Compounds Consisting of Quinazoline, Ibuprofen, and Amino Acids with Cytotoxic and Anti-Inflammatory Effects.

In this research work, a series of 16 quinazoline derivatives bearing ibuprofen and an amino acid were designed as inhibitors of epidermal growth factor receptor tyrosine kinase domain (EGFR-TKD) and cyclooxygenase-2 (COX-2) with the intention of presenting dual action in their biological behavior. The designed compounds were synthesized and assessed for cytotoxicity on epithelial cancer cells lines (AGS, A-431, MCF-7, MDA-MB-231) and epithelial non-tumorigenic cell line (HaCaT). From this evaluation, derivative 6 was observed to exhibit higher cytotoxic potency (IC50) than gefitinib (reference drug) on three cancer cell lines (0.034 µM in A-431, 2.67 µM in MCF-7, and 3.64 µM in AGS) without showing activity on the non-tumorigenic cell line (>100 µM). Furthermore, assessment of EGFR-TKD inhibition by 6 showed a discreet difference compared to gefitinib. Additionally, 6 was used to conduct an in vivo anti-inflammatory assay using the 12-O-tetradecanoylphorbol-3-acetate (TPA) method, and it was shown to be 5 times more potent than ibuprofen. Molecular dynamics studies of EGFR-TKD revealed interactions between compound 6 and M793. On the other hand, one significant interaction was observed for COX-2, involving S531. The RMSD graph indicated that the ligand remained stable in 50 ns.

N-Methyl-(2S,4R)-trans-4-hydroxy-L-proline isolated Sideroxylon obtusifolium attenuates TPA-induced irritant contact dermatitis in mice.

Dermatitis is defined as a set of inflammatory diseases that affect the skin, with varied causes. Among the different types of dermatitis, contact dermatitis is the most prevalent. Although the current therapy is often effective, it is associated with adverse effects and the possibility of drug tolerance. N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline is a L-proline amino acid derivative found in the leaves of Sideroxylon obtusifolium, a species traditionally used to treat inflammatory diseases. The aim of this study was to investigate the topical anti-inflammatory effect of N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline (NMP) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritant contact dermatitis in mice. Topically administered NMP, at doses of 0.03 - 0.50 mg/ear, reduced TPA-induced ear edema and neutrophil migration, as evidenced by low tissue myeloperoxidase activity and verified by histological examination. In addition, NMP (0.06 mg/ear) reduced tissue levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, INF-γ and MCP-1) and of the anti-inflammatory cytokine IL-10, and reduced gene expression of TNF-α, IL-6 and IL-1ß increased by TPA. The data suggest that N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline acts as a topical anti-inflammatory agent that decreases the expression of inflammatory cytokines, making it useful for the treatment of skin inflammation. Further investigations are necessary for its development as a therapeutic agent.

Lysophosphatidic acid receptor LPA1 trafficking and interaction with Rab proteins, as evidenced by Förster resonance energy transfer.

LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 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 LPA1-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA1-Rab5 interaction and receptor internalization. LPA-induced LPA1-Rab9 interaction was only observed at 60 min, and LPA1-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA1-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA1-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA1 receptor internalization varies with the stimuli.

Neutrophil Activated by the Famous and Potent PMA (Phorbol Myristate Acetate).

This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, ß and δ contribute to ROS production while PKC ßII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1ß release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine proteases are inhibited, NETs are formed by citrullination through the PAD4 pathway. This review puts together results from the last 31 years of research on the effects of PMA on the neutrophil and proposes new insights on their interpretation.

Quercetin inhibits neutrophil extracellular traps release and their cytotoxic effects on A549 cells, as well the release and enzymatic activity of elastase and myeloperoxidase.

Neutrophil extracellular traps (NETs) were first reported as a microbicidal strategy for activated neutrophils. Through an immunologic response against several stimuli, neutrophils release their DNA together with proteins from granules, nucleus, and cytoplasm (e.g., elastase and myeloperoxidase). To date, NETs have been implicated in tissue damage during intense inflammatory processes, mainly when their release is dependent on oxygen radical generation. Flavonoids are antioxidant and anti-inflammatory agents; of these, quercetin is commonly found in our daily diet. Therefore, quercetin could exert some protective activity against tissue damage induced by NETs. In our in vitro assays, quercetin reduced NETs, myeloperoxidase (MPO), and elastase release from neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). The activity of these enzymes also decreased in the presence of quercetin. Quercetin also reduced the cytotoxic effect of NETs on alveolar cells (A549 cell line). Further, in silico assays indicated favorable interactions between quercetin and NET proteins (MPO and elastase). Overall, our results demonstrate that quercetin decreases deleterious cellular effects of NETs by reducing their release from activated neutrophils, and diminishing the enzymatic activity of MPO and elastase, possibly through direct interaction.

Detection of Reactive Oxygen Species in Human Neutrophils Under Various Conditions of Exposure to Galectin.

Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.

PMA treatment fosters rat retinal ganglion cell survival via TNF signaling.

An insult can trigger a protective response or even cell death depending on different factors that include the duration and magnitude of the event and the ability of the cell to activate protective intracellular signals, including inflammatory cytokines. Our previous work showed that the treatment of Lister Hooded rat retinal cell cultures with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, increases the survival of retinal ganglion cells (RGCs) kept in culture for 48 h after axotomy. Here we aim to analyze how PMA modulates the levels of TNF-α and IL-1ß (both key inflammatory mediators) and the impact of this modulation on RGCs survival. We hypothesize that the increase in RGCs survival mediated by PMA treatment depends upon modulation of the levels of IL-1ß and TNF-α. The effect of PMA treatment was assayed on cell viability, caspase 3 activation, TNF-α and IL-1ß release and TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) levels. PMA treatment increases IL-1ß and TNF-α levels in 15 min in culture and increases the release of both cytokines after 30 min and 24 h, respectively. Both IL-1ß and TNF-α levels decrease after 48 h of PMA treatment. PMA treatment also induces an increase in TNFRII levels while decreasing TNFRI after 24 h. PMA also inhibited caspase-3 activation, and decreased ROS production and EthD-1/calcein ratio in retinal cell cultures leading to an increase in cell viability. The neutralization of IL-1ß (anti-IL1ß 0,1ng/mL), the neutralization of TNF-α (anti-TNF-α 0,1ng/mL) and the TNF-α inhibition using a recombinant soluble TNFRII abolished PMA effect on RGCs survival. These data suggest that PMA treatment induces IL1ß and TNF-α release and modulation of TNFRI/TNFRII expression promoting RGCs survival after axotomy.

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules.

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

Effect of docosahexaenoic acid, phorbol myristate acetate, and insulin on the interaction of the FFA4 (short isoform) receptor with Rab proteins.

Human embryonic kidney (HEK) 293 cells were co-transfected with plasmids for the expression of mCherry fluorescent protein-tagged FFA4 receptors and the enhanced green fluorescent protein-tagged Rab proteins involved in retrograde transport and recycling, to study their possible interaction through Förster Resonance Energy Transfer (FRET), under the action of agents that induce FFA4 receptor phosphorylation and internalization through different processes, i.e., the agonist, docosahexaenoic acid, the protein kinase C activator phorbol myristate acetate, and insulin. Data indicate that FFA4 receptor internalization varied depending on the agent that induced the process. Agonist activation (docosahexaenoic acid) induced an association with early endosomes (as suggested by interaction with Rab5) and rapid recycling to the plasma membrane (as indicated by receptor interaction with Rab4). More prolonged agonist stimulation also appears to allow the FFA4 receptors to interact with late endosomes (interaction with Rab9), slow recycling (interaction with Rab 11), and target to degradation (Rab7). Phorbol myristate acetate, triggered a rapid association with early endosomes (Rab5), slow recycling to the plasma membrane (Rab11), and some receptor degradation (Rab7). Insulin-induced FFA4 receptor internalization appears to be associated with interaction with early endosomes (Rab5) and late endosomes (Rab9) and fast and slow recycling to the plasma membrane (Rab4, Rab11). Additionally, we observed that agonist- and PMA-induced FFA4 internalization was markedly reduced by paroxetine, which suggests a possible role of G protein-coupled receptor kinase 2.

Lymphocyte activation after a high-intensity street dance class.

Intense dance training leads to inflammation, which may impair the health and performance of the practitioners. Herein, we evaluate the effect of a single street dancing class on the profile of muscle enzymes, lymphocyte activation, and cell surface CD62L expression. We also investigated the correlation between muscle enzymes, adhesion molecules, and lymphocyte activation in dancers. Fifteen male participants (mean ± standard error: age 22.4 ± 1.08 years, body mass index 24.8 ± 0.69 kg/m2, body fat 12.3 ± 1.52%), who were amateur dancers, had blood samples collected previously and subsequent to a high-intensity street dance class. After the class, dancers showed an increase in total lymphocyte count (2.0-fold), creatine kinase (CK)-NAC (4.87%), and CK-MB (3.36%). We also observed a decrease (2.5-fold) in reactive oxygen species (ROS) produced by lymphocytes, under phorbol myristate acetate-stimulated environments. Following the dance class, CD62L expression in lymphocytes decreased (51.42%), while there was a negative correlation between the intensity of the exercise and CD62L expression (r = -0.73; p = 0.01). Lymphocytes were less responsive to stimuli after a single bout of street dancing, indicating transient immunosuppression.

Effects of agonists and phorbol esters on α1A-adrenergic receptor-Rab protein interactions.

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.

Identification and characterization of human PEIG-1/GPRC5A as a 12-O-tetradecanoyl phorbol-13-acetate (TPA) and PKC-induced gene.

Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 µM), and the Ca2+ chelator BAPTA-AM (150 µM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 µM), and the MEK1/2 inhibitor U0126 (10 µM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 µM. The IL-1ß autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation.

The Diethylcarbamazine Delays and Decreases the NETosis of Polymorphonuclear Cells of Humans with DM Type 2.

Type 2 diabetes mellitus (DM2) is a disease that reports high morbidity and mortality rates worldwide. Between its complications, one of the most important is the development of plantar ulcers. The role of the polymorphonuclear cells (PMNs) is affected by metabolic diseases like DM2. Fifteen years ago, reports about a new mechanism of innate immune response where PMNs generate some kind of webs with their chromatin were published. This mechanism was called NETosis. Also, some researchers have demonstrated that NETosis is responsible for the delay of the ulcer healing both in patients with DM2 and in animal models of DM2. Purified PMNs from healthy and DM2 human volunteers were incubated with diethylcarbamazine (DEC) and then induced to NETosis using phorbol 12-myristate 13-acetate (PMA). In a randomized blind study model, the NETosis was documented by confocal microscopy. On microphotographs, the area of each extracellular neutrophil trap (NET) formed at different times after stimuli with PMA was bounded, and the intensity of fluorescence (IF) from the chromatin dyed with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was quantified. PMNs from healthy volunteers showed the development of NETs at expected times according to the literature. The same phenomenon was seen in cultures of PMNs from metabolically controlled DM2 volunteers. The use of DEC one hour before of the challenge with PMA delayed the NETosis in both groups. The semiquantitative morphometric analysis of the IF from DAPI, as a measure of PMN's capacity to forming NETs, is consistent with these results. The ANOVA test demonstrated that NETosis was lower and appeared later than expected time, both in PMNs from healthy (p ≤ 0.000001) and from DM2 (p ≤ 0.000477) volunteers. In conclusion, the DEC delays and decreases the NETosis by PMNs from healthy as well as DM2 people.

Neutrophil extracellular trap-enriched supernatants carry microRNAs able to modulate TNF-α production by macrophages.

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.

Limited expression of TLR9 on T cells and its functional consequences in patients with nonalcoholic fatty liver disease.

BACKGROUND/AIMS: Toll-like receptors (TLRs) modulate T cell responses in diverse diseases. Co-stimulation of T cell activation via TLR9 induces production of interferon gamma (IFN-γ), priming of which is critical for differentiation of pro-inflammatory macrophages. These macrophages have a crucial role in nonalcoholic fatty liver disease (NAFLD). We aimed to evaluate the expression of TLR9 protein on T cells and the consequences of TLR9-mediated triggering of these cells in patients with NAFLD. METHODS: Our study included 34 patients with simple steatosis, 34 patients with nonalcoholic steatohepatitis, eight patients with NAFLD who met general diagnostic criteria but lacked histological diagnosis, and 51 control subjects. We used a synthetic TLR9 ligand to co-stimulate T cells. We measured TLR9 expression in liver and peripheral T cells and CD69 and IFN-γ as phenotypic markers of T cell activation and differentiation by flow cytometry. RESULTS: TLR9 expression on liver and peripheral T cells was lowest in patients with simple steatosis and was positively associated with anthropometric, biochemical, and histopathological features of NAFLD. In vitro co-stimulation of T cells from patients with simple steatosis induced a limited number of IFN-γ-producing CD8+ T cells. At baseline, these patients showed a low frequency of circulating type 1 CD8+ cells. CONCLUSION: The positive associations between TLR9 and anthropometric, clinical, and histological features and the crucial role of IFN-γ-in NAFLD suggest that limited TLR9 expression and production of IFN-γ play a protective role in patients with simple steatosis.

BAY 41-2272 inhibits human T lymphocyte functions.

BAY 41-2272 increases guanosine 3', 5'-cyclic monophosphate (cGMP) levels by stimulating soluble guanylate cyclase (sGC). In this study, we evaluated the effect of BAY 41-2272 on human T lymphocyte functions. Pretreating T cells for 24 h with BAY 41-2272 at 3 µM and 30 µM, followed by activation with 90 nM phorbol myristate acetate (PMA), inhibited interferon-gamma (IFN-γ) production, with 3 µM and 30 µM BAY causing 16.5-fold and 12.1-fold inhibition, respectively, compared to PMA alone (p < 0.05, one-way ANOVA followed by Tukey's test). We also observed suppressive effects on the expression of CD69, with 30 µM BAY causing 3.55-fold lower expression than PMA/ionomycin (p < 0.001 one-way ANOVA followed by Tukey's test), and T-bet, with 30 µM BAY causing 1.47-fold lower expression than PMA/ionomycin (p < 0.05, one-way ANOVA test followed by Tukey's test). Additionally, T lymphocyte proliferation was reduced 2.13-fold and 4.3-fold, respectively, by 3 µM BAY and 30 µM BAY compared to PMA/ionomycin (p < 0.01, p < 0.001, one-way ANOVA followed by Tukey's test). BAY 41-2272 inhibits human T lymphocyte function and may be explored as an immunomodulatory drug in patients with autoimmune/inflammatory diseases and lymphoproliferative syndromes.

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A2A and Muscarinic M1 Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters.

BACKGROUND/AIMS: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A2A and muscarinic M1 receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. METHODS: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500-750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. RESULTS: Activation of A2A and M1 receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 µmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 µmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 µmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 µmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 µmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 µmol/L) was also prevented by HC-3 (4 µmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 µmol/L). CONCLUSION: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A2A and M1 receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

S1P1 receptor phosphorylation, internalization, and interaction with Rab proteins: effects of sphingosine 1-phosphate, FTY720-P, phorbol esters, and paroxetine.

Sphingosine 1-phosphate (S1P) and FTY720-phosphate (FTYp) increased intracellular calcium in cells expressing S1P1 mCherry-tagged receptors; the synthetic agonist was considerably less potent. Activation of protein kinase C by phorbol myristate acetate (PMA) blocked these effects. The three agents induced receptor phosphorylation and internalization, with the action of FTYp being more intense. S1P1 receptor-Rab protein (GFP-tagged) interaction was studied using FRET. The three agents were able to induce S1P1 receptor-Rab5 interaction, although with different time courses. S1P1 receptor-Rab9 interaction was mainly increased by the phorbol ester, whereas S1P1 receptor-Rab7 interaction was only increased by FTYp and after a 30-min incubation. These actions were not observed using dominant negative (GDP-bound) Rab protein mutants. The data suggested that the three agents induce interaction with early endosomes, but that the natural agonist induced rapid receptor recycling, whereas activation of protein kinase C favored interaction with late endosome and slow recycling and FTYp triggered receptor interaction with vesicles associated with proteasomal/lysosomal degradation. The ability of bisindolylmaleimide I and paroxetine to block some of these actions suggested the activation of protein kinase C was associated mainly with the action of PMA, whereas G protein-coupled receptor kinase (GRK) 2 (GRK2) was involved in the action of the three agents.

Regulatory roles of miR-155 and let-7b on the expression of inflammation-related genes in THP-1 cells: effects of fatty acids.

The main aim of this investigation was to study the regulatory roles of let-7b and miR-155-3p on the expression of inflammation-associated genes in monocytes, macrophages, and lipopolysaccharide (LPS)-activated macrophages (AcM). A second goal was to analyze the potential modulatory roles of different fatty acids, including oleic, palmitic, eicosapentaenoic (EPA), and docosahexaenoic (DHA), on the expression of these miRNAs in the three cell types. This hypothesis was tested in human acute monocytic leukemia cells (THP-1), which were differentiated into macrophages with 2-O-tetradecanoylphorbol-13-acetate (TPA) and further activated with LPS for 24 h. Monocytes, macrophages, and AcM were transfected with a negative control, or mimics for miR-155-3p and miR-let-7b-5p. The expression of both miRNAs and some proinflammatory genes was analyzed by qRT-PCR. Interestingly, let-7b mimic reduced the expression of IL6 and TNF in monocytes, and SERPINE1 expression in LPS-activated macrophages. However, IL6, TNF, and SERPINE1 were upregulated in macrophages by let-7b mimic. IL6 expression was higher in the three types of cells after transfecting with miR-155-3p mimic. Similarly, expression of SERPINE1 was increased by miR-155-3p mimic in monocytes and macrophages. However, TLR4 was downregulated by miR-155-3p in monocytes and macrophages. Regarding the effects of the different fatty acids, oleic acid increased the expression of let-7b in macrophages and AcM and also increased the expression of miR-155 in monocytes when compared with DHA but not when compared with non-treated cells. Overall, these results suggest anti- and proinflammatory roles of let-7b and miR-155-3p in THP-1 cells, respectively, although these outcomes are strongly dependent on the cell type. Noteworthy, oleic acid might exert beneficial anti-inflammatory effects in immune cells (i.e., non-activated and LPS-activated macrophages) by upregulating the expression of let-7b.

PKC delta activation increases neonatal rat retinal cells survival in vitro: Involvement of neurotrophins and M1 muscarinic receptors.

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.