAA rather than a metabolite is responsible for cytokine induction and suggest that the microenvironment of the tumour may be responsible for the observed selective induction of cytokines in tumour tissue. [Copyright &y& Elsevier]

In this study, we determined the effect of inhaled nitric oxide (NO) on the acute lung injury induced by phorbol myristate acetate (PMA) in isolated rat lung. Typical acute lung injury was induced successfully by PMA during 60 min of observation. PMA (2 μg/kg) elicited a significant increase in microvascular permeability, (measured using the capillary filtration coefficient Kfc), lung weight gain, lung weight/body weight ratio, pulmonary arterial pressure (PAP) and protein concentration of the bronchoalveolar lavage fluid. Pretreatment with inhaled NO (30 ppm) significantly exacerbated acute lung injury. All of the parameters reflective of lung injury increased significantly except PAP (P<0.05). Coadministration of Nω-nitro-l-arginine methyl ester (l-NAME) (5 mM) attenuated the detrimental effect of inhaled NO in PMA-induced lung injury, except for PAP. In addition, l-NAME (5 mM) significantly attenuated PMA-induced acute lung injury except for PAP. These experimental data suggest that inhaled NO significantly exacerbated acute lung injury induced by PMA in rats. l-NAME attenuated the detrimental effect of inhaled NO. [Copyright &y& Elsevier]

Summary 1. In the present study, we determined whether the endogenous nitric oxide (NO) synthase (NOS) inhibitor N ω -nitro-l-arginine methyl ester (l-NAME) could ameliorate the acute lung injury (ALI) induced by phorbol myristate acetate (PMA) in rat isolated lung. 2. Typical ALI was induced successfully by PMA during 60 min of observation. At 2 µg/kg, PMA elicited a significant increase in microvascular permeability (measured using the capillary filtration coefficient Kfc ), lung weight gain, lung weight/bodyweight ratio, pulmonary arterial pressure (PAP) and protein concentration of bronchoalveolar lavage fluid. 3. Pretreatment with the NOS inhibitor l-NAME (5 mmol/L) significantly attenuated ALI. None of the parameters reflective of lung injury showed significant increase, except for PAP (P < 0.001). The addition of l-arginine (4 mmol/L) blocked the protective effective of l-NAME. Pretreatment with l-arginine exacerbated PMA-induced lung injury. 4. These data suggest that l-NAME significantly ameliorates ALI induced by PMA in rats, indicating that endogenous NO plays a key role in the development of lung oedema in PMA-induced lung injury. [ABSTRACT FROM AUTHOR]

Glutamate release from microglial cells may cause neuronal damage. To elucidate the mechanism of glutamate release, we examined the possible regulation by nitric oxide and protein kinase C. Cultured microglia prepared from the whole brains of newborn rats released glutamate by the stimulation with lipopolysaccharide (LPS) dose dependently. The time course study revealed that glutamate release showed a long lag time about 6 h after LPS stimulation, whereas about 3 h lag time was observed in LPS-induced NO production. An inhibitor for NO synthase, NG-nitro-l-arginine, could effectively inhibit the glutamate release. Glutamate release induced by LPS was enhanced by 1 nM phorbol myristate acetate (PMA). Furthermore, high concentrations of PMA (>10 nM) induced glutamate release even without LPS stimulation. Glutamate release stimulated either by 100 ng/ml LPS or 100 nM PMA was inhibited by staurosporine, and also by α-aminoadipate. These results provide insight into the pathways regulating microglial pathological activation by protein kinase C and may be a base for the protection against microglia-evoked neurotoxicity. [Copyright &y& Elsevier]