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Abstract
Transient receptor potential ankyrin 1 (TRPA1) is a sensor of nociceptive stimuli, expressed predominantly in a subpopulation of peptidergic sensory neurons which co‐express the noxious heat‐sensor transient receptor potential vanilloid 1. In this study, we describe a spinal cord synaptosome‐calcitonin gene‐related peptide (CGRP) release assay for examining activation of TRPA1 natively expressed on the central terminals of dorsal root ganglion neurons. We have shown for the first time that activation of TRPA1 channels expressed on spinal cord synaptosomes by a selection of agonists evokes a concentration‐dependent release of CGRP which is inhibited by TRPA1 antagonists. In addition, our results demonstrate that depolarization of spinal cord synaptosomes by a high concentration of KCl induces CGRP release via a T‐type calcium channel‐dependent mechanism whilst TRPA1‐induced CGRP release functions independently of voltage‐gated calcium channel activation. Finally, we have shown that pre‐treatment of synaptosomes with the opioid agonist, morphine, results in a reduction of depolarization‐induced CGRP release. This study has demonstrated the use of a dorsal spinal cord homogenate assay for investigation of natively expressed TRPA1 channels and for modulation of depolarizing stimuli at the level of the dorsal spinal cord.
Highlights
Neuropeptides released from primary afferent fibres relay nociceptive information in the spinal cord and play an important role in pain hypersensitivity (Gangadharan and Kuner 2013)
In order to examine whether activation of Transient receptor potential ankyrin 1 (TRPA1) evokes calcitonin gene-related peptide (CGRP) release from rat spinal cord synaptosomes, spinal cord homogenate was stimulated with TRPA1 agonists and the resulting CGRP release examined by enzyme-linked immunosorbent assay (ELISA)
Stimulation of ventral spinal cord homogenate by KCl (40 mmol/L) only evoked a small increase in CGRP release which was significantly lower than the release evoked by dorsal spinal cord homogenate (6 Æ 4% over basal release; P < 0.01; t-test; n = 3 experiments; Fig. 2). These findings suggest that cinnamaldehyde-evoked CGRP release is dependent on synaptosomes which originate from the dorsal spinal cord
Summary
Neuropeptides (e.g., calcitonin gene-related peptide [CGRP] and Substance P) released from primary afferent fibres relay nociceptive information in the spinal cord and play an important role in pain hypersensitivity (Gangadharan and Kuner 2013). TRPA1 has been suggested to play a role in spinal processing of nociceptive input. Activation of TRPA1 expressed on central terminals of primary afferent fibers has been shown to increase the frequency of miniature excitatory postsynaptic currents (mEPSCs) and diminish the amplitude of eEPSCs in subpopulations of laminae I and II neurons (Wrigley et al 2009). Pharmacology Research & Perspectives published by 2015 | Vol 3 | Iss. 6 | e00191
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