Evaluation of the efficacy and safety of artemether emulsion on localized senile pruritus: A randomized pilot study.

BACKGROUND: Localized senile pruritus is a continued health problem for the elderly. This study aimed to evaluate the efficacy and safety of artemether emulsion on localized senile pruritus. METHODS: Sixty patients diagnosed with senile pruritus were randomized into the artemether emulsion (1%) group or emulsion base group in a 1:1 ratio (the artemether group vs the control group). The patients used artemether emulsion or emulsion base for pruritus twice daily for 2 weeks. The pruritus visual analog scale (VAS) and the rate of adverse events were evaluated in week 0 and week 2. RESULTS: The VAS scores in week 2 after treatment decreased significantly compared with those before treatment in both groups (P < .05). After treatment, patients receiving the artemether emulsion had significantly lower mean VAS scores compared to those who received the emulsion base (1.21 ±â€…1.64 vs 3.67 ±â€…2.97, P < .05). When the VAS scores were compared between the 2 groups before treatment, the effective rate of the artemether group was significantly higher than that of the control group (χ2 = 55, P < .05) in week 2 after treatment. Besides, no adverse events occurred in both groups. CONCLUSIONS: Both artemether emulsion and emulsion base were effective in treating localized senile pruritus, and artemether emulsion was superior to emulsion base.

Low-dose intralesional injection of 5-fluorouracil and triamcinolone reduces tissue resident memory T cells in chronic eczema.

BACKGROUND: Tissue resident memory T (TRM) cells have been reported to play a significant role in the pathogenesis and relapse of chronic eczema. AIM: To compare the efficacy and safety of the intralesional injection of 5-fluorouracil (5-FU) and triamcinolone (TA) with those associated with TA alone for the treatment of chronic eczema. METHODS: A total of 168 patients were randomized to 5-FU+TA or TA groups and received a one-time intralesional injection of 5-FU+TA or TA only. Biopsies were collected before and 2 wk after treatment for evaluation of histopathological changes. All patients were followed up monthly for up to 1 year. RESULTS: No serious adverse event was observed in either group. Although the mean atopic dermatitis severity index scores and effective rates were comparable between the two groups after 2 wk of treatment, the relapse rate was significantly lower in the 5-FU+TA group than in the TA group. Histological examination showed significantly fewer CD8+ and CD103+ T cells but not CD4+ T cells in the 5-FU+TA group. CONCLUSION: One-time intralesional injection of 5-FU+TA is effective and safe for chronic eczema treatment and can further reduce the retention of TRM cells in the lesional skin and the relapse rate of chronic eczema.

Efficacy and safety of artemether emulsion for the treatment of mild-to-moderate acne vulgaris: a randomized pilot study.

OBJECTIVE: To evaluate the efficacy and safety of artemether emulsion treating patients with mild-to-moderate acne vulgaris. METHODS: A total of 73 (randomized 1:1) patients were externally administered either artemether emulsion (1%) or fusidic acid emulsion (5g: 0.1g) twice daily for 12 weeks. Efficacy and safety evaluations were performed at weeks 0 and 12 by Global acne Grading System (GAGS), the number of acne and papule, as well as the rate of clinical respond. RESULTS: After 12 weeks, patients randomized to the artemether emulsion group received artemether emulsion had significantly lower GAGS scores (5.08 ± 1.99 versus 13.75 ± 4.87, p < .001) compared to patients who received fusidic acid emulsion. Patients in the artemether emulsion group had comparable baseline acne scores (11.11 ± 3.73 versus 10.75 ± 4.66, p = .626) and papule score (16.11 ± 5.58 versus 17.03 ± 6.34, p = .356), but significantly lower acne score (3.00 ± 1.55 versus 9.08 ± 4.90, p < .001) and comparable papule score (2.81 ± 1.61 versus 12.69 ± 5.45, p < .001) compared to the fusidic acid emulsion group at 12 weeks. No major adverse events were noted in either treatment group through 12 weeks. CONCLUSIONS: Artemether emulsion had better effect in improving mild-to-moderate AV compared to fusidic acid emulsion with barely AEs.

Evaluation of the efficacy and tolerance of artemether emulsion for the treatment of papulopustular rosacea: a randomized pilot study.

Objective: To assess the efficacy and safety of artemether emulsion in patients with papulopustular rosacea. Methods: A total of 130 (randomized 1:1) were externally administered either artemether emulsion (1%) or metronidazole emulsion (3%) twice daily for 4 weeks with an open-label 8-week follow-up. The primary endpoints included the proportion of patients who achieved clinical effective responses, as well as erythema and papule and pustule score at week 4. Results: Numerically more patients achieved an effective response at week 4 with artemether emulsion (87.1%) than metronidazole emulsion (80.0%) (p > .05). Patients with artemether emulsion had comparable baseline erythema score (2.45 ± 0.67 versus 2.42 ± 0.70, p = .809) and papule and pustule score (2.11 ± 0.96 versus 2.32 ± 0.83, p = .264), but significantly lower papule and pustule score (0.21 ± 0.52 versus 0.42 ± 0.83, p = .001) and comparable erythema score (0.53 ± 0.88 versus 0.62 ± 0.88, p = .999) compared to patients with metronidazole emulsion at week 4. There was a significantly higher proportion of patients with metronidazole emulsion relapse compared to metronidazole emulsion during the open-label 8-week follow-up period (21.6% versus 2.4%, p < .01). Conclusions: Artemether emulsion improved papulopustular rosacea in the metronidazole emulsion group as early as 4 weeks, but its beneficial effect was maintained through the 8-week follow-up period compared to metronidazole emulsion.

Isoalantolactone suppresses LPS-induced inflammation by inhibiting TRAF6 ubiquitination and alleviates acute lung injury.

Isoalantolactone (IAL) is a sesquiterpene lactone extracted from roots of Inula helenium L and has shown anti-inflammatory effects. In this study we investigated the therapeutic effects of IAL on acute lung injury (ALI) and elucidated the mechanisms underlying its anti-inflammation potential in vitro and in vivo. Treatment with lipopolysaccharide (LPS, 100 ng/mL) drastically stimulated production of inflammatory mediators such as NO, TNF-α, IL-1ß, and IL-6 in mouse bone marrow-derived macrophages (BMDMs), which was dose-dependently suppressed by pretreatment with IAL (2.5, 5, 10, 20 µM). We further revealed that IAL suppressed LPS-induced NF-κB, ERK, and Akt activation. Moreover, the downregulation of non-degradable K63-linked polyubiquitination of TRAF6, an upstream transcription factor of NF-κB, contributed to the anti-inflammatory effects of IAL. ALI was induced in mice by intratracheal injection of LPS (5 mg/kg). Administration of IAL (20 mg/kg, i.p.) significantly suppressed pulmonary pathological changes, neutrophil infiltration, pulmonary permeability, and pro-inflammatory cytokine expression. Our results demonstrate that IAL is a potential therapeutic reagent against inflammation and ALI.

A Role for MK2 in Enhancing Neutrophil-Derived ROS Production and Aggravating Liver Ischemia/Reperfusion Injury.

Increased inflammatory responses and enhanced reactive oxygen species contribute to hepatic ischemia/reperfusion (I/R) injury, however the modulatory mechanisms haven't been completely unveiled. Here, we report that genetic deficiency of MAPK-activated protein kinase 2 (MK2) protected against hepatic I/R injury and decreased hepatic neutrophil accumulation in MK2-/- mice. Depletion of neutrophil attenuated hepatic I/R injury in wide type mice. In response to C5a stimulation, MK2-/- neutrophils generated less superoxide in which both NADPH oxidase activation and p47phox phosphorylation were decreased. Furthermore, Ser329 of p47phox was identified for enhancement of superoxide production. The Ser329 phosphorylation was reduced in MK2-/- neutrophils. To determine whether MK2 modulates hepatic I/R injury via activating neutrophils, we generated myeloid-specific MK2 deletion mice (MK2Lyz2-KO) and liver I/R injury was reduced in MK2Lyz2-KO mice. Our results indicate that MK2 augments hepatic I/R injury and induces ROS production with increased p47phox phosphorylation and MK2 is a potential drug target for treating hepatic I/R injury.

Tabersonine attenuates lipopolysaccharide-induced acute lung injury via suppressing TRAF6 ubiquitination.

Sepsis caused by Gram-negative bacteria is one of major causes for the progression of acute lung injury (ALI) with limited treatment and effective medicines. Tabersonine is an indole alkaloid mainly isolated from Catharanthus roseus, and a potential drug candidate for treatment of cancer and Alzheimer's disease (AD), however, its anti-inflammatory effect has not been revealed. In this study, we reported that tabersonine ameliorated lipopolysaccharides (LPS)-induced ALI in vivo and inhibited LPS-mediated macrophage activation in vitro. By using murine ALI model, we found that tabersonine significantly attenuated LPS-induced pathological injury in the lung. Tabersonine also inhibited LPS-mediated neutrophil infiltration, elevation of MPO activity and the production of TNF-α, IL-6 and IL-1ß. Furthermore, tabersonine inhibited LPS-induced the production of pro-inflammatory mediators such as iNOS, NO and cytokines by suppressing NF-κB and p38 MAPK/MK2 signaling cascades. Tabersonine reduced the K63-linked polyubiquitination of TRAF6. Taken together, these results suggested that tabersonine has anti-inflammatory activities in vitro and in vivo, and is a potential therapeutic candidate for the treatment of ALI/ARDS.

MK2 mediates macrophage activation and acute lung injury by regulating let-7e miRNA.

MAPK-activated protein kinase 2 (MK2) plays a critical role in the development of inflammation. However, the modulatory mechanisms in macrophage activation and acute lung injury (ALI) have not been completely defined. Here, we reported that MK2-deficient mice (MK2-/-) protected against sepsis-induced ALI. In response to lipopolysaccharide (LPS) challenge, MK2-/- mice and myeloid cell-specific MK2 conditional knockout mice (MK2Lyz2-KO) exhibited attenuated inflammatory response, especially producing fewer amounts of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and macrophage inflammatory protein 2 (MIP-2). LPS treatment in vitro resulted in reduced cytokine expression in MK2-/- bone marrow-derived macrophages (BMDMs). Furthermore, we found that LPS-induced microRNA lethal-7e ( let-7e) expression was significantly increased in MK2-/- macrophages. Transfection of let-7e antagomirs into MK2-/- BMDM rescued LPS-induced expression of TNF-α, IL-6, and MIP-2. In contrast, transfection of let-7e mimics into MK2+/+BMDM decreased cytokine expression. Meanwhile, LPS-induced phosphorylation of cAMP response element-binding (CREB) protein, a substrate of MK2, was downregulated in MK2-/- BMDMs. Lin28, an inhibitory molecule of let-7, was significantly reduced in MK2-/- macrophages. Our results suggested that MK2 boosts LPS-induced macrophage activation and ALI via increasing activation of CREB and consequently, the expression of Lin28 and downregulation of let-7e.

Lanatoside C inhibits cell proliferation and induces apoptosis through attenuating Wnt/ß-catenin/c-Myc signaling pathway in human gastric cancer cell.

Gastric cancer is the third common cause of cancer mortality in the world with poor prognosis and high recurrence due to lack of effective medicines. Our studies revealed that lanatoside C, a FDA-approved cardiac glycoside, had an anti-proliferation effect on different human cancer cell lines (MKN-45; SGC-7901; HN4; MCF-7; HepG2) and gastric cell lines MKN-45 and SGC-7901 were the most sensitive cell lines to lanatoside C. MKN-45 cells treated with lanatoside C showed cell cycle arrest at G2/M phase and inhibition of cell migration. Meanwhile, upregulation of cleaved caspase-9 and cleaved PARP and downregulation of Bcl-xl were accompanied with the loss of mitochondrial membrane potential (MMP) and induction of intracellular reactive oxygen species (ROS). Lanatoside C inhibited Wnt/ß-catenin signaling with downregulation of c-Myc, while overexpression of c-Myc reversed the anti-tumor effect of lanatoside C, confirming that c-Myc is a key drug target of lanatoside C. Furthermore, we discovered that lanatoside C prompted c-Myc degradation in proteasome-ubiquitin pathway with attenuating the binding of USP28 to c-Myc. These findings indicate that lanatoside C targeted c-Myc ubiquitination to inhibit MKN-45 proliferation and support the potential value of lanatoside C as a chemotherapeutic candidate.

Protostemonine effectively attenuates lipopolysaccharide-induced acute lung injury in mice.

Protostemonine (PSN) is the main anti-inflammatory alkaloid extracted from the roots of Stemona sessilifolia (known as "Baibu" in traditional Chinese medicine). Here, we reported the inhibitory effects of PSN on lipopolysaccharide (LPS)-induced macrophage activation in vitro and LPS-induced acute lung injury in mice. Macrophage cell line RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs) were treated with PSN (1, 3, 10, 30 and 100 µmol/L) for 0.5 h and then challenged with LPS (0.1 µg/mL) for 24 h. Pretreatment with PSN significantly inhibited LPS-induced phosphorylation of MAPKs and AKT, iNOS expression and NO production in the macrophages. C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) to induce acute lung injury (ALI). The mice were subsequently treated with PSN (10 mg/kg, ip) at 4 and 24 h after LPS challenge. PSN administration significantly attenuated LPS-induced inflammatory cell infiltration, reduced pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) production and eliminated LPS-mediated lung edema. Furthermore, PSN administration significantly inhibited LPS-induced pulmonary MPO activity. Meanwhile, LPS-induced phosphorylation of p38 MAPK, iNOS expression and NO production in the lungs were also suppressed. The results demonstrate that PSN effectively attenuates LPS-induced inflammatory responses in vitro and in vivo; the beneficial effects are associated with the decreased phosphorylation of MAPK and AKT and the reduced expression of pro-inflammatory mediators, such as iNOS, NO and cytokines. These data suggest that PSN may be a potential therapeutic agent in the treatment of ALI.

AKT2 Regulates Pulmonary Inflammation and Fibrosis via Modulating Macrophage Activation.

Idiopathic pulmonary fibrosis (IPF) is a highly lethal pathological process that is characterized by inflammation, fibroblast accumulation, and excessive collagen deposition. Although AKT2-mediated signaling pathways modulate inflammatory responses, their role in IPF has not been defined. We report that AKT2 deficiency (Akt2-/-) protected against bleomycin-induced pulmonary fibrosis and inflammation. Adoptive transfer of wild-type macrophages or administration of IL-13 to Akt2-/- mice could restore pulmonary fibrosis. In response to IL-33 treatment, Akt2-/- macrophages displayed decreased production of IL-13 and TGF-ß1 and attenuated phosphorylation of FoxO3a compared with Akt2+/+ macrophages. Furthermore, the expression of IL-13 was increased by small interfering RNA knockdown of FoxO3a or in FoxO3a-deficient macrophages. By evaluating lung sections from pulmonary fibrosis patients, we found that the phosphorylation of AKT2 and FoxO3a was remarkably upregulated. Collectively, these results indicate that AKT2 modulates pulmonary fibrosis through inducing TGF-ß1 and IL-13 production by macrophages, and inhibition of AKT2 may be a potential strategy for treating IPF.

The Formyl Peptide Receptors: Diversity of Ligands and Mechanism for Recognition.

The formyl peptide receptors (FPRs) are G protein-coupled receptors that transduce chemotactic signals in phagocytes and mediate host-defense as well as inflammatory responses including cell adhesion, directed migration, granule release and superoxide production. In recent years, the cellular distribution and biological functions of FPRs have expanded to include additional roles in homeostasis of organ functions and modulation of inflammation. In a prototype, FPRs recognize peptides containing N-formylated methionine such as those produced in bacteria and mitochondria, thereby serving as pattern recognition receptors. The repertoire of FPR ligands, however, has expanded rapidly to include not only N-formyl peptides from microbes but also non-formyl peptides of microbial and host origins, synthetic small molecules and an eicosanoid. How these chemically diverse ligands are recognized by the three human FPRs (FPR1, FPR2 and FPR3) and their murine equivalents is largely unclear. In the absence of crystal structures for the FPRs, site-directed mutagenesis, computer-aided ligand docking and structural simulation have led to the identification of amino acids within FPR1 and FPR2 that interact with several formyl peptides. This review article summarizes the progress made in the understanding of FPR ligand diversity as well as ligand recognition mechanisms used by these receptors.

LYRM03, an ubenimex derivative, attenuates LPS-induced acute lung injury in mice by suppressing the TLR4 signaling pathway.

Toll-like receptor 4 (TLR4)-mediated signaling plays a critical role in sepsis-induced acute lung injury (ALI). LYRM03 (3-amino-2-hydroxy-4-phenyl-valyl-isoleucine) is a novel derivative of ubenimex, a widely used antineoplastic medicine. We previously found that LYRM03 has anti-inflammatory effects in cecal ligation puncture mouse model. In this study we determined whether LYRM03 attenuated LPS-induced ALI in mice. LPS-induced ALI mouse model was established by challenging the mice with intratracheal injection of LPS (5 mg/kg), which was subsequently treated with LYRM03 (10 mg/kg, ip). LYRM03 administration significantly alleviated LPS-induced lung edema, inflammatory cell (neutrophils and macrophages) infiltration and myeloperoxidase (MPO) activity, decreased pro-inflammatory and chemotactic cytokine (TNF-α, IL-6, IL-1ß, MIP-2) generation and reduced iNOS and COX-2 expression in the lung tissues. In cultured mouse alveolar macrophages in vitro, pretreatment with LYRM03 (100 µmol/L) suppressed LPS-induced macrophage activation by reducing Myd88 expression, increasing IκB stability and inhibiting p38 phosphorylation. These results suggest that LYRM03 effectively attenuates LPS-induced ALI by inhibiting the expression of pro-inflammatory mediators and Myd88-dependent TLR4 signaling pathways in alveolar macrophages. LYRM03 may serve as a potential treatment for sepsis-mediated lung injuries.

Shikonin Derivative DMAKO-05 Inhibits Akt Signal Activation and Melanoma Proliferation.

DMAKO-05((S)-1-((5E,8E)-5,8-bis(hydroxyimino)-1,4-dimethoxy-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-methylbutanoate) is a novel oxime derivative of shikonin, the major component extracted from Chinese herb Lithospermun erythrorhizon. Here, we report that DMAKO-05 had an antitumor activity against mouse melanoma cell line B16F0. Our studies indicated that DMAKO-05 not only inhibited B16F0 proliferation and migration but also led to cell cycle arrest at G1 phase and cell apoptosis, in which DMAKO-05 triggered mitochondrial-mediated apoptosis signal including caspase-9/3 and PARP. In response to DMAKO-05 treatment, the Akt-mediated survival signals were remarkably attenuated in B16F0 cells. Collectively, DMAKO-05 has a strong cytotoxicity in B16F0 cells via inhibiting Akt activation, inducing G1 arrest, and promoting B16F0 cell apoptosis. DMAKO-05 might serve as a potential candidate lead compound for melanoma.

The chemerin receptor CMKLR1 is a functional receptor for amyloid-ß peptide.

Amyloid-ß peptides such as Aß1-42 (Aß42) play a pivotal role in the progression of Alzheimer's disease (AD). Aß42 is neurotoxic and can activate microglial cells. These cells in turn migrate toward senile (neuritic) plaques and help to clear Aß deposits through an endocytotic mechanism. It is of potential significance to characterize the Aß42 receptors that mediate microglia chemotaxis and Aß42 uptake. We found that the transcript of the chemerin receptor CMKLR1 was upregulated in the brain of AD patients and in mouse brain tissue following systemic LPS administration. CMKLR1 and Aß42 colocalized in hippocampus and cortex of AßPP/PS1 transgenic mice. Moreover, Aß42 bound specifically to CMKLR1 in stably transfected rat basophilic leukemia (RBL) cells (CMKLR1-RBL), suggesting that CMKLR1 is a receptor for Aß42. Aß42 induced migration of primary microglia, the mouse microglial cell line N9, and CMKLR1-RBL cells, but not untransfected RBL-2H3 cells. Mechanistic studies showed that Aß42 induced CMKLR1-dependent cell migration through activation of the ERK1/2, PKA, and Akt pathways, but not Ca2+ mobilization. Aß42 stimulation of CMKLR1-RBL cells and primary glial cells led to internalization of the Aß42-CMKLR1 complex, suggesting a potential role for CMKLR1 in Aß42 clearance. Taken together, these results indicate that Aß42 activates CMKLR1, leading to glia cell migration and clearance of Aß42. CMKLR1 is a new addition to the repertoire of cell surface molecules that are responsible for Aß processing and clearance.

Chemerin C9 peptide induces receptor internalization through a clathrin-independent pathway.

AIM: The chemerin receptor CMKLR1 is one type of G protein-coupled receptors abundant in monocyte-derived dendritic cells and macrophages, which plays a key role in the entry of a subset of immunodeficiency viruses including HIV/SIV into lymphocytes and macrophages. The aim of this work was to investigate how CMKLR1 was internalized and whether its internalization affected cell signaling in vitro. METHODS: Rat basophilic leukemia RBL-2H3 cells, HEK 293 cells, and HeLa cells were used. CMKLR1 internalization was visualized by confocal microscopy imaging or using a FACScan flow cytometer. Six potential phosphorylation sites (Ser337, Ser343, Thr352, Ser344, Ser347, and Ser350) in CMKLR1 were substituted with alanine using site-directed mutagenesis. Heterologous expression of wild type and mutant CMKLR1 allowed for functional characterization of endocytosis, Ca(2+) flux and extracellular signal-regulated kinase (ERK) phosphorylation. RESULTS: Chemerin and the chemerin-derived nonapeptide (C9) induced dose-dependent loss of cell surface CMKLR1-GFP fusion protein and increased its intracellular accumulation in HEK 293 cells and RBL-2H3 cells stably expressing CMKLR1. Up to 90% of CMKLR1 was internalized after treatment with C9 (1 µmol/L). By using different agents, it was demonstrated that clathrin-independent mechanism was involved in CMKLR1 internalization. Mutations in Ser343 for G protein-coupled receptor kinase phosphorylation and in Ser347 for PKC phosphorylation abrogated CMKLR1 internalization. Loss of CMKLR1 internalization partially enhanced the receptor signaling, as shown by increased Ca(2+) flux and a shorter latency to peak level of ERK phosphorylation. CONCLUSION: CMKLR1 internalization occurs in a clathrin-independent manner, which negatively regulated the receptor-mediated Ca(2+) flux and ERK phosphorylation.

Structural determinants for the interaction of formyl peptide receptor 2 with peptide ligands.

Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-281(7.32) is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-281(7.32) might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D281(7.32)G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides.

Functional characterization of three mouse formyl peptide receptors.

The evolutionary relationship and functional correlation between human formyl peptide receptors (FPRs) and their mouse counterparts remain incompletely understood. We examined three members of the mouse formyl peptide receptor subfamily (mFprs) and found that they differ in agonist preference and cellular distributions. When stably expressed in transfected rat basophilic leukemia (RBL-2H3) cells, mFpr1 was readily activated by N-formylated peptides derived from Listeria monocytogenes (fMIVTLF), Staphylococcus aureus (fMIFL), and mitochondria (fMMYALF). In contrast, the Escherichia coli-derived fMLF was 1000-fold less potent. The aforementioned peptides were much less efficacious at mFpr2, which responded better to the synthetic hexapeptide WKYMVm, the synthetic agonists Quin-C1 (a substituted quinazolinone), and compound 43 (a nitrosylated pyrazolone derivative). Saturation binding assays showed that mFpr1 and mFpr2 were expressed at similar levels on the cell surface, although their affinity for N-formyl-Met-Leu-Phe-Ile-Ile-Lys-fluorescein isothiocyanate varied by more than 1000-fold [dissociation constant (K(d)) values of 2.8 nM for mFpr1 and 4.8 µM for mFpr2]). Contrary to these receptors, mFpr-rs1 responded poorly to all the previously mentioned peptides that were tested. Fluorescent microscopy revealed an intracellular distribution pattern of mFpr-rs1. On the basis of these results, we conclude that mFpr1 is an ortholog of human FPR1 with certain pharmacologic properties of human FPR2/ALX, whereas mFpr2 has much lower affinity for formyl peptides. The intracellular distribution of mFpr-rs1 suggests an evolutionary correlation with human FPR3.

Pharmacological kinetics of BmK AS, a sodium channel site 4-specific modulator on Nav1.3.

OBJECTIVE: In this study, the pharmacological kinetics of Buthus martensi Karsch (BmK) AS, a specific modulator of voltage-gated sodium channel site 4, was investigated on Na(v)1.3 expressed in Xenopus oocytes. METHODS: Two-electrode voltage clamp was used to record the whole-cell sodium current. RESULTS: The peak currents of Na(v)1.3 were depressed by BmK AS over a wide range of concentrations (10, 100, and 500 nmol/L). Most remarkably, BmK AS at 100 nmol/L hyperpolarized the voltage-dependence and increased the voltage-sensitivity of steady-state activation/inactivation. In addition, BmK AS was capable of hyperpolarizing not only the fast inactivation but also the slow inactivation, with a greater preference for the latter. Moreover, BmK AS accelerated the time constant and increased the ratio of recovery in Na(v)1.3 at all concentrations. CONCLUSION: This study provides direct evidence that BmK AS facilitates steady-state activation and inhibits slow inactivation by stabilizing both the closed and open states of the Na(v)1.3 channel, which might result from an integrative binding to two receptor sites on the voltage-gated sodium channels. These results may shed light on therapeutics against Na(v)1.3-targeted pathology.

Localization of receptor site on insect sodium channel for depressant ß-toxin BmK IT2.

BACKGROUND: BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized. PRINCIPAL FINDINGS: 2 µM BmK IT2 strongly shifted the activation of DmNa(v)1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNa(v)1.2, rNa(v)1.3 and mNa(v)1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu(896), Leu(899), Gly(904) in extracellular loop Domain II S3-S4 of DmNa(v)1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNa(v)1 could be conferred by Domain III. Analysis of subsequent DmNa(v)1 mutants highlighted the residues in Domain III pore loop, esp. Ile(1529) was critical for recognition and binding of BmK IT2. CONCLUSIONS/SIGNIFICANCE: In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNa(v)1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Na(v)1.2, Na(v)1.3 and Na(v)1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.