Research reportTRPM8 protein localization in trigeminal ganglion and taste papillae
Introduction
Cold sensation is derived from activation of the somatosensory system by a cold stimulus. Cold stimulus elicits afferent impulses in nerve fibers of sensory neurons with a small diameter in dorsal root ganglia (DRG) or trigeminal ganglia (TG), innervating the trunk and head, respectively [5], [16], [21]. Cold fibers are comprised of both A delta and C fibers, have activation thresholds approximately <30 °C, and have maximal activation between 20 and 30 °C [18]. In contrast, noxious cold (cold pain) stimuli (<15 °C) activate C fibers [18]. Calcium-imaging and patch-clamp studies in dissociated DRG or TG neurons have revealed that cold stimuli induced calcium influx, suggesting the direct opening of calcium-permeable ion channels by cold [16], [21], [25].
Recently, two cold activated cation channels called transient receptor potential melastatin-8 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1) were cloned [13], [23]. TRPM8 and TRPA1 are activated by cold temperature (<23–28 °C) and noxious cold temperature (<15 °C), respectively [8], [13], [19], [23]. Co-expression analysis showed that TRPA1 mRNA was mostly found in transient receptor potential vanilloid-1 (TRPV1: a heat-, acid-, or capsaicin-activated cation channel)-positive neurons, whereas cells expressing TRPM8 mRNA were few in TRPV1-positive neurons [19], [23]. The analysis of TRPV1 knockout mice revealed that TRPV1 is involved in pain sensation and hypersensitivity to noxious stimuli [1]. These reports indicate that TRPA1 is involved in cold pain (noxious cold) sensation. Therefore, TRPM8 is thought to be a candidate protein for cold reception in the sensory system. Cooling-evoked increase of intracellular calcium in DRG and TG, and TRPM8 mRNA expression in a subset of both DRG and TG cells, supported the expression of TRPM8 protein in DRG and TG neurons [16], [25]. However, there is no evidence that TRPM8 protein is expressed in TG neurons or in their peripheral nerve terminals.
The tongue is one of the well-characterized sensory organs. Various stimuli, including chemical, thermal, and touch stimuli, on the tongue elicit responses in sensory nerve fibers. The lingual branch of the trigeminal nerve (V) and the chorda tympani branch of the facial nerve (VII) innervate the anterior two-thirds of the tongue, and the glossopharyngeal nerve (IX) innervates the posterior one-third of the tongue [3], [4], [26]. Most lingual nerve fibers terminate in the epithelial layers or papillary layers and do not form synapses with taste bud cells [11]. Chorda tympani nerve fibers make synapses with the taste bud cells in the fungiform or the foliate papillae [10]. Glossopharyngeal nerve innervates taste buds in the circumvallate and foliate papillae [14]. Chemical stimuli initially interact with taste receptor on taste cells in taste papilla and activate nerve fibers which make synapse with taste cells [22]. Chemical and thermal stimuli on the tongue elicit activation of lingual nerve fibers predominantly [20], indicating warm and cold receptors were expressed in the lingual branch of trigeminal neuron. Immunohistochemical studies revealed that TRPV1-immunoreactive nerve fibers reached the outer epithelial layer, but were absent around the taste buds in circumvallate, foliate, and fungiform papillae [7], [9], indicating that the TRPV1 protein locates on the lingual branch of the trigeminal nerve. These reports lead us to the hypothesis that cold receptor protein TRPM8 is present in the sensory nerve fibers that innervate the tongue. In this study, we produced a rabbit antiserum against TRPM8 and examined the localization of TRPM8 protein in TG and their nerve endings in tongue.
TRPM8 mRNA was found in a subset of TRPV1-immunoreactive cells in the primary culture of DRG [15]. Sensory neurons and their nerve terminals expressed with both heat and cold receptors might conduct a paradoxical cold sensation [15]. Calcitonin gene-related peptide (CGRP)-positive neurons are present in approximately 10% of the TRPV1-positive neurons in human TG, suggesting that TRPV1 activation might modulate pain by CGRP release in CGRP and TRPV1 co-expressed cells [6]. From these observations, we hypothesize that TRPM8 is expressed in CGRP-positive neurons in TG and their nerve terminals. Neurons expressing both TRPM8 and CGRP might evoke cold-induced pain sensation [18]. We also examine the possibility of colocalization of TRPM8 and TRPV1/CGRP in TG neurons and nerve fibers in sensory organ using double staining analysis.
Section snippets
Antibodies
The cDNA encoding rat TRPM8 was kindly provided by Dr. D. Julius [13]. Antibody against TRPM8 was obtained by immunizing rabbits using glutathione S-transferase (GST)-tagged recombinant C-terminus (1005–1104) of rat TRPM8. Anti-TRPM8 antiserum was affinity purified with His-tagged recombinant TRPM8 protein (1025–1104). Each construct was checked by sequencing. Anti-c-Myc (Pierce), anti-PGP 9.5 (Biogenesis), anti-neurofilament-200 (NF200; a marker of neurons forming A-fibers [12], Sigma),
Antibody against TRPM8
To examine the localization of TRPM8, we generated antiserum against recombinant protein that corresponded to the carboxyl terminus of rat TRPM8. Specificity of the raised antiserum was examined by immunoblotting and immunofluorescence analysis (Fig. 1). Both anti-TRPM8 and anti-c-Myc antibody recognized a single specific 130-kDa band on the Western blot of proteins from HEK293 cells transfected with pCMV-Myc-TRPM8, but not mock-transfected cells (Fig. 1A, lanes 1–4). The signal obtained by
Discussion
TRPM8 expression is restricted in a subset of neurons with a small diameter (Fig. 2). This observation is consistent with previous reports that neurons responding to decreasing temperature (<30 °C) are found in a cell group with small diameter in TG or DRG [19], [24], [25], and that expression of TRPM8 mRNA was observed in a group of neurons with relatively small diameter in TG [13]. Temperature-sensitive neurons are known to have C fibers or A delta fibers [18]. Double labeling experiments
Acknowledgment
This work was supported by a grant-in-aid from Japan Society for the Promotion of Science.
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2020, European Journal of PharmacologyCitation Excerpt :A growing body of evidence reported TRPM8 expression in animal models. In rats, TRPM8 mRNA and protein were detected in the prostate (Stein et al., 2004), bladder (Stein et al., 2004), skin (Chen et al., 2010), dorsal root ganglia (DRG) (McKemy et al., 2002; Stein et al., 2004) and trigeminal ganglia (TG) (10–13%) (Abe et al., 2005; McKemy et al., 2002), also in femoral, tail, mesenteric and intralobar pulmonary arteries, and thoracic aorta (Johnson et al., 2009; Yang et al., 2006). Transcripts were more prevalent in TG than DRG (McKemy et al., 2002).