Molecular identification and functional characterization of a temperature-sensitive transient receptor potential channel (TRPM8) from canine

TRPM8 belongs to the family of transient receptor potential channels and is activated by cooling and cooling agents, such as icilin and menthol. It is expressed in a subset of sensory neurons and is thought to be involved in thermosensation. Here, we report the cloning and functional characterization of canine TRPM8 (cTRPM8). cTRPM8 shares 95.1%, 94.1%, and 93.9% protein sequence identity with human, rat and mouse TRPM8, respectively. Similar to these mammalian orthologs, cTRPM8 was activated by menthol and icilin with strong outward rectification and little cation selectivity. Menthol and icilin also caused calcium-dependent desensitization. Interestingly, cTRPM8 was activated at <17 degrees C, a temperature threshold lower than that reported for the other orthologs. At 22 degrees C, the EC(50) for activation of cTRPM8 expressed in HEK293 cells by icilin and menthol was 0.06 and 4.3 microM determined by Fluorometric Imaging Plate Reader (FLIPR) and 0.4 and 85 microM by patch clamp, respectively. Mustard oil also activated cTRPM8 (FLIPR EC(50) = 490 microM). Menthol activation was more potent at +60 mV than at -60 mV (EC(50) = 53 and 124 microM, respectively, in Xenopus ooctyes). Icilin-, menthol- and mustard oil-induced intracellular Ca(2+) increases were similarly blocked by N-(4-tertiarybutyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (BCTC) with IC(50) = 2.3, 2.8 and 1.8 microM, respectively. Cooling-activated current was also inhibited by BCTC. Extracellular calcium blocked cTRPM8 in a concentration- and voltage-dependent manner (half maximal blocking [Ca(2+)] = 1.6 mM at -100 mV). These results constitute the first study of cTRPM8 and support the idea that cTRPM8 functions as a transducer of cold stimuli in vivo.     

Lack of transient receptor potential melastatin 8 activation by phthalate esters that enhance contact hypersensitivity in mice

We studied the involvement of sensory neurons in skin sensitization to allergens using a mouse model in which the T-helper type 2 response is essential. Skin sensitization to fluorescein isothiocyanate (FITC) has been shown to be enhanced by several phthalate esters, including dibutyl phthalate (DBP). For different types of phthalate esters, we found a correlation between the ability of transient receptor potential (TRP) A1 activation and that of enhancing skin sensitization. A TRPA1-specific antagonist, HC-030031, was shown to suppress skin sensitization in the presence of DBP. However, since phthalate esters also activate TRPV1, phthalate esters could activate other types of TRP channels non-selectively. Furthermore, sensitization to FITC is also enhanced by menthol, which activates TRPA1 and TRPM8. Here we established an in vitro system for measuring TRPM8 activation. The selectivity for TRPM8 was established by the fact that two TRPM8 agonists (menthol and icilin) induced calcium mobilization, whereas agonists of TRPA1 and TRPV1 did not. We demonstrated that phthalate esters do not activate TRPM8. TRPA1-antagonist HC-030031 did not inhibit TRPM8 activation induced by menthol or icilin. These results show that phthalate esters activate TRPA1 and TRPV1 with selectivity. TRPM8 activation is not likely to be involved in the sensitization to FITC.     

Design and optimization of benzimidazole-containing transient receptor potential melastatin 8 (TRPM8) antagonists

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermoresponsive to cool to cold temperatures (8-28 °C) and also may be activated by chemical agonists such as menthol and icilin. Antagonism of TRPM8 activation is currently under investigation for the treatment of painful conditions related to cold, such as cold allodynia and cold hyperalgesia. The design, synthesis, and optimization of a class of selective TRPM8 antagonists based on a benzimidazole scaffold is described, leading to the identification of compounds that exhibited potent antagonism of TRPM8 in cell-based functional assays for human, rat, and canine TRPM8 channels. Numerous compounds in the series demonstrated excellent in vivo activity in the TRPM8-selective "wet-dog shakes" (WDS) pharmacodynamic model and in the rat chronic constriction injury (CCI)-induced model of neuropathic pain. Taken together, the present results suggest that the in vivo antagonism of TRPM8 constitutes a viable new strategy for treating a variety of disorders associated with cold hypersensitivity, including certain types of neuropathic pain.     

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1)

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.     

TRPM8 biology and medicinal chemistry

TRPM8 belongs to the TRPM Melastatin subfamily of Transient Receptor Potential (TRP) ion channels. Activated by cool temperatures and mimetic ligands, such as menthol and icilin, TRPM8 has been shown to play a role in thermoreception and is expressed in peripheral nerves. TRPM8 is also expressed in other tissues which are not exposed to temperature fluctuations, such as the prostate. The recent advancement of a TRPM8 agonist into the clinic for the treatment of prostate cancer suggests that the channel plays a role in some human pathologies. As more drug-like and selective agonists and antagonists of TRPM8 become available, in vivo pharmacology studies will complement already published knockout data to further our understanding of the role of TRPM8 in human disease.     

Differential behavioral effect of the TRPM8/TRPA1 channel agonist icilin (AG-3-5)

Molecular identification of two new transient receptor potential (TRP) channels, TRPM8 and TRPA1, has prompted an intense interest in their functional roles. We report that an acute exposure to the TRPM8/TRPA1 agonist icilin (0.01-100 microM), but not TRPV1 agonist capsaicin (10 microM), causes an atypical dose-related increase in planarian motility. This is the first demonstration of a TRPM8/TRPA1 channel subtype agonist-induced differential pharmacological effect in invertebrates and provides a novel sensitive, quantifiable end-point for studying TRP channel pharmacology.     

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1)

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC(50) values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+/-0.20 microM; human=1.90+/-0.30 microM: CHO cells: rat=0.20+/-0.06 microM; human=0.19+/-0.08 microM). In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC(50) values for capsaicin-induced responses were similar in both cell lines (rat=0.35+/-0.06 microM, human=0.53+/-0.03 microM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC(50) pH 5.49+/-0.04 and pH 5.78+/-0.09, respectively). The threshold for heat activation was identical (42 degrees C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC(50) between 3 and 10 microM) but was virtually inactive at the human VR1 (EC(50)>10 microM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.     

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.     

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.     

Primary afferents with TRPM8 and TRPA1 profiles target distinct subpopulations of rat superficial dorsal horn neurones

Background and purpose:

The transient receptor potential (TRP) channels, transient receptor potential melastatin-1 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1), are expressed in subpopulations of sensory neurones and have been proposed to mediate innocuous and noxious cold sensation respectively. The aim of this study was to compare TRPM8 and TRPA1 modulation of glutamatergic afferent transmission within the spinal dorsal horn.

Experimental approach:

Whole cell patch clamp recordings were made from rat spinal cord slices in vitro to examine the effect of TRP agonists and temperature on glutamatergic excitatory postsynaptic currents (EPSCs).

Key results:

Icilin (3 or 100 µmol·L⁻¹), menthol (200 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) reduced the amplitude of primary afferent evoked EPSCs in subpopulations of lamina I and II neurones. In a subpopulation of superficial neurones, innocuous cold (threshold 29°C), 3 µmol·L⁻¹ icilin (EC₅₀ 1.5 µmol·L⁻¹) and menthol (EC₅₀ 263 µmol·L⁻¹) increased the rate of spontaneous miniature EPSCs. In the majority of lamina I and II neurones, 100 µmol·L⁻¹ icilin (EC₅₀ 79 µmol·L⁻¹), allyl isothiocyanate (EC₅₀ 226 µmol·L⁻¹), cinnamaldehyde (EC₅₀ 38 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) increased miniature EPSC rate. The response to 100 µmol·L⁻¹, but not 3 µmol·L⁻¹ icilin, was abolished by ruthenium red, while neither was affected by iodoresiniferatoxin. Responsiveness to 3 µmol·L⁻¹, but not to 100 µmol·L⁻¹ icilin, was highly predictive of innocuous cold responsiveness. Neurones responding to 3 µmol·L⁻¹ icilin and innocuous cold were located more superficially than those responding to 100 µmol·L⁻¹ icilin.

Conclusions and implications:

Activation of TRPM8 and TRPA1 presynaptically modulated glutamatergic transmission onto partially overlapping but distinct populations of superficial dorsal horn neurones. Spinal TRPM8 and TRPA1 channels may therefore provide therapeutic targets in cold hyperesthesia.     

Fused piperidines as a novel class of potent and orally available transient receptor potential melastatin type 8 (TRPM8) antagonists

The transient receptor potential melastatin type 8 (TRPM8) is a nonselective cation channel primarily expressed in a subpopulation of sensory neurons that can be activated by a wide range of stimuli, including menthol, icilin, and cold temperatures (<25 °C). Antagonism of TRPM8 is currently under investigation as a new approach for the treatment of pain. As a result of our screening efforts, we identified tetrahydrothienopyridine 4 as an inhibitor of icilin-induced calcium influx in CHO cells expressing recombinant rat TRPM8. Exploration of the structure-activity relationships of 4 led to the identification of a potent and orally bioavailable TRPM8 antagonist, tetrahydroisoquinoline 87. Compound 87 demonstrated target coverage in vivo after oral administration in a rat pharmacodynamic model measuring the prevention of icilin-induced wet-dog shakes (WDS).     

Icilin induces a hyperthermia in rats that is dependent on nitric oxide production and NMDA receptor activation

Icilin (AG-3-5) is a cold-inducing agent that activates the transient receptor potential channels TRPM8 and TRPA1. Both channels are members of the transient receptor potential (TRP) superfamily of ion channels and are activated by cold. Despite the key role of cold-activated TRPM8 and TRPA1 channels in temperature sensation and other physiological processes, the significance of these channels in thermoregulation in conscious animals is poorly understood. Therefore, in the present study we investigated the effects of icilin on body temperature in rats and tested the hypothesis that cold-activated TRP channel activation by icilin causes a hyperthermia which requires nitric oxide (NO) production and NMDA receptor stimulation. Our experiments revealed that icilin (2.5, 5, 7.5 and 10 mg/kg, i.m.) elicits a dose-related hyperthermia that is rapid in onset and of long duration. Pretreating rats with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) (10, 25 and 50 mg/kg, i.p.), a non-selective NO synthase inhibitor, attenuated the hyperthermia associated with icilin (7.5 mg/kg, i.m.). Pretreatment with (-)-6-[phosphonomethyl-1,2,3,4,4a,5,6,7,8,8a-decahydro-isoquinoline-2-carboxylate] (LY 235959) (0.25, 0.5 and 1 mg/kg, i.p.), a selective NMDA receptor antagonist, also attenuated the icilin-evoked hyperthermia. The administration of icilin (5 and 100 microg) into the lateral cerebroventricle of rats did not affect body temperature, thus indicating a peripheral site of action. These results indicate that icilin, a TRPM8/TRPA1 agonist, produces a dose-related hyperthermia in rats which requires both NO production and NMDA receptor activation.     

NMDA receptor dependence of mGlu-mediated depression of synaptic transmission in the CA1 region of the rat hippocampus.

1. The depression of synaptic transmission by the specific metabotropic glutamate receptor (mGlu) agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD) was investigated in area CA1 of the hippocampus of 4-10 week old rats, by use of grease-gap and intracellular recording techniques. 2. In the presence of 1 mM Mg2+, (1S,3R)-ACPD was a weak synaptic depressant. In contrast, in the absence of added Mg2+, (1S,3R)-ACPD was much more effective in depressing both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components of synaptic transmission. At 100 microM, (1S,3R)-ACPD depressed the slope of the field excitatory postsynaptic potential (e.p.s.p.) by 96 +/- 1% (mean +/- s.e.mean; n = 7) compared with 23 +/- 4% in 1 mM Mg(2+)-containing medium (n = 17). 3. The depressant action of 100 microM (1S,3R)-ACPD in Mg(2+)-free medium was reduced from 96 +/- 1 to 46 +/- 6% (n = 7) by the specific NMDA receptor antagonist (R)-2-amino-5-phosphonopentanoate (AP5; 100 microM). 4. Blocking both components of GABA receptor-mediated synaptic transmission with picrotoxin (50 microM) and CGP 55845A (1 microM) in the presence of 1 mM Mg2+ also enhanced the depressant action of (1S,3R)-ACPD (100 microM) from 29 +/- 5 to 67 +/- 6% (n = 6). 5. The actions of (1S,3R)-ACPD, recorded in Mg(2+)-free medium, were antagonized by the mGlu antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG). Thus, depressions induced by 30 microM (1S,3R)-ACPD were reversed from 48 +/- 4 to 8 +/- 6% (n = 4) by 1 mM (+)-MCPG. 6. In Mg(2+)-free medium, a group I mGlu agonist, (RS)-3, 5-dihydroxyphenylglycine (DHPG; 100 microM) depressed synaptic responses by 74 +/- 2% (n = 18). In contrast, neither the group II agonists ((2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine; L-CCG-1; 10 microM; n = 4) and ((2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine; DCG-IV; 100 nM; n = 3) nor the group III agonist ((S)-2-amino-4-phosphonobutanoic acid; L-AP4; 10 microM; n = 4) had any effect. 7. The depolarizing action of (1S,3R)-ACPD, recorded intracellularly, was similar in the presence and absence of Mg(2+)-AP5 did not affect the (1S,3R)-ACPD-induced depolarization in Mg(2+)-free medium. Thus, 50 microM (1S,3R)-ACPD induced depolarizations of 9 +/- 3 mV (n = 5), 10 +/- 2 mV (n = 4) and 8 +/- 2 mV (n = 5) in the three respective conditions. 8. On resetting the membrane potential in the presence of 50 microM (1S,3R)-ACPD to its initial level, the e.p.s.p. amplitude was enhanced by 8 +/- 3% in 1 mM Mg2+ (n = 5) compared with a depression of 37 +/- 11% in the absence of Mg2+ (n = 4). Addition of AP5 prevented the (1S,3R)-ACPD-induced depression of the e.p.s.p. (depression of 4 +/- 5% (n = 5)). 9. It is concluded that activation by group 1 mGlu agonists results in a depression of excitatory synaptic transmission in an NMDA receptor-dependent manner.     

Cloning and functional characterization of the guinea pig vanilloid receptor 1

We have cloned a guinea pig Vanilloid receptor 1 (VR1) from a dorsal root ganglion cDNA library and expressed it in CHO cells. The receptor has been functionally characterized by measuring changes in intracellular calcium produced by capsaicin, low pH and noxious heat.Capsaicin produced a concentration-dependent increase in intracellular calcium in guinea pig VR1-CHO cells with an estimated EC(50) of 0.17 +/- 0.0065 micro M, similar to that previously reported for rat and human VR1. Olvanil and resiniferatoxin were also effective agonists (EC(50) values of 0.0087 +/- 0.0035 micro M and 0.067 +/- 0.014 micro M, respectively), but 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) and anandamide showed little agonist activity up to 10 micro M. As with human and rat VR1, guinea pig VR1 was also activated by pH below 6.0 and by noxious heat (>42 degrees C). Capsazepine acted as an antagonist of capsaicin responses in guinea pig VR1-CHO cells (IC(50) of 0.324 +/- 0.041 micro M ), as seen at rat VR1. However, in contrast to its lack of activity against pH and heat responses at rat VR1, capsazepine was an effective antagonist of these responses at guinea pig VR1. Capsazepine displayed an IC(50) of 0.355 +/- 25 micro M against pH 5.5, and provided complete blockade of heat responses at 1 micro M. Thus, capsazepine can significantly inhibit calcium influx due to heat and pH 5.5 at guinea pig VR1 and human VR1 but is inactive against these activators at rat VR1.     

Mu and kappa opioid receptor agonists antagonize icilin-induced wet-dog shaking in rats

Icilin is a cooling agent that precipitates vigorous wet-dog shakes in rats after acute i.p. administration. Recent research has emphasized the peripheral agonist properties (e.g. activation of transient receptor potential channels, TRPM8 and TRPA1) of icilin rather than its unusual and pronounced behavioral effects, often classified as quasi-morphine withdrawal. We tested selective opioid receptor agonists against icilin-induced wet-dog shakes in rats. Shaking was antagonized following s.c. pretreatment with the mu agonists, morphine (1, 2, 3 mg/kg) and buprenorphine (0.10 mg/kg) or the kappa agonists, nalfurafine (0.02, 0.04 mg/kg) and U50,488H (5 mg/kg). Pretreatment with ICI 204,448 (1, 5, 10 mg/kg), the peripherally directed kappa agonist, or the delta agonist, SNC 80 (0.30, 1, 3, 10 mg/kg), had no marked effect on the incidence of shaking. We conclude that (a) icilin can trigger shaking via interactions within the central nervous system and (b) mu and kappa opioid receptors are involved in suppressing this stimulant behavior.     

Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids

Effects of common monoterpenoid alcohols and ketones were investigated on recombinant human gamma-aminobutyric acid A (GABAA; alpha1beta2gamma2s) and glycine (alpha1 homomers) receptors expressed in Xenopus oocytes. GABA currents were enhanced by coapplications of 10-300 microM: (+)-menthol>(-)-menthol>(-)-borneol>(-)-menthone=camphor enantiomers>carvone enantiomers, with menthol acting stereoselectively. By contrast, thujone diastereomers inhibited GABAA receptor currents while glycine currents were only markedly potentiated by menthol. Positive modulation by (+)-menthol was explored given its pronounced effects (e.g., at 100 microM, GABA and glycine EC20 responses increased by 496+/-113% and 135+/-56%, respectively). (+)-Menthol, 100 microM, reduced EC50 values for GABA and glycine from 82.8+/-9.9 to 25.0+/-1.8 microM, and from 98.7+/-8.6 to 75.7+/-9.4 microM respectively, with negligible effects on maximal currents. This study reveals a novel neuroactive role for menthol as a stereoselective modulator of inhibitory ligand-gated channels.     

Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells.

1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 microM) and WAY 100635 (5-HT1A; 1 microM). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pKb = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pKb = 10.44 +/- 0.06). 5. Pretreatment with PTX (200 ng ml-1 for 4 h) completely attenuated the Ca2+ response to 100 microM 5-HT. 6. In untransfected CHO-K1 cells, 5-HT (1 microM), RU 24969 (1 microM), and 5-CT (1 microM) elicited increases in [Ca2+]i similar to those observed in CHO-A1 cells. 7. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s).     

Structural and in Vitro Functional Characterization of a Menthyl TRPM8 Antagonist Indicates Species-Dependent Regulation

TRPM8 antagonists derived from its cognate ligand, (−)-menthol, are underrepresented. We determine the absolute stereochemistry of a well-known TRPM8 antagonist, (−)-menthyl 1, using VCD and 2D NMR. We explore 1 for its antagonist effects of the human TRPM8 (hTRPM8) orthologue to uncover species-dependent inhibition versus rat channels. (−)-Menthyl 1 inhibits menthol- and icilin-evoked Ca²⁺ responses at hTRPM8 with IC₅₀ values of 805 ± 200 nM and 1.8 ± 0.6 μM, respectively, while more potently inhibiting agonist responses at the rat orthologue (rTRPM8 IC₅₀ (menthol) = 117 ± 18 nM, IC₅₀ (icilin) = 521 ± 20 nM). Whole-cell patch-clamp recordings of hTRPM8 confirm the 1 inhibition of menthol-stimulated currents, with an IC₅₀ of 700 ± 200 nM. We demonstrate that 1 possesses ≥400-fold selectivity for hTRPM8 versus hTRPA1/hTRPV1. (−)-menthyl 1 can be used as a novel chemical tool to study hTRPM8 pharmacology and differences in species commonly used in drug discovery.     

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.