ABSTRACT
Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.
Subject(s)
Eicosapentaenoic Acid , Neuralgia , Nucleotide Transport Proteins , Adenosine Triphosphate/metabolism , Animals , Eicosapentaenoic Acid/pharmacology , Eicosapentaenoic Acid/therapeutic use , Humans , Insulin Resistance , Mice , Neuralgia/drug therapy , Neuralgia/genetics , Nociception , Nucleotide Transport Proteins/antagonists & inhibitors , Nucleotide Transport Proteins/genetics , Nucleotide Transport Proteins/metabolismABSTRACT
Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT-/- mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.
Subject(s)
Adenosine Triphosphate/metabolism , Neutrophil Infiltration , Neutrophils/metabolism , Nucleotide Transport Proteins/metabolism , Secretory Vesicles/metabolism , Adjuvants, Immunologic/pharmacology , Animals , Biological Transport/drug effects , Biomarkers/metabolism , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Movement/drug effects , Freund's Adjuvant/pharmacology , Gene Expression Regulation , Humans , Male , Matrix Metalloproteinase 9/metabolism , Membrane Transport Modulators/pharmacology , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Activation/drug effects , Neutrophil Infiltration/drug effects , Neutrophils/cytology , Neutrophils/drug effects , Neutrophils/immunology , Nucleotide Transport Proteins/antagonists & inhibitors , Nucleotide Transport Proteins/genetics , Protein Transport/drug effects , Secretory Vesicles/drug effects , Secretory Vesicles/immunologyABSTRACT
Vesicular nucleotide transporter (VNUT) is responsible for vesicular ATP storage in ATP-secreting cells. In the present study, we examined the effects on VNUT-mediated transport of ATP release inhibitors such as ATP-binding cassette (ABC) proteins, hemichannels, maxi anion channels and P2X7 receptor. The ATP transport activity of proteoliposomes containing purified human VNUT was blocked by glibenclamide, carbenoxolone, 18 α-glycyrrhetinic acid, flufenamic acid, arachidonic acid and A438079 without the formation of Δψ (positive inside) as a driving force being affected. Thus, inhibitors of ATP release may inhibit VNUT and subsequent ATP release, since the previous works proved that inhibitors of ATP release blocked VNUT-mediated ATP release at the cell level.