Ruth Jostock

Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay

TRPM8 (CMR1) is a Ca2+‐permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type‐1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. The effects of 70 odorants and menthol‐related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR® assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS‐3, WS‐23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. The EC50 values of the agonists defined their relative potencies: icilin (0.2±0.1 μM)>FrescolatML (3.3±1.5 μM) > WS‐3 (3.7±1.7 μM) >(−)menthol (4.1±1.3 μM) >frescolatMAG (4.8±1.1 μM) > cooling agent 10 (6±2.2 μM) >(+)menthol (14.4±1.3 μM) > PMD38 (31±1.1 μM) > WS‐23 (44±7.3 μM) > Coolact P (66±20 μM) > geraniol (5.9±1.6 mM) > linalool (6.7±2.0 mM) > eucalyptol (7.7±2.0 mM) > hydroxycitronellal (19.6±2.2 mM). Known VR1 antagonists (BCTC, thio‐BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC50: 0.8±1.0, 3.5±1.1 and 18±1.1 μM, respectively). The Ca2+ response of hVR1‐transfected HEK293 cells to the endogenous VR1 agonist N‐arachidonoyl‐dopamine was potentiated by low pH. In contrast, menthol‐ and icilin‐activated TRPM8 currents were suppressed by low pH. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.

Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1

To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED(50) value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.

The cAMP Pathway Sensitizes VR1 Expressed in Oocytes from Xenopus laevis and in CHO Cells

The vanilloid receptor 1 (VR1) is a heat-activated cation channel which also responds to capsaicin and other chemical stimuli. Protein kinase C has a stimulatory effect on VR1 activity, either alone or after activation with capsaicin. The influence of the cAMP-signaling pathway on the effects of capsaicin is controversial. To clarify this, the actions of capsaicin and the modulatory effects of forskolin, pCPT-cAMP, and isobutylmethylxanthine were studied in Xenopus laevis oocytes expressing rat VR1 and in CHO cells expressing human VR1. Capsaicin activated the VR1 channel and increased the intracellular calcium concentration. The effects of capsaicin were enhanced by forskolin, pCPT-cAMP, and isobutylmethylxanthine. A modulatory function of the cAMP system on VR1 activation could, therefore, modulate heat sensation and pain.

Nociceptin/orphanin FQ opioid peptide-receptor expression in pachyonychia congenita

Nociceptin/orphanin FQ opioid peptide (NOP)‐receptor (NOP‐R) is a member of the opioid receptor family. NOP‐R activation has demonstrated analgesic effects in preclinical pain models without the addiction risks associated with other opiate targets. Pachyonychia congenita (PC) is a palmoplantar keratoderma characterized by neuropathic pain in affected skin. A cohort of KRT6A gene mutation PC patients with no other explanation for their neuropathic pain offered a unique opportunity to assess potential of NOP‐R as a therapeutic target. Plantar biopsies from 10 PC patients and 10 age/gender matched controls were performed at the ball (PC‐affected) and the arch (PC‐unaffected) of the foot. NOP‐R expression was assessed by immunohistochemistry. Localization of NOP‐R in subsets of epidermal nerve fibers was investigated using the pan‐neuronal marker PGP9.5, markers for unmyelinated peptidergic fibers (calcitonin gene‐related peptide [CGRP] and substance P [SP]), as well as for myelinated Aδ and Aβ fibers (neurofilament H [NFH]). Robust NOP‐R expression was detected in epidermal keratinocytes and in a subset of PGP9.5+ fibers in both epidermis and dermis, confirmed by western blot and absorption experiments with NOP‐R peptide. NOP‐R expression in keratinocytes was significantly reduced in PC‐affected plantar skin compared with PC‐unaffected skin. In addition, NOP‐R expression occurred in dermal NFH+ myelinated fibers in all groups, although few CGRP+ fibers co‐expressed NOP‐R. Furthermore, most SP+ fibers also co‐expressed NOP‐R. These findings indicate that NOP‐R is expressed on epidermal keratinocytes, as well as on epidermal and dermal nerve fibers and has potential as a promising target to treat neuropathic pain in PC.