Melittin Treatment Research Articles

Hydrogels Embedded With Melittin and Tobramycin Are Effective Against Pseudomonas aeruginosa Biofilms in an Animal Wound Model.

We demonstrate that the antimicrobial peptide, melittin, is effective alone and in combination with the aminoglycosides tobramycin to kill Pseudomonas aeruginosa growing as biofilms both in vitro and in vivo. Melittin and tobramycin show enhanced in vitro activity in combination at micromolar concentrations, resulting in a 2-log10 reduction in the number of cells within mature PAO1 P. aeruginosa biofilms after 6-h of treatment. Alternatively, either agent alone resulted in half-a-log10 reduction. Time-killing assays demonstrated that the combination of melittin and tobramycin was effective at 2-h whereas tobramycin was not effective until after 6-h of treatment. We also found the combination was more effective than tobramycin alone against biofilms of 7 P. aeruginosa cystic fibrosis clinical isolates, resulting in a maximum 1.5-log10 cellular reduction. Additionally, melittin alone was effective at killing biofilms of 4 Staphylococcus aureus isolates, resulting in a maximum 2-log10 cellular reduction. Finally, melittin in combination with tobramycin embedded in an agarose-based hydrogel resulted in a 4-fold reduction in bioluminescent P. aeruginosa colonizing mouse wounds by 4-h. In contrast, tobramycin or melittin treatment alone did not cause a statistically significant reduction in bioluminescence. These data demonstrate that melittin in combination with tobramycin embedded in a hydrogel is a potential treatment for biofilm-associated wound infections.

Melittin – A bee venom component – Enhances muscle regeneration factors expression in a mouse model of skeletal muscle contusion

Melittin is a major peptide component of sweet bee venom that possesses anti-allergic, anti-inflammatory, anti-arthritis, anti-cancer, and neuroprotective properties. However, the therapeutic effects of melittin on muscle injury have not been elucidated. We investigated the therapeutic effects of melittin on muscle injury in a mouse model of muscle contusion. The biceps femoris muscle of the mice was injured using drop mass method, and the animals were treated with melittin (4, 20, or 100 μg/kg) for 7 days. Melittin significantly increased: locomotor activity in open field test, and treadmill running activity in a dose-dependent manner to level comparable to the positive control, diclofenac (30 mg/kg). Melittin treatment attenuated the pro-inflammatory cytokine MCP-1, TNF-α and IL-6. The expression of muscle regeneration biomarkers, including MyoD (muscle differentiation marker), myogenin, smooth muscle actin, and myosin heavy chain was markedly increased in the injured muscle tissue of melittin-treated mice, as determined by western blotting and quantitative real-time polymerase chain reaction. These results demonstrate that melittin inhibits inflammatory response and improves muscle damage by regenerating muscles in a mouse model of muscle contusion. Taken together, the results of present study suggest that melittin is a promising candidate for the muscle injury treatment.

Melittin Exerts Beneficial Effects on Paraquat-Induced Lung Injuries In Mice by Modifying Oxidative Stress and Apoptosis.

Melittin (MEL) is a 26-amino acid peptide with numerous biological activities. Paraquat (PQ) is one of the most widely used herbicides, although it is extremely toxic to humans. To date, PQ poisoning has no effective treatment, and therefore the current study aimed to assess for the first time the possible effects of MEL on PQ-induced lung injuries in mice. Mice received a single intraperitoneal (IP) injection of PQ (30 mg/kg), followed by IP treatment with MEL (0.1 and 0.5 mg/kg) twice per week for four consecutive weeks. Histological alterations, oxidative stress, and apoptosis in the lungs were studied. Hematoxylin and eosin (H&E) staining indicated that MEL markedly reduced lung injuries induced by PQ. Furthermore, treatment with MEL increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity, and decreased malonaldehyde (MDA) and nitric oxide (NO) levels in lung tissue homogenates. Moreover, immunohistochemical staining showed that B-cell lymphoma-2 (Bcl-2) and survivin expressions were upregulated after MEL treatment, while Ki-67 expression was downregulated. The high dose of MEL was more effective than the low dose in all experiments. In summary, MEL efficiently reduced PQ-induced lung injuries in mice. Specific pharmacological examinations are required to determine the effectiveness of MEL in cases of human PQ poisoning.

Melittin Inhibits Hypoxia-Induced Vasculogenic Mimicry Formation and Epithelial-Mesenchymal Transition through Suppression of HIF-1α/Akt Pathway in Liver Cancer.

In this study, we investigated whether melittin could suppress hypoxia-induced vasculogenic mimicry (VM) formation in liver cancer and explored the underlying mechanisms. Melittin significantly inhibited the proliferation of liver cancer cells with or without CoCl2 presence. Melittin also significantly inhibited CoCl2-induced migration, invasion, and VM formation of liver cancer cells. CoCl2 treatment suppressed the expression of E-cadherin and elevated the expression of N-cadherin and Vimentin. Melittin reversed the changes in the protein and mRNA levels of these epithelial-mesenchymal transition (EMT) markers. CoCl2-induced accumulation of HIF-1α increased the level of phosphorylated Akt and upregulated the expression of VEGF and MMP-2/9. Melittin decreased the HIF-1α level and thereby suppressed the levels of p-Akt, VEGF, and MMP-2/9. In addition, the inhibitor of PI3K/Akt also suppressed CoCl2-induced EMT and liver cancer cells migration, and the activator of Akt, SC-79, partly blocked the effect of melittin on CoCl2-induced EMT and liver cancer cells migration. In the xenograft tumor model in nude mice, melittin treatment significantly suppressed the tumor growth, VM formation, and HIF-1α expression in the tumor. In conclusion, this study indicates melittin may inhibit hypoxia-induced VM formation and EMT in liver cancer through inhibiting HIF-1α/Akt pathway.

Melittin-induced long non-coding RNA NONHSAT105177 inhibits proliferation and migration of pancreatic ductal adenocarcinoma

Long non-coding RNAs (lncRNAs) play crucial roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Previously, we found that melittin treatment suppressed PDAC tumor growth. However, it is unclear whether lncRNAs have any role in the melittin-induced suppression of PDAC. In this study, we used microarray data to identify 844 lncRNAs that were significantly differentially expressed in response to melittin treatment. Of these lncRNAs, we focused on the lncRNA NONHSAT105177, which had about a 22-fold increase in expression with melittin treatment. We found that melittin treatment increased NONHSAT105177 expression in PDAC cell lines but not in normal pancreatic ductal epithelial cell line. NONHSAT105177 expression was significantly lower in PDAC cancer tissues than in adjacent noncancerous tissues. Additionally, overexpression of NONHSAT105177 inhibited PDAC cell proliferation, migration, and the epithelial–mesenchymal transition (EMT), both in vitro and in vivo. Consistent with the mechanism of action of melittin, NONHSAT105177 significantly downregulated cholesterol pathway genes, including Clusterin (CLU). Moreover, we found that NONHSAT105177 trafficking was mediated by exosomes. The combined findings of our current and previous studies suggest that NONHSAT105177 mediated the melittin-induced inhibition of PDAC cell growth and metastasis, which indicated a potential target for developing new strategies.

Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1\u03b2 release

The nucleotide-binding domain and leucine-rich repeat-containing receptor with a pyrin domain 3 (NLRP3) inflammasome is a sensor for different types of infections and alterations of homeostatic parameters, including abnormally high levels of the extracellular nucleotide ATP or crystallization of different metabolites. All NLRP3 activators trigger a similar intracellular pathway, where a decrease in intracellular K+ concentration and permeabilization of plasma membrane are key steps. Cationic amphipathic antimicrobial peptides and peptide toxins permeabilize the plasma membrane. In fact, some of them have been described to activate the NLRP3 inflammasome. Among them, the bee venom antimicrobial toxin peptide melittin is known to elicit an inflammatory reaction via the NLRP3 inflammasome in response to bee venom. Our study found that melittin induces canonical NLRP3 inflammasome activation by plasma membrane permeabilization and a reduction in the intracellular K+ concentration. Following melittin treatment, the apoptosis-associated speck-like protein, an adaptor protein with a caspase recruitment domain (ASC), was necessary to activate caspase-1 and induce IL-1β release. However, cell death induced by melittin prevented the formation of large ASC aggregates, amplification of caspase-1 activation, IL-18 release and execution of pyroptosis. Therefore, melittin-induced activation of the NLRP3 inflammasome results in an attenuated inflammasome response that does not result in caspase-1 dependent cell death.

Melittin-Containing Hybrid Peptide Hydrogels for Enhanced Photothermal Therapy of Glioblastoma.

The design of biocompatible and efficacious anticancer biomaterials to achieve relatively low tumor recurrence rates is the main pursuit of cancer photothermal therapy (PTT). RADA16-I is a synthetic amphiphilic peptide with the sequence RADARADARADARADA that can self-assemble into a peptide nanofiber hydrogel. In this study, we synthesized a novel melittin-RADA32-indocyanine green (ICG) hydrogel ("MRI hydrogel"), which contains melittin in the peptide hydrogel backbone and ICG in the hydrogel matrix, for enhanced PTT of glioblastomas. The MRI hydrogel exhibited physiologic characteristics similar to those of the RADA16 hydrogel, while displaying concentration-dependent cytotoxicity to C6 glioma cells and photothermal effects. The in vivo biodistribution of the MRI hydrogel was visualized by near-infrared fluorescence and photoacoustic imaging. More importantly, in vivo PTT provided by the MRI hydrogel significantly reduced the tumor size and the tumor recurrence rate compared with the RADR16-ICG hydrogel and other controls, suggesting a synergistic effect of MRI hydrogel-carried melittin and ICG-based PTT treatment. Thus, MRI provides an alternative tool for the safe and efficient PTT treatment of tumors.

Melittin suppresses tumor progression by regulating tumor-associated macrophages in a Lewis lung carcinoma mouse model.

Tumor-associated macrophages (TAM) are a major component of tumor stroma. It has been reported that TAMs have M2-like phenotype and facilitate tumor progression by promoting angiogenesis and immunosuppression. Melittin, a major polypeptide of bee venom, has been widely studied as an anti-cancer drug due to its cytotoxicity to malignant cells. However, very little is known regarding the effect of melittin on immune cells in the tumor microenvironment. This study focuses on the effect of melittin on TAMs in a Lewis lung carcinoma mouse model. Melittin inhibited the rapid tumor growth compared to the control in vivo. Melittin increased the M1/M2 ratio of TAMs by selectively reducing the number of CD206+ M2-like TAMs while not altering the population of CD86+ M1-like TAMs. Melittin also preferentially binds to M2 macrophages, and this binding was not associated with phagocytosis. Gene and protein expression of vascular endothelial growth factor (Vegf) and mannose receptor C type 1 (Mrc1/CD206) was reduced in M2-like bone marrow-derived macrophages by melittin treatment, but there was no significant change in the gene level of Vegf and FMS-like tyrosine kinase 1 (Flt1/VEGFR1) in tumor cells in vitro. Additionally, the levels of VEGF and CD31, markers of angiogenesis, were significantly decreased by melittin treatment in tumor tissues. This study revealed a novel role for melittin in tumor treatment and suggested that melittin could be a promising therapeutic agent for targeting M2-like TAMs.

Melittin suppresses tumor progression by regulating tumor-associated macrophages in a Lewis lung carcinoma mouse model

Abstract Tumor-associated macrophages (TAM) are a major component of tumor stroma. It has been reported that TAMs have M2-like phenotype and facilitate tumor progression by promoting angiogenesis and immunosuppression. Melittin, a major polypeptide of bee venom, has been widely studied as an anti-cancer drug due to its cytotoxicity to malignant cells. However, very little is known regarding the effect of melittin on immune cells in the tumor microenvironment. We demonstrated here that melittin inhibited the rapid tumor growth compared to the control in vivo. Melittin increased the M1/M2 ratio of TAMs by selectively reducing the number of CD206+ M2-like TAMs, while not affecting the number of CD86+ M1-like TAMs. Melittin also preferentially bound to CD11b+ cells and this binding was not associated with phagocytosis. Gene expression of vascular endothelial growth factor (Vegf) and mannose receptor C type 1 (Mrc1/CD206) was reduced in M2-like bone marrow-derived macrophages by melittin treatment, but there was no significant change in the gene level of Vegf and FMS-like tyrosine kinase 1 (Flt1/VEGFR1) in tumor cells. Additionally, the levels of VEGF and CD31, markers of angiogenesis, were significantly decreased by melittin treatment in tumor tissues. This study revealed a novel role for melittin in tumor treatment and suggested that melittin could be a promising therapeutic agent for targeting M2-like TAMs.

The Protective Effect of Melittin on Renal Fibrosis in an Animal Model of Unilateral Ureteral Obstruction.

Renal fibrosis is the principal pathological process underlying the progression of chronic kidney disease that leads to end-stage renal disease. Melittin is a major component of bee venom, and it has anti-bacterial, anti-viral, and anti-inflammatory properties in various cell types. Thus, this study examined the therapeutic effects of melittin on the progression of renal fibrosis using the unilateral ureteral obstruction (UUO) model. In addition, the effects of melittin on inflammation and fibrosis in renal fibroblast cells were explored using transforming growth factor-β1 (TGF-β1). Histological observation revealed that UUO induced a considerable increase in the number of infiltrated inflammatory cells. However, melittin treatment markedly reduced these reactions compared with untreated UUO mice. The expression levels of inflammatory cytokines and pro-fibrotic genes were significantly reduced in melittin-treated mice compared with UUO mice. Melittin also effectively inhibited fibrosis-related gene expression in renal fibroblasts NRK-49F cells. These findings suggest that melittin attenuates renal fibrosis and reduces inflammatory responses by the suppression of multiple growth factor-mediated pro-fibrotic genes. In conclusion, melittin may be a useful therapeutic agent for the prevention of fibrosis that characterizes the progression of chronic kidney disease.

Effect of melittin treatment in a mouse model of xenobiotic-induced cholangitis and biliary fibrosis

Effect of melittin treatment in a mouse model of xenobiotic-induced cholangitis and biliary fibrosis

Effects of Melittin Treatment in Cholangitis and Biliary Fibrosis in a Model of Xenobiotic-Induced Cholestasis in Mice.

Cholangiopathy is a chronic immune-mediated disease of the liver, which is characterized by cholangitis, ductular reaction and biliary-type hepatic fibrosis. There is no proven medical therapy that changes the course of the disease. In previous studies, melittin was known for attenuation of hepatic injury, inflammation and hepatic fibrosis. This study investigated whether melittin provides inhibition on cholangitis and biliary fibrosis in vivo. Feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to mice is a well-established animal model to study cholangitis and biliary fibrosis. To investigate the effects of melittin on cholangiopathy, mice were fed with a 0.1% DDC-containing diet with or without melittin treatment for four weeks. Liver morphology, serum markers of liver injury, cholestasis markers for inflammation of liver, the degree of ductular reaction and the degree of liver fibrosis were compared between with or without melittin treatment DDC-fed mice. DDC feeding led to increased serum markers of hepatic injury, ductular reaction, induction of pro-inflammatory cytokines and biliary fibrosis. Interestingly, melittin treatment attenuated hepatic function markers, ductular reaction, the reactive phenotype of cholangiocytes and cholangitis and biliary fibrosis. Our data suggest that melittin treatment can be protective against chronic cholestatic disease in DDC-fed mice. Further studies on the anti-inflammatory capacity of melittin are warranted for targeted therapy in cholangiopathy.

Neuroprotective effects of melittin on hydrogen peroxide-induced apoptotic cell death in neuroblastoma SH-SY5Y cells.

BackgroundFree radicals are involved in neuronal cell death in human neurodegenerative diseases. Since ancient times, honeybee venom has been used in a complementary medicine to treat various diseases and neurologic disorders. Melittin, the main component of honeybee venom, has various biologic effects, including anti-bacterial, anti-viral, and anti-inflammatory activities.MethodsWe investigated the neuroprotective effects of melittin against H2O2-induced apoptosis in the human neuroblastoma cell line SH-SY5Y. The neuroprotective effects of melittin on H2O2-induced apoptosis were investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide assay, caspase 3 activity, 4,6-diamidino-2-phenylindole staining, a lactate dehydrogenase release assay, Western blots, and reverse transcription-polymerase chain reaction.ResultsThe H2O2-treated cells had decreased cell viability with apoptotic features and increased production of caspase-3. On the other hand, melittin treatment increased cell viability and decreased apoptotic DNA fragmentation. Melittin attenuated the H2O2-induced decrease in mRNA and protein production of the anti-apoptotic factor Bcl-2. In addition, melittin inhibited both the H2O2-induced mRNA and protein expression of Bax-associated pro-apoptotic factor and caspase-3.ConclusionsThese findings suggest that melittin has potential therapeutic effects as an agent for the prevention of neurodegenerative diseases.

Protective effect of melittin against inflammation and apoptosis on Propionibacterium acnes-induced human THP-1 monocytic cell

Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). It has been used in treatment of various chronic inflammatory diseases. However, the cellular mechanism and the anti-apoptotic effect of melittin in Propionibactierium acnes (P. acnes)-induced THP-1 cells have not been explored. In the present study, we investigated the anti-inflammatory and anti-apoptotic mechanism by examining the effect of melittin on P. acnes-induced THP-1 monocytic cells. THP-1 monocytic cells were stimulated by heat-killed P. acnes in the presence of melittin. The expression levels of pro-inflammatory cytokines, NF-κB signaling, caspase family, and PARP signaling were measured by ELISA or Western blot analysis. The number of apoptotic cells and changes of cell morphology were examined using fluorescence microscopy and flow cytometry. Heat-killed P. acnes increased the secretion of pro-inflammatory cytokines and cleavage of caspase-3 and -8 in heat-killed P. acnes-induced THP-1 cells. However, treatment with melittin inhibited the pro-inflammatory cytokines and cleavage of the caspase-3 and -8. Moreover, the cleaved PARP appeared after 8h of heat-killed P. acnes treatment and its cleavage was reduced by melittin treatment. These results demonstrate that 1.0×107CFU/ml of heat-killed P. acnes induces THP-1 cell apoptosis and secretion of inflammatory cytokines. Also, administration of melittin significantly decreases the expression of various inflammatory cytokines in heat-killed P. acnes-treated THP-1 monocytic cells. In particular, melittin exerts anti-apoptotic effects against 1.0×107CFU/ml of heat-killed P. acnes injury to THP-1 cells.

The Protective Effects of Melittin on Propionibacterium acnes–Induced Inflammatory Responses In Vitro and In Vivo

Melittin is the main component in the venom of the honey bee (Apis mellifera). It has multiple effects including antibacterial, antiviral, and anti-inflammatory activities in various cell types. However, the anti-inflammatory mechanisms of melittin have not been elucidated in Propionibactierium acnes (P. acnes)-induced keratinocyte or inflammatory skin disease animal models. In this study, we examined the effects of melittin on the production of inflammatory cytokines in heat-killed P. acnes-induced HaCaT cells. Heat-killed P. acnes-treated keratinocytes increased the expression of pro-inflammatory cytokines and Toll-like receptor 2. However, melittin treatment significantly suppressed the expression of these cytokines through regulation of the NF-κB and MAPK signaling pathways. Subsequently, the living P. acnes (1 × 10(7) CFU) were intradermally injected into the ear of mice. Living P. acnes-injected ears showed cutaneous erythema, swelling, and granulomatous response at 24 hours after injection. However, melittin-treated ears showed markedly reduced swelling and granulomatous responses compared with ears injected with only living P. acnes. These results demonstrate the feasibility of applying melittin for the prevention of inflammatory skin diseases induced by P. acnes.

Protective effects of melittin on tumor necrosis factor-α induced hepatic damage through suppression of apoptotic pathway and nuclear factor-kappa B activation.

Melittin, a major polypeptide in honeybee venom, have been used to treat inflammatory disease. Various studies have demonstrated the anti-bacterial, anti-viral, anti-inflammatory and anticancer effects of bee venom and melittin. However, the precise mechanism of melittin in liver disease is not yet known. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis. In the present study, we investigated the anti-apoptotic effect of melittin on tumor necrosis factor (TNF)-α/actinomycin (Act) D-induced apoptosis in hepatocytes. Our results show significant protection from DNA damage by melittin treatment compared with corresponding TNF-α/Act D-treated hepatocytes without melittin. Melittin inhibited TNF-α/Act D-induced activation of the caspase, bcl-2 family of proteins and poly ADP-ribose polymerase (PARP)-1. Our results also indicate that melittin decreased nuclear factor-kappa B (NF-κB) by degradation of phosphorylation of IκB kinase (p-IKK) and NF-κB DNA binding activity in TNF-α/Act D-treated hepatocytes. These results suggest that melittin possesses a potent suppressive effect on apoptotic responses in TNF-α/Act D-treated hepatocytes via the NF-κB pathway.

Melittin ameliorates the inflammation of organs in an amyotrophic lateral sclerosis animal model.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disorder characterized by a selective loss of motor neurons in the spinal cord, brainstem, and motor cortex, leading to weakness of the limb and bulbar muscles. Although the immediate cause of death in ALS is the destruction of motor neurons, ALS is a multi-organ disease that also affects the lungs, spleen, and liver. Melittin is one of components of bee venom and has anti-neuroinflammatory effects in the spinal cord, as shown in an ALS animal model. To investigate the effects of melittin on inflammation in the lungs and spleen, we used hSOD1G93A transgenic mice that are mimic for ALS. Melittin treatment reduced the expression of inflammatory proteins, including Iba-1 and CD14 by 1.9- and 1.3-fold (p<0.05), respectively, in the lungs of symptomatic hSOD1G93A transgenic mice. In the spleen, the expression of CD14 and COX2 that are related to inflammation were decreased by 1.4 fold (p<0.05) and cell survival proteins such as pERK and Bcl2 were increased by 1.3- and 1.5-fold (p<0.05) in the melittin-treated hSOD1G93A transgenic mice. These findings suggest that melittin could be a candidate to regulate the immune system in organs affected by ALS.

Comparative proteome analysis of Tumor necrosis factor α-stimulated human Vascular Smooth Muscle Cells in response to melittin

BackgroundBee venom has been used to relieve pain and to treat inflammatory diseases, including rheumatoid arthritis, in humans. To better understand the mechanisms of the anti-inflammatory and anti-atherosclerosis effect of bee venom, gel electrophoresis and mass spectrometry were used to identify proteins whose expression was altered in human Vascular Smooth Muscle Cells (hVSMCs) stimulated by tumor necrosis factor alpha after 12 h in the presence of melittin.ResultsTo obtain valuable insights into the anti-inflammatory and anti-atherosclerosis mechanisms of melittin, two-dimensional (2-D) gel electrophoresis and MALDI-TOF/TOF were used. The proteome study, we showed 33 significant proteins that were differentially expressed in the cells treated with tumor necrosis factor alpha and melittin. Thirteen proteins were significantly increased in the cells treated with tumor necrosis factor alpha, and those proteins were reduced in the cells treated with melittin. Five of the proteins that showed increased expression in the cells treated with tumor necrosis factor alpha are involved in cell migration, including calreticulin, an essential factor of development that plays a role in transcription regulation. The proteins involved in cell migration were reduced in the melittin treated cells. The observed changes in the expression of GRP75, prohibitin, and a select group of other proteins were validated with reverse transcribed-PCR. It was confirmed that the observed change in the protein levels reflected a change in the genes level. In addition, the phosphorylation of EGFR and ERK was validated by analyzing the protein pathway.ConclusionTaken together, these data established that the expression of some proteins was significantly changed by melittin treatment in tumor necrosis factor alpha stimulated the cells and provided insights into the mechanism of the melittin function for its potential use as an anti-inflammatory agent.

Involvement of Peripheral NMDA Receptor in Melittin-Induced Thermographic Flare

Intradermal injection of an active compound of European honeybee toxin, melittin, into the forearm in humans produces temporary pain and evokes sustained increase of local skin temperature. This increase of skin temperature is suppressed by the pretreatment of a voltage gated sodium channel blocker, lidocaine, suggesting that neurogenic inflammation is involved in the skin temperature increase after the melittin treatment. In this study, we tested a hypothesis that the melittin-induced skin temperature increase is augmented by an N-methyl-D-aspartate (NMDA) glutamate receptor that is present on the peripheral terminals of cutaneous primary afferents. Skin temperature was examined after the local application of incremental doses of melittin by a computer-assisted-thermography in pentobarbital-anesthetized rats. Local subcutaneous glutamate was collected through a microdialysis probe and glutamate levels were measured by a high pressure liquid chromatography with electrochemical detection method. Intraplantar injection of melittin resulted in the increase of subcutaneous glutamate levels and the increase of local skin temperature, which was partially attenuated by co-injection of an NMDA receptor antagonist, MK-801. In addition, intraplantar injection of NMDA itself increased the local skin temperature. Our data suggest that melittin-induced increase of skin temperature is enhanced through the activation of peripheral NMDA receptors by locally released glutamate. We suggest that topical administration of NMDA receptor antagonists could be an effective treatment of neuro-inflammatory pain.

Melittin initiates dopamine transporter internalization and recycling in transfected HEK-293 cells

The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked by cocaine and other ligands. Melittin inhibits dopamine transporter function and causes internalization of the recombinant transporter in stably transfected HEK-293 cells, but the specific pathways for internalization and disposition of the transporter are unknown. Here we report that melittin treatment increased both transporter internalization and colocalization with clathrin, effects that were blocked by pretreatment with cocaine. Density gradient centrifugation revealed that melittin treatment caused the dopamine transporter to associate with a density fraction containing the early endosome marker Rab 5A. Confocal microscopy revealed that melittin treatment also increased transporter colocalization with Rab 5A and decreased colocalization with the late endosome marker Rab 7 and the recycling endosome marker Rab 11. Following 60min of melittin treatment, the transporter was trafficked back to the membrane. By comparison, phorbol ester treatment increased transporter colocalization with early endosome antigen 1 and Rab 7 in a time-dependent manner. Cocaine treatment alone does not affect transporter trafficking in these cells. Results indicate multiple dopamine transporter internalization and recycling pathways that depend on transporter-ligand interactions and post-translational modifications.