ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons

BACKGROUND: ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1) ASIC3 might trigger ischemic pain in heart and muscle; 2) it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses. RESULTS: Less than half (40%) of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small (<25 μm) sensory neurons that innervate muscle are more likely to express ASIC3 than those that innervate skin (51% of small muscle afferents vs. 28% of small skin afferents). Over 80% of ASIC3+ muscle afferents co-express CGRP (a vasodilatory peptide). Remarkably few (9%) ASIC3+ cells express P2X3 receptors (an ATP-gated ion channel), whereas 31% express TRPV1 (the noxious heat and capsaicin-activated ion channel also known as VR1). ASIC3+/CGRP+ sensory nerve endings were observed on muscle arterioles, the blood vessels that control vascular resistance; like the cell bodies, the endings are P2X3- and can be TRPV1+. The TrkC+/ASIC3+ cell bodies are uniformly large, possibly consistent with non-nociceptive mechanosensation. They are not proprioceptors because they fail two other tests: ASIC3+ cells do not express parvalbumin and they are absent from the mesencephalic trigeminal nucleus. CONCLUSION: Our data indicates that: 1) ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2) co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen

Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists

Agonists of TRPA1 such as mustard oil and its key component AITC cause pain and neurogenic inflammation in humans and pain behavior in rodents. TRPA1 is activated by numerous reactive compounds making it a sensor for reactive compounds in the body. Failure of AITC, formalin and other reactive compounds to trigger pain behavior in TRPA1 knockout mice, as well as the ability of TRPA1 antisense to alleviate cold hyperalgesia after spinal nerve ligation, suggest that TRPA1 is a potential target for novel analgesic agents. Here, we have characterized CHO cells expressing human and rat TRPA1 driven by an inducible promoter. As reported previously, both human and rat TRPA1 are activated by AITC and inhibited by ruthenium red. We have also characterized noxious cold response of these cell lines and show that noxious cold activates both human and rat TRPA1. Further, we have used CHO cells expressing human TRPA1 to screen a small molecule compound library and discovered that 'trichloro(sulfanyl)ethyl benzamides' (AMG2504, AMG5445, AMG7160 and AMG9090) act as potent antagonists of human TRPA1 activated by AITC and noxious cold. However, trichloro(sulfanyl)ethyl benzamides' (TCEB compounds) displayed differential pharmacology at rat TRPA1. AMG2504 and AMG7160 marginally inhibited rat TRPA1 activation by AITC, whereas AMG5445 and AMG9090 acted as partial agonists. In summary, we conclude that both human and rat TRPA1 channels show similar AITC and noxious cold activation profiles, but TCEB compounds display species-specific differential pharmacology at TRPA1

Pharmacologic Characterization of AMG 334, a Potent and Selective Human Monoclonal Antibody against the Calcitonin Gene-Related Peptide Receptor

ABSTRACT Therapeutic agents that block the calcitonin gene-related peptide (CGRP) signaling pathway are a highly anticipated and promising new drug class for migraine therapy, especially after reports that small-molecule CGRP-receptor antagonists are efficacious for both acute migraine treatment and migraine prevention. Using XenoMouse technology, we successfully generated AMG 334, a fully human monoclonal antibody against the CGRP receptor. Here we show that AMG 334 competes with [ 125 I]-CGRP binding to the human CGRP receptor, with a K i of 0.02 nM. AMG 334 fully inhibited CGRP-stimulated cAMP production with an IC 50 of 2.3 nM in cell-based functional assays (human CGRP receptor) and was 5000-fold more selective for the CGRP receptor than other human calcitonin family receptors, including adrenomedullin, calcitonin, and amylin receptors. The potency of AMG 334 at the cynomolgus monkey (cyno) CGRP receptor was similar to that at the human receptor, with an IC 50 of 5.7 nM, but its potency at dog, rabbit, and rat receptors was significantly reduced (.5000-fold). Therefore, in vivo target coverage of AMG 334 was assessed in cynos using the capsaicin-induced increase in dermal blood flow model. AMG 334 dose-dependently prevented capsaicin-induced increases in dermal blood flow on days 2 and 4 postdosing. These results indicate AMG 334 is a potent, selective, full antagonist of the CGRP receptor and show in vivo dose-dependent target coverage in cynos. AMG 334 is currently in clinical development for the prevention of migraine

Trichloro(sulfanyl)ethyl benzamides inhibit AITC (80 μM) induced increase in intracellular calcium and inward currents in CHO cells stably expressing human TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Effect of AMG9090 , AMG5445 , AMG2504 , and AMG7160 on AITC-induced increase in intracellular calcium in CHO cells expressing human TRPA1 measured in an aequorin-readout assay. Open circles represent the response of cells to the compound itself in the absence of agonist. Each point in the graph is an average ± SEM of an experiment conducted in triplicate

Effect of human TRPA1 antagonists on CHO cells stably expressing rat TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Concentration-response curves generated in aequorin-readout assay Note, AMG9090 and AMG5445 acted as partial agonists. Each point in the graph is presented as percent of AITC response and is an average ± SEM of an experiment conducted in triplicate. Representative traces of inward currents induced by AITC, AMG9090 and AMG5445 are shown in , , and respectively. AITC-induced increase in intracellular calcium in CHO cells expressing rat TRPA1 in the absence (100%) or presence of different concentrations of AMG2504 and AMG 7160. Representative current traces induced by AITC in the presence of AMG2504 and AMG 7160 are shown in and , respectively

Characterization of CHO cell lines stably expressing human and rat TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> FLASH luminometer was used to measure concentration-dependent AITC-induced increase in intracellular calcium, and concentration-dependent inhibition of AITC (80 μM) activation by ruthenium red in CHO cells expressing human and rat TRPA1. Each point in the graph is an average ± SEM of an experiment conducted in triplicate. Maximum response of AITC (80 μM) was normalized to 100%. Cold activation profiles of CHO cells expressing human and rat TRPA1 were characterized utilizing cold-induced Cauptake assay. Cauptake by CHO cells and CHO cells transfected with human TRPA1 in response to stimulation with temperatures between 3.5 and 25°C. Cold temperature (4°C) activation of un-induced and tetracycline-induced CHO cells transfected with either human or rat TRPA1. Concentration-dependent inhibition of cold (4°C) activation by ruthenium red in CHO cells expressing human and rat TRPA1. Each point in the graph is an average ± SEM of an experiment conducted in duplicate

Trichloro(sulfanyl)ethyl benzamides inhibit AITC (80 μM) induced inward currents in CHO cells stably expressing human TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Representative traces of inward currents evoked by AITC in the absence or presence of AMG9090 , AMG5445 , AMG2504 , and AMG7160 are shown. ICvalue for each compound was determined from their concentration-dependent inhibition of AITC-induced currents using a PatchXpress 7000A workstation

Trichloro(sulfanyl)ethyl benzamides inhibit cold temperature (4°C) induced Cauptake in CHO cells stably expressing human TRPA1

<p><b>Copyright information:</b></p><p>Taken from "Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists"</p><p>http://www.molecularpain.com/content/3/1/39</p><p>Molecular Pain 2007;3():39-39.</p><p>Published online 17 Dec 2007</p><p>PMCID:PMC2222611.</p><p></p> Concentration-response curves were generated utilizing Cauptake assays as described under . Each point in the graph is an average ± SEM of an experiment conducted in duplicate