Inhibition of TRPM8 by the urinary tract analgesic drug phenazopyridine.

BACKGROUND: and purpose: Phenazopyridine (PAP) is an over-the-counter drug widely used to provide symptomatic relief of bladder pain in conditions such as cystitis or bladder pain syndrome (BPS). Whereas the analgesic effect of PAP has been attributed to a local effect on the mucosa of the lower urinary tract (LUT), the molecular targets of PAP remain unknown. We investigated the effect of PAP on pain-related Transient Receptor Potential (TRP) channels expressed in sensory neurons that innervate the bladder wall. EXPERIMENTAL APPROACH: The effects of PAP on the relevant TRP channels (TRPV1, TRPA1, TRPM8, TRPM3) expressed in HEK293 or CHO cells was investigated using Fura-2-based calcium measurements and whole-cell patch-clamp recordings. Activity of PAP on TRPM8 was further analysed using Fura-2-based calcium imaging on sensory neurons isolated from lumbosacral dorsal root ganglia (DRG) of mice. KEY RESULTS: PAP rapidly and reversibly inhibits responses of TRPM8 expressed in HEK293 cells to cold and menthol, with IC50 values between 2 and 10 µM. It acts by shifting the voltage dependence of channel activation towards positive potentials, opposite to the effect of menthol. PAP also inhibits TRPM8-mediated, menthol-evoked calcium responses in lumbosacral DRG neurons. At a concentration of 10 µM, PAP did not significantly affect TRPA1, TRPV1, or TRPM3. CONCLUSION AND IMPLICATIONS: PAP inhibits TRPM8 in a concentration range consistent with PAP levels in the urine of treated patients. Since TRPM8 is expressed in bladder afferent neurons and upregulated in patients with painful bladder disorders, TRPM8 inhibition may underlie the analgesic activity of PAP.

Bradykinin induces peripheral antinociception in PGE2-induced hyperalgesia in mice.

BACKGROUND: Bradykinin (BK) is an endogenous peptide involved in vascular permeability and inflammation. It has opposite effects (inducing hyperalgesia or antinociception) when administered directly in the central nervous system. The aim of this study was to evaluate whether BK may also present this dual effect when injected peripherally in a PGE2-induced nociceptive pain model, as well as to investigate the possible mechanisms of action involved in this event in mice. METHODS: Male Swiss and C57BL/6 knockout mice for B1 or B2 bradykinin receptors were submitted to a mechanical paw pressure test and hyperalgesia was induced by intraplantar prostaglandin E2 (2 µg/paw) injection. RESULTS: Bradykinin (20, 40 and 80 ng/paw) produced dose-dependent peripheral antinociception against PGE2-induced hyperalgesia. This effect was antagonized by bradyzide (8, 16 and 32 µg/paw), naloxone (12.5, 25 and 50 µg/paw), nor-binaltorphimine (50, 100 and 200 µg/paw) and AM251 (20, 40 and 80 µg/paw). Bestatin (400 µg/paw), MAFP (0.5 µg/paw) and VDM11 (2.5 µg/paw) potentiated the antinociception of a lower 20 ng BK dose. The knockout of B1 or B2 bradykinin receptors partially abolished the antinociceptive action of BK (80 ng/paw), bremazocine (1 µg/paw) and anandamide (40 ng/paw) when compared with wild-type animals, which show complete antinociception with the same dose of each drug. CONCLUSION: The present study is the first to demonstrate BK-induced antinociception in peripheral tissues against PGE2-induced nociception in mice and the involvement of κ-opioid and CB1 cannabinoid receptors in this effect.

Pharmacological properties of TRPM3 isoforms are determined by the length of the pore loop.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that plays a pivotal role in the peripheral nervous system as a transducer of painful heat signals. Alternative splicing gives rise to several TRPM3 variants. The functional consequences of these splice isoforms are poorly understood. Here, the pharmacological properties of TRPM3 variants arising from alternative splicing in the pore-forming region were compared. EXPERIMENTAL APPROACH: Calcium microfluorimetry and patch clamp recordings were used to compare the properties of heterologously expressed TRPM3α1 (long pore variant) and TRPM3α2-α6 (short pore variants). Furthermore, site-directed mutagenesis was done to investigate the influence of the length of the pore loop on the channel function. KEY RESULTS: All short pore loop TRPM3α variants (TRPM3α2-α6) were activated by the neurosteroid pregnenolone sulphate (PS) and by nifedipine, whereas the long pore loop variant TRPM3α1 was insensitive to either compound. In contrast, TRPM3α1 was robustly activated by clotrimazole, a compound that does not directly activate the short pore variants but potentiates their responses to PS. Clotrimazole-activated TRPM3α1 currents were largely insensitive to established TRPM3α2 antagonists and were only partially inhibited upon activation of the µ opioid receptor. Finally, by creating a set of mutant channels with pore loops of intermediate length, we showed that the length of the pore loop dictates differential channel activation by PS and clotrimazole. CONCLUSION AND IMPLICATIONS: Alternative splicing in the pore-forming region of TRPM3 defines the channel's pharmacological properties, which depend critically on the length of the pore-forming loop. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Why the emperor penguin reigns where elephants shiver.

Accurate sensing of the environmental temperature is crucial for the survival and wellbeing of organisms. In vertebrates, the cold- and menthol-activated ion channel TRPM8 acts as the prime molecular sensor of cool temperatures. By comparing TRPM8 in vertebrates with different habitat temperatures, from elephants to penguins, Yang et al. identify key residues within the pore domain that determine the channel's cold sensitivity. Strikingly, mice engineered to express penguin TRPM8 show a remarkable tolerance to cold.

A spider derived peptide, PnPP-19, induces central antinociception mediated by opioid and cannabinoid systems.

BACKGROUND: Some peptides purified from the venom of the spider Phoneutria nigriventer have been identified as potential sources of drugs for pain treatment. In this study, we characterized the antinociceptive effect of the peptide PnPP-19 on the central nervous system and investigated the possible involvement of opioid and cannabinoid systems in its action mechanism. METHODS: Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. All drugs were administered by the intracerebroventricular route. RESULTS: PnPP-19 induced central antinociception in mice in the doses of 0.5 and 1 µg. The non-selective opioid receptor antagonist naloxone (2.5 and 5 µg), µ-opioid receptor antagonist clocinnamox (2 and 4 µg), δ-opioid receptor antagonist naltrindole (6 and 12 µg) and CB1 receptor antagonist AM251 (2 and 4 µg) partially inhibited the antinociceptive effect of PnPP-19 (1 µg). Additionally, the anandamide amidase inhibitor MAFP (0.2 µg), the anandamide uptake inhibitor VDM11 (4 µg) and the aminopeptidase inhibitor bestatin (20 µg) significantly enhanced the antinociception induced by a low dose of PnPP-19 (0.5 µg). In contrast, the κ-opioid receptor antagonist nor-binaltorphimine (10 µg and 20 µg) and the CB2 receptor antagonist AM630 (2 and 4 µg) do not appear to be involved in this effect. CONCLUSIONS: PnPP-19-induced central antinociception involves the activation of CB1 cannabinoid, µ- and δ-opioid receptors. Mobilization of endogenous opioids and cannabinoids might be required for the activation of those receptors, since inhibitors of endogenous substances potentiate the effect of PnPP-19. Our results contribute to elucidating the action of the peptide PnPP-19 in the antinociceptive pathway.

A spider derived peptide, PnPP-19, induces central antinociception mediated by opioid and cannabinoid systems

Some peptides purified from the venom of the spider Phoneutria nigriventer have been identified as potential sources of drugs for pain treatment. In this study, we characterized the antinociceptive effect of the peptide PnPP-19 on the central nervous system and investigated the possible involvement of opioid and cannabinoid systems in its action mechanism. Methods Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. All drugs were administered by the intracerebroventricular route. Results PnPP-19 induced central antinociception in mice in the doses of 0.5 and 1 g. The non-selective opioid receptor antagonist naloxone (2.5 and 5 g), -opioid receptor antagonist clocinnamox (2 and 4 g), -opioid receptor antagonist naltrindole (6 and 12 g) and CB1 receptor antagonist AM251 (2 and 4 g) partially inhibited the antinociceptive effect of PnPP-19 (1 g). Additionally, the anandamide amidase inhibitor MAFP (0.2 g), the anandamide uptake inhibitor VDM11 (4 g) and the aminopeptidase inhibitor bestatin (20 g) significantly enhanced the antinociception induced by a low dose of PnPP-19 (0.5 g). In contrast, the -opioid receptor antagonist nor-binaltorphimine (10 g and 20 g) and the CB2 receptor antagonist AM630 (2 and 4 g) do not appear to be involved in this effect. Conclusions PnPP-19-induced central antinociception involves the activation of CB1 cannabinoid, - and -opioid receptors. Mobilization of endogenous opioids and cannabinoids might be required for the activation of those receptors, since inhibitors of endogenous substances potentiate the effect of PnPP-19. Our results contribute to elucidating the action of the peptide PnPP-19 in the antinociceptive pathway.

A spider derived peptide, PnPP-19, induces central antinociception mediated by opioid and cannabinoid systems

Background: Some peptides purified from the venom of the spider Phoneutria nigriventer have been identified as potential sources of drugs for pain treatment. In this study, we characterized the antinociceptive effect of the peptide PnPP-19 on the central nervous system and investigated the possible involvement of opioid and cannabinoid systems in its action mechanism. Methods: Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. All drugs were administered by the intracerebroventricular route.Results: PnPP-19 induced central antinociception in mice in the doses of 0.5 and 1 µg. The non-selective opioid receptor antagonist naloxone (2.5 and 5 µg), µ-opioid receptor antagonist clocinnamox (2 and 4 µg), δ-opioid receptor antagonist naltrindole (6 and 12 µg) and CB1 receptor antagonist AM251 (2 and 4 µg) partially inhibited the antinociceptive effect of PnPP-19 (1 µg). Additionally, the anandamide amidase inhibitor MAFP (0.2 µg), the anandamide uptake inhibitor VDM11 (4 µg) and the aminopeptidase inhibitor bestatin (20 µg) significantly enhanced the antinociception induced by a low dose of PnPP-19 (0.5 µg). In contrast, the κ-opioid receptor antagonist nor-binaltorphimine (10 µg and 20 µg) and the CB2 receptor antagonist AM630 (2 and 4 µg) do not appear to be involved in this effect. Conclusions: PnPP-19-induced central antinociception involves the activation of CB1 cannabinoid, µ- and δ-opioid receptors. Mobilization of endogenous opioids and cannabinoids might be required for the activation of those receptors, since inhibitors of endogenous substances potentiate the effect of PnPP-19. Our results contribute to elucidating the action of the peptide PnPP-19 in the antinociceptive pathway.(AU)