ABSTRACT
Molecular and behavioral evidence suggests that acid-sensing ion channels (ASICs) contribute to pain processing, but an understanding of their precise role remains elusive. Existing ASIC knock-out mouse experiments are complicated by the heteromultimerization of ASIC subunits. Therefore, we have generated transgenic mice that express a dominant-negative form of the ASIC3 subunit that inactivates all native neuronal ASIC-like currents by oligomerization. Using whole-cell patch-clamp recordings, we examined the response properties of acutely isolated dorsal root ganglion neurons to protons (pH 5.0). We found that whereas 33% of the proton-responsive neurons from wild-type mice exhibited an ASIC-like transient response, none of the neurons from the transgenic mice exhibited a transient inward current. Capsaicin-evoked responses mediated by the TRPV1 receptor were unaltered in transgenic mice. Adult male wild-type and transgenic mice were subjected to a battery of behavioral nociceptive assays, including tests of thermal, mechanical, chemical/inflammatory, and muscle pain. The two genotypes were equally sensitive to thermal pain and to thermal hypersensitivity after inflammation. Compared with wild types, however, transgenic mice were more sensitive to a number of modalities, including mechanical pain (von Frey test, tail-clip test), chemical/inflammatory pain (formalin test, 0.6% acetic acid writhing test), mechanical hypersensitivity after zymosan inflammation, and mechanical hypersensitivity after intramuscular injection of hypotonic saline. These data reinforce the hypothesis that ASICs are involved in both mechanical and inflammatory pain, although the increased sensitivity of transgenic mice renders it unlikely that they are direct transducers of nociceptive stimuli.
Subject(s)
Gene Expression Regulation/physiology , Inflammation/physiopathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/physiology , Protein Structure, Tertiary/physiology , Sodium Channels/genetics , Sodium Channels/metabolism , Acid Sensing Ion Channels , Animals , Behavior, Animal , Blotting, Northern/methods , Capsaicin/pharmacology , Cell Line , Chlorocebus aethiops , Cloning, Molecular/methods , Cricetinae , Ganglia, Spinal/cytology , Humans , Hydrogen-Ion Concentration , Inflammation/genetics , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutagenesis/physiology , Neurons/drug effects , Oocytes , Pain Measurement/methods , Patch-Clamp Techniques/methods , Physical Stimulation/methods , Protein Structure, Tertiary/genetics , Protein Subunits/genetics , Protein Subunits/metabolism , Protons , RNA, Messenger/metabolism , Reaction Time/drug effects , Reaction Time/physiology , Reaction Time/radiation effects , Reverse Transcriptase Polymerase Chain Reaction/methods , Time Factors , Transfection/methods , XenopusABSTRACT
Acid Sensing Ion Channels (ASICs) are a group of sodium-selective ion channels that are activated by low extracellular pH. The role of ASIC in disease states remains unclear partly due to the lack of selective pharmacological agents. In this report, we describe the effects of A-317567, a novel non-amiloride blocker, on three distinct types of native ASIC currents evoked in acutely dissociated adult rat dorsal root ganglion (DRG) neurons. A-317567 produced concentration-dependent inhibition of all pH 4.5-evoked ASIC currents with an IC50 ranging between 2 and 30muM, depending upon the type of ASIC current activated. Unlike amiloride, A-317567 equipotently blocked the sustained phase of ASIC3-like current, a biphasic current akin to cloned ASIC3, which is predominant in DRG. When evaluated in the rat Complete Freud's Adjuvant (CFA)-induced inflammatory thermal hyperalgesia model, A-317567 was fully efficacious at a dose 10-fold lower than amiloride. A-317567 was also potent and fully efficacious when tested in the skin incision model of post-operative pain. A-317567 was entirely devoid of any diuresis or natriuresis activity and showed minimal brain penetration. In summary, A-317567 is the first reported small molecule non-amiloride blocker of ASIC that is peripherally active and is more potent than amiloride in vitro and in vivo pain models. The discovery of A-317567 will greatly help to enhance our understanding of the physiological and pathophysiological role of ASICs.
Subject(s)
Acids/pharmacology , Amiloride/analogs & derivatives , Ganglia, Spinal/cytology , Membrane Proteins/drug effects , Nerve Tissue Proteins/drug effects , Neurons/drug effects , Sodium Channels/drug effects , Acid Sensing Ion Channels , Amiloride/pharmacology , Amiloride/therapeutic use , Animals , Cell Count/methods , Cell Size , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Interactions , Freund's Adjuvant , Hydrogen-Ion Concentration , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Isoquinolines/pharmacology , Isoquinolines/therapeutic use , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Proteins/classification , Naphthalenes/pharmacology , Naphthalenes/therapeutic use , Nerve Tissue Proteins/classification , Pain Measurement/methods , Pain Threshold/drug effects , Pain, Postoperative/chemically induced , Pain, Postoperative/diet therapy , Patch-Clamp Techniques/methods , Rats , Rats, Sprague-Dawley , Sodium Channels/classificationABSTRACT
C-fiber nociceptors can be divided into two groups based on growth factor dependency and isolectin B4 (IB4) binding. IB4-negative nociceptors have been proposed to contribute to inflammatory pain. Since the TRPV1 receptor is critical for inflammatory heat hyperalgesia, we hypothesized that inflammation would sensitize IB4 negative but not IB4-positive small-diameter neurons to TRPV1 stimuli. Two days after complete Freund's adjuvant (CFA)-induced inflammation in the hind paw of mice, lumbar 4/5 ganglia were dissociated and small-diameter (=26 microm) neurons were quantified for responsiveness to the TRPV1 agonists, capsaicin and protons using patch clamp recordings. Surprisingly, inflammation did not alter the responsiveness of IB4-negative neurons to capsaicin or protons. Conversely, inflammation increased the percentage of IB4-positive neurons that responded to 1 microM capsaicin from 24 to 80% and increased the percentage that responded to pH 5.0 from 54 to 85%. In parallel, inflammation increased the percentage of IB4-positive neurons that was TRPV1-immunoreactive. The inflammation-induced increase in capsaicin- and proton-responsiveness was entirely mediated by TRPV1 because IB4-positive neurons from inflamed TRPV1-/- mice were capsaicin-insensitive and unaltered in proton-responsiveness. Interestingly, comparison of neurons from TRPV1+/+ and TRPV1-/- mice revealed that the sustained proton-evoked currents in IB4-positive neurons were independent of TRPV1 whereas the sustained-only proton currents in IB4-negative neurons were TRPV1-dependent. Together, these data indicate that TRPV1 function and expression are selectively increased in IB4-positive neurons during inflammation in mouse and suggest a novel role for IB4-positive C-fibers during inflammation.
Subject(s)
Glycoproteins/metabolism , Hyperalgesia/physiopathology , Inflammation/physiopathology , Ion Channels/metabolism , Nociceptors/physiopathology , Animals , Capsaicin/administration & dosage , Cells, Cultured , Freund's Adjuvant , Hydrogen-Ion Concentration , Hyperalgesia/chemically induced , Inflammation/chemically induced , Ion Channels/drug effects , Men , Mice , Mice, Inbred C57BL , Nociceptors/drug effects , TRPV Cation ChannelsABSTRACT
Microglia, as phagocytes and antigen-presenting cells in the central nervous system, are activated in such disease processes as stroke and multiple sclerosis. Because peripheral macrophages are capable of producing endocannabinoids, we have examined endocannabinoid production in a macrophage-colony stimulating factor (M-CSF)-dependent rat microglial cell line (RTMGL1) using reversed phase high-pressure liquid chromatography and liquid chromatography-mass spectroscopy. We determined that cultured microglial cells produce the endocannabinoid 2-arachidonylglycerol (2-AG) as well as anandamide in smaller quantities. When 2-AG, but not anandamide, is added exogenously, RTMGL1 microglia increase their proliferation. This increased proliferation is blocked by an antagonist of the CB(2) receptor N-[(1S)endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) and mimicked by the CB(2) receptor-specific agonist 1,1-dimethylbutyl-1-deoxy-Delta(9)-tetrahydrocannabinol (JWH133). Accompanying the increase in proliferation seen with 2-AG is an increase in active ERK1 that is also blocked with SR144528. The RTMGL1 microglial cells, which exist in a primed state, express the CB(1) and CB(2) receptors as demonstrated by reverse transcription-polymerase chain reaction and immunostaining. The CB(2) receptor in untreated cells is expressed both at the cell surface and internally, and exposure of the cells to 2-AG significantly increases receptor internalization. These data suggest that 2-AG activation of CB(2) receptors may contribute to the proliferative response of microglial cells, as occurs in neurodegenerative disorders.
Subject(s)
Arachidonic Acids/metabolism , Cannabinoid Receptor Modulators/metabolism , Endocannabinoids , Glycerides/metabolism , Microglia/metabolism , Analysis of Variance , Animals , Cell Division , Cells, Cultured , Enzyme Activation , Flow Cytometry , Macrophage Colony-Stimulating Factor/metabolism , Microglia/cytology , Microglia/enzymology , Mitogen-Activated Protein Kinases/metabolism , Rats , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/metabolismABSTRACT
We have established a clonal cell line derived from rat microglia that proliferates in response to macrophage-colony stimulating factor (CSF-1). Like primary neonatal microglia, these cells (named RTMGL1) exhibit a ramified morphology, bind isolectin B4, express CD68 and are weakly positive for CD11b and MHC class II. CSF-1-dependent proliferation requires intact signal transduction through several pathways. RTMGL1 synthesize multiple cyclooxygenase (COX) products including 11- and 15-hydroxyeicosatetraenoic acid (HETE) and express COX-2. RTMGL1 synthesize 5-HETE from arachidonic acid (AA) likely via a 5-lipoxygenase (LO). Thus, RTMGL1 have morphological and histological characteristics of primary microglia and metabolize AA via both COX and LO pathways.
Subject(s)
Hydroxyeicosatetraenoic Acids/metabolism , Microglia/metabolism , Prostaglandins/metabolism , Animals , Animals, Newborn , Blotting, Western/methods , Cell Differentiation/drug effects , Cell Line , Chromatography, High Pressure Liquid/methods , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry/methods , Lectins/metabolism , Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Macrophage Colony-Stimulating Factor/pharmacology , Mass Spectrometry/methods , Prostaglandin-Endoperoxide Synthases/metabolism , Rats , Rats, Sprague-Dawley , Thymidine/pharmacokinetics , Time Factors , Tritium/pharmacokinetics , Tumor Necrosis Factor-alpha/analysisABSTRACT
Clinically, chronic pain and hyperalgesia induced by muscle injury are disabling and difficult to treat. Cellular and molecular mechanisms underlying chronic muscle-induced hyperalgesia are not well understood. For this reason, we developed an animal model where repeated injections of acidic saline into one gastrocnemius muscle produce bilateral, long-lasting mechanical hypersensitivity of the paw (i.e. hyperalgesia) without associated tissue damage. Since acid sensing ion channels (ASICs) are found on primary afferent fibers and respond to decreases in pH, we tested the hypothesis that ASICs on primary afferent fibers innervating muscle are critical to development of hyperalgesia and central sensitization in response to repeated intramuscular acid. Dorsal root ganglion neurons innervating muscle express ASIC3 and respond to acidic pH with fast, transient inward and sustained currents that resemble those of ASICs. Mechanical hyperalgesia produced by repeated intramuscular acid injections is prevented by prior treatment of the muscle with the non-selective ASIC antagonist, amiloride, suggesting ASICs might be involved. ASIC3 knockouts do not develop mechanical hyperalgesia to repeated intramuscular acid injection when compared to wildtype littermates. In contrast, ASIC1 knockouts develop hyperalgesia similar to their wildtype littermates. Extracellular recordings of spinal wide dynamic range (WDR) neurons from wildtype mice show an expansion of the receptive field to include the contralateral paw, an increased response to von Frey filaments applied to the paw both ipsilaterally and contralaterally, and increased response to noxious pinch contralaterally after the second intramuscular acid injection. These changes in WDR neurons do not occur in ASIC3 knockouts. Thus, activation of ASIC3s on muscle afferents is required for development of mechanical hyperalgesia and central sensitization that normally occurs in response to repeated intramuscular acid. Therefore, interfering with ASIC3 might be of benefit in treatment or prevention of chronic hyperalgesia.