Transient Receptor Potential M8 Research Articles

Comprehensive metabolite and biological profile of “Sulmona Red Garlic” ecotype’s aerial bulbils

“Sulmona Red Garlic” is a well-known Italian traditional product. Bulbs, used for culinary purposes, have been largely investigated for their medicinal properties whereas aerial bulbils are usually removed as waste material. Here, for the first time, chemical composition and biological properties of the hydroalcoholic extract from aerial bulbils were investigated. Complementary information on metabolite composition were obtained using both NMR based untargeted and HPLC-DAD targeted methodologies. The NMR analysis revealed the presence of sugars, organic acids, amino acids, organosulphur compounds (methiin, alliin, allicin and cycloalliin), and other secondary metabolites. In particular, methiin and alliin were identified for the first time in the NMR spectra of aerial bulbil garlic extracts. Polyphenol content was determined by HPLC-DAD analysis: catechin, chlorogenic acid, and gallic acid turned out to be the most abundant phenolics.Hydroalcoholic extract blocked cell proliferation of colon cancer cell line HCT116 with an IC50 of 352.07 µg/mL, while it was non-toxic to myoblast cell line C2C12. In addition, it caused seedling germination reduction of two edible and herbaceous dicotyledon species, namely Cichorium intybus and C. endivia. Moreover, the same extract reduced the gene expression of TNF-α (tumor necrosis factor), HIF1-α (hypoxia-inducible factor), VEGFA (vascular endothelial growth factor), and transient receptor potential (TRP) M8 (TRPM8) indicating the ability to contrast cancer development through the angiogenic pathway. Final, in silico experiments were also carried out supporting the biological effects of organosulphur compounds, particularly alliin, which may directly interact with TRPM8.The results here reported suggest the potential use of garlic aerial bulbils often considered a waste product as a source in phytotherapeutic remedies.

A grape (Vitis vinifera L.) pomace water extract modulates inflammatory and immune response in SW-480 cells and isolated mouse colon.

Grape (Vitis vinifera L.) pomace is a residue derived from the winemaking process, which contains bioactive compounds displaying noteworthy health-promoting properties. The aim of the present study was to investigate the phenolic composition and protective effects of a water extract of grape pomace (WEGP) in colorectal cancer cell line SW480 and in isolated mouse colon exposed to Escherichia coli lipopolysaccharide (LPS). The extract decreased SW-480 cell viability, as well as vascular endothelial factor A (VEGFA), hypoxia-induced factor 1α (HIF1α), and transient receptor potential M8 (TRPM8) LPS-induced gene expression. Moreover, the extract inhibited mRNA levels of nuclear factor kB (NFkB), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)α, interleukin (IL)-6, IL-1β, IL-10, inducible nitric oxide synthase (iNOS), and interferon (IFN)γ, in isolated colon. Conversely, WEGP increased the gene expression of antioxidant catalase (CAT) and superoxide dismutase (SOD), in the same model. The modulatory effects exerted by WEGP could be related, at least in part, to the phenolic composition, with particular regards to the catechin level. Docking calculations also predicted the interactions of catechin toward TRPM8 receptor, deeply involved in colon cancer; thus further suggesting the grape pomace as a valuable source of bioactive extracts and phytochemicals with protective effects in the colon.

A Neuron-Glial Model of Exosomal Release in the Onset and Progression of Alzheimer's Disease.

Exosomes are nano-sized extracellular vesicles that perform a variety of biological functions linked to the pathogenesis of various neurodegenerative disorders. In Alzheimer's disease (AD), for examples, exosomes are responsible for the release of Aβ oligomers, and their extracellular accumulation, although the underpinning molecular machinery remains elusive. We propose a novel model for Alzheimer's Aβ accumulation based on Ca2+-dependent exosome release from astrocytes. Moreover, we exploit our model to assess how temperature dependence of exosome release could interact with Aβ neurotoxicity. We predict that voltage-gated Ca2+ channels (VGCCs) along with the transient-receptor potential M8 (TRPM8) channel are crucial molecular components in Alzheimer's progression.

Reduced cardiac ischemia/reperfusion injury by hypothermic reperfusion via activation of transient receptor potential M8 channel

AimsTo investigate the cardioprotective effects of hypothermic (25 °C) reperfusion on ischemia/reperfusion injury and the role of transient potential channel M8 (TRPM8) in this process. Main methodsWestern blot and real-time PCR were used to monitor the expression of TRPM8 in myocardium. Myocardial ischemia/reperfusion injury was induced by 30 min of global ischemia followed by 120 min of reperfusion in Langendorff-perfused hearts from Sprague-Dawley rats. The reperfusion was either normothermic (37 °C) or hypothermic (25 °C). Infarct size and left ventricular function were assessed, and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) in the coronary effluent were measured spectrophotometrically, and cardiomyocyte apoptosis was detected by TUNEL assay. The expression of TRPM8, Bcl-2, Bax, cleaved capspase-3, RhoA, and ROCK2 was quantified. Key findingsTRPM8 protein and mRNA were expressed in rat myocardium. Hypothermic reperfusion decreased the infarct size, LDH activity, MDA content, apoptosis, and expression of Bax, cleaved caspase-3, RhoA, and ROCK2 compared with normothermic reperfusion. These effects were associated with improved recovery of left ventricular contractility, and were reduced by BCTC, a TRPM8 antagonist. Ischemia/reperfusion injury and the increased expression of Bax, caspase-3, RhoA, and ROCK2 induced by normothermic reperfusion were reduced by Icilin, a TRPM8 agonist. SignificanceHypothermic reperfusion at 25 °C has cardioprotective effects against ischemia/reperfusion injury via activation of TRPM8 to inhibit the oxidative stress-related RhoA/ROCK2 signal pathway.

Signaling Pathways Relevant to Nerve Growth Factor-induced Upregulation of Transient Receptor Potential M8 Expression

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that primarily detects the innocuous cold. In pathological conditions, TRPM8 plays a role in the development of cold hyperalgesia/allodynia. Nerve growth factor (NGF) is an important mediator involved in various pain disorders. In the present study, the NGF-TrkA pathway increased TRPM8 expression by stabilizing TRPM8 mRNA through the actions of phosphatidylinositol 3-kinase and p38 MAP kinase. Moreover, c-Jun N-terminal kinase and Src tyrosine kinase were identified as a positive and negative regulator of TRPM8 expression, respectively, via post-transcriptional mechanisms independent of mRNA stabilization. PTEN activity was found to increase protein TRPM8 expression. Calcium imaging confirmed that NGF induced TRPM8 functional upregulation. Time-lapse fluorescence microscopic analysis and a cell fractionation assay revealed that NGF promoted the trafficking of TRPM8 to the plasma membrane. In the presence of NGF, lysosome-associated membrane protein-2 (LAMP-2) was localized to TRPM8-positive dot-like and linear structures, the latter of which were observed in the periphery of the cytoplasm. It was inferred that LAMP-2 was involved in the vesicular transport of TRPM8. Pharmacological blockade of the proteasome with MG132 led to a further increase in NGF-induced TRPM8 expression, indicating that the proteasome system played a pivotal role in the degradation of TRPM8. Our findings provide novel insight into the signaling pathways involved in NGF-mediated TRPM8 upregulation and its reversion to the normal state.

Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

BackgroundRecent genome-wide association studies have identified transient receptor potential M8 (TRPM8) as a migraine susceptibility gene. TRPM8 is a nonselective cation channel that mediates cool perception. However, its precise role in migraine pathophysiology is elusive. Transient receptor potential V1 (TRPV1) is a nonselective cation channel activated by noxious heat. Both TRPM8 and TRPV1 are expressed in trigeminal ganglion (TG) neurons.MethodsWe investigated the functional roles of TRPM8 and TRPV1 in a meningeal inflammation-based migraine model by measuring the effects of facial TRPM8 activation on thermal allodynia and assessing receptor coexpression changes in TG neurons. We performed retrograde tracer labeling to identify TG neurons innervating the face and dura.ResultsWe found that pharmacological TRPM8 activation reversed the meningeal inflammation-induced lowering of the facial heat pain threshold, an effect abolished by genetic ablation of TRPM8. No significant changes in the heat pain threshold were seen in sham-operated animals. Meningeal inflammation caused dynamic alterations in TRPM8/TRPV1 coexpression patterns in TG neurons, and colocalization was most pronounced when the ameliorating effect of TRPM8 activation on thermal allodynia was maximal. Our tracer assay disclosed the presence of dura-innervating TG neurons sending collaterals to the face. Approximately half of them were TRPV1-positive. We also demonstrated functional inhibition of TRPV1 by TRPM8 in a cell-based assay using c-Jun N-terminal kinase phosphorylation as a surrogate marker.ConclusionsOur findings provide a plausible mechanism to explain how facial TRPM8 activation can relieve migraine by suppressing TRPV1 activity. Facial TRPM8 appears to be a promising therapeutic target for migraine.

Oral treatment with essential oil of Hyptis spicigera Lam. (Lamiaceae) reduces acute pain and inflammation in mice: Potential interactions with transient receptor potential (TRP) ion channels

Ethnopharmacological relevanceThe genus Hyptis comprehends almost 400 species widespread in tropical and temperate regions of America. The use of Hyptis spicigera Lam. (Lamiaceae) is reported in traditional medicine due to its gastroprotective, anti-inflammatory and analgesic properties. Aim of the studyThe rationale of this study was to investigate the potential use of the essential oil of H. spicigera (EOHs) as analgesic. Material and methodsThe antinociceptive effect of EOHs was verified analyzing acute nocifensive behavior of mice induced by chemical noxious stimuli [i.e., formalin and transient receptor potential (TRP) channels agonists]. We also verified the effects of EOHs on locomotor activity and motor performance in mice. Finally, we investigate the involvement of central afferent C-fibers with EOHs analgesic effect. ResultsEOHs presented antinociceptive effect at 300 and 1000mg/kg on formalin-induced pain behavior model, presenting 50% and 72% of inhibition during the first phase (ED50 =292mg/kg), and 85% and 100% during de second phase (ED50 =205mg/kg), respectively. Temperature of the hind paw was reduced by EOHs treatment in a dose-dependent manner; oedema was diminished only by EOHs 1000mg/kg. EOHs does not impaired locomotor activity or motor performance. For mice injected with capsaicin, a TRPV1 activator, EOHs (1000mg/kg, ED50 =660mg/kg) showed decreased (63%) nociceptive behavior. When injected with cinnamaldehyde (TRPA1 activator), mice treated with EOHs showed 23%, 43% and 66% inhibition on nociceptive behavior (100, 300 and 1000mg/kg, respectively; ED50 402mg/kg). When mice were injected with menthol (TRPM8 activator), EOHs showed 29%, 59% and 98% inhibition of nociceptive behavior (100, 300 and 1000mg/kg, respectively; with ED50 =198mg/kg. Finally, when desensitized mice were injected with menthol, EOHs (300mg/kg) does not show antinociceptive effect. ConclusionsThis study demonstrated the efficacy of EOHs on experimental models of nociception. We have found the involvement of TRP channels V1, A1 and M8 with EOHs activity, which was remarkably potent and efficient in inhibiting pain evoked by menthol, a TRPM8 channel activator. TRPM8 channels from TRPV1+ C-fibers, but not TRPM8+ C-fibers nor TRPM8+ Aδ mechanosensory fibers, mediate EOHs analgesic effects.

TRPM8 and Migraine.

Migraine is among the most common diseases on earth and one of the most disabling, the latter due in large part to poor treatment efficacy. Development of new therapeutics is dependent on the identification of mechanisms contributing to migraine and discovery of targets for new drugs. Numerous genome-wide association studies (GWAS) have implicated the transient receptor-potential M8 (TRPM8) channel in migraine. This channel is predominantly expressed on peripheral sensory neurons and is known as the sensor for cold temperature in cutaneous tissue but is also expressed on deep visceral afferents where cold is not likely a stimulus. Consequently, a number of alternative endogenous agonists have been proposed. Apart from its role in cold sensation, TRPM8 also contributes to cold allodynia after nerve injury or inflammation, and it is necessary for cooling/menthol-based analgesia. How it might contribute to migraine is less clear. The purpose of this review is to discuss the anatomical and physiological mechanisms by which meningeal TRPM8 may play a role in migraine as well as the potential of TRPM8 as a therapeutic target. TRPM8 is expressed on sensory afferents innervating the meninges, and these neurons are subject to developmental changes that may influence their contribution to migraine. As in viscera, meningeal TRPM8 channels are unlikely to be activated by temperature fluctuations and their endogenous ligands remain unknown. Preclinical migraine studies show that activation of meningeal TRPM8 by exogenous agonists can both cause and alleviate headache behaviors, depending on whether other meningeal afferents concurrently receive noxious stimuli. This is reminiscent of the fact that cold can trigger migraine in humans but menthol can also alleviate headache. We propose that both TRPM8 agonists and antagonists may be potential therapeutics, depending on how migraine is triggered in individual patients. In this regard, TRPM8 may be a novel target for personalized medicine in migraine treatment.

Genetic variants affecting human TRPA1 or TRPM8 structure can be classified in vitro as 'well expressed', 'poorly expressed' or 'salvageable'.

Multiple mis-sense variants of TRPA1 (transient receptor potential A1) and TRPM8 (transient receptor potential M8) are recorded in the human genome single nt polymorphism (SNP) database, but their potential impact on channel signalling in patho-physiology is not fully explored. Variants, mostly quite rare in the general human population, alter sites in different structural domains of these homo-tetrameric ion channel proteins. The effects of individual SNPs affecting the large cytoplasmic N-terminal domain have not been completely documented for TRPM8 or TRPA1. We examined the Ca(2+) signalling properties of a short-list of eight variants affecting the N-terminal domain by individual expression in human embryonic kidney HEK293 or neuroblastoma (SH-SY5Y) cell lines (four SNP variants for TRPM8: G150R, K423N, R475C, R485W and four for TRPA1: Y69C, A366D, E477K, D573A). These were compared with TRPA1 SNP variants affecting intracellular loops located beyond the N-terminal domain and associated with gain of function (such as increased sensitivity to agonists: TRPA1 R797T and N855S). A substitution in TRPA1 (Y69C) exhibited high expression/sensitivity to agonists (high iCa(2+)max (maximum level of intracellular calcium ion), similar to R797T, but less sensitive than N855S), whereas each of the other non-conservative substitutions exhibited poor signalling response (low iCa(2+)max). Responses from these poorly expressed variants could be salvaged, to different extents, by pre-treating cells with the Src (Src protein) family inhibitor protein kinase inhibitor PP2 (PP2: 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), or with micromolar Zn(2+). The TRPA1 variants and several experimental mutants (TRPA1 Y97F, Y226F and YY654-655FF) expressed poorly in SH-SY5Y compared with HEK293 cells. More in-depth studies are needed to identify SNP variants eliciting gain of function in these TRP (transient receptor potential) channels and to assess their roles in medical conditions.

Low-level laser therapy alleviates mechanical and cold allodynia induced by oxaliplatin administration in rats.

Cold and mechanical allodynia caused by oxaliplatin-induced acute peripheral neuropathy frequently occur after drug infusion. Low-level laser therapy (LLLT) has been used to improve pain symptoms associated with various conditions and may have potential as a therapy for oxaliplatin-induced allodynia. The purpose of the present study was to investigate the antiallodynic effect of LLLT in an oxaliplatin-treated animal model by assessing sensory behavioral responses, levels of nerve growth factor (NGF), and transient receptor potential M8 (TRPM8) in dorsal root ganglia (DRG) neurons, as well as substance P (SP) in the spinal dorsal horn. Adult male Sprague-Dawley rats each received a total of four doses of oxaliplatin (4 mg/kg, i.p.), injected at 3-day intervals. Following oxaliplatin administration, LLLT (7.5 J/cm(2)) was applied for 12 consecutive days to the skin surface directly above sites where the sciatic nerve is distributed. Behavioral assessments were then performed, followed by immunoassays for NGF, TRPM8, and SP proteins. LLLT relieved both cold and mechanical allodynia induced by oxaliplatin in rats. Oxaliplatin-related increases in protein levels of NGF and TRPM8 in DRG and SP in the dorsal horn were also reduced after LLLT. The findings of this study support LLLT as a potential treatment for oxaliplatin-induced neuropathy. Moreover, our findings suggest that SP, TRPM8, and NGF proteins in the superficial dorsal horn and DRG may be involved in an antiallodynic effect for LLLT.

Essential Role of Transient Receptor Potential M8 (TRPM8) in a Model of Acute Cold-induced Urinary Urgency

BackgroundAcute exposure of part of the skin to cold stimuli can evoke urinary urgency, a phenomenon termed acute cold-induced urgency (ACIU). Despite its high prevalence, particularly in patients with overactive bladder, little is known about the mechanisms that induce ACIU. ObjectiveTo develop an animal model of ACIU and test the involvement of cold-activated ion channels transient receptor potential (TRP) M8 and TRPA1. Design, setting, and participantsIntravesical pressure and micturition were monitored in female mice (wild-type C57BL/6J, Trpa1−/−, Trpm8+/+, and Trpm8−/−) and Sprague Dawley rats. InterventionsAn intravesical catheter was implanted. Localized cooling of the skin was achieved using a stream of air or topical acetone. The TRPM8 antagonist (N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide (AMTB) or vehicle was injected intraperitoneally. Outcome measurements and statistical analysisFrequencies of bladder contractions and voids in response to sensory stimuli were compared using the Mann-Whitney or Kruskal-Wallis test. Results and limitationsBrief, innocuously cold stimuli applied to different parts of the skin evoked rapid bladder contractions and voids in anesthetized mice and rats. These responses were strongly attenuated in Trpm8−/− mice and in rats treated with AMTB. As rodent bladder physiology differs from that of humans, it is difficult to directly extrapolate our findings to human patients. ConclusionsOur findings indicate that ACIU is an evolutionarily conserved reflex rather than subconscious conditioning, and provide a useful in vivo model for further investigation of the underlying mechanisms. Pharmacological inhibition of TRPM8 may be useful for treating ACIU symptoms in patients. Patient summaryBrief cold stimuli applied to the skin can evoke a sudden desire to urinate, which can be highly bothersome in patients with overactive bladder. We developed an animal model to study this phenomenon, and found that it depends on a specific molecular cold sensor, transient receptor potential M8 (TRPM8). Pharmacological inhibition of TRPM8 may alleviate acute cold-induced urinary urgency in humans.

Menthol facilitates the intravenous self-administration of nicotine in rats.

Menthol is preferred by ~25% of smokers and is the most common flavoring additive in tobacco and electronic cigarettes. Although some clinical studies have suggested that menthol facilitates the initiation of smoking and enhances the dependence on nicotine, many controversies remain. Using licking as the operant behavior, we found that adolescent rats self-administering nicotine (30μg/kg/infusion, free base, i.v.) with contingent oral menthol (60μl, 0.01% w/v) obtained significantly more infusions than rats receiving a vehicle cue or rats self-administering i.v. saline with a menthol cue. Rats yoked to their menthol-nicotine masters emitted significantly fewer licks on the active spouts, indicating that contingent pairing between nicotine and menthol is required for sustained nicotine intake. Rats that self-administered nicotine with a menthol cue also exhibited a long-lasting extinction burst and robust reinstatement behavior, neither of which were observed in rats that self-administered saline with a menthol cue. The cooling sensation of menthol is induced by activating the transient receptor potential M8 (TRPM8) channel. When WS-23, an odorless agonist of the TRPM8 channel, was used as a contingent cue for nicotine, the rats obtained a similar number of nicotine infusions as the rats that were provided a menthol cue and exhibited a strong preference for the active spout. In contrast, highly appetitive taste and odor cues failed to support nicotine self-administration. These data indicated that menthol, likely by inducing a cooling sensation, becomes a potent conditioned reinforcer when it is contingently delivered with nicotine. Together, these results provide a key behavioral mechanism by which menthol promotes the use of tobacco products or electronic cigarettes.

Colon carcinogenesis is inhibited by the TRPM8 antagonist cannabigerol, a Cannabis-derived non-psychotropic cannabinoid.

Cannabigerol (CBG) is a safe non-psychotropic Cannabis-derived cannabinoid (CB), which interacts with specific targets involved in carcinogenesis. Specifically, CBG potently blocks transient receptor potential (TRP) M8 (TRPM8), activates TRPA1, TRPV1 and TRPV2 channels, blocks 5-hydroxytryptamine receptor 1A (5-HT1A) receptors and inhibits the reuptake of endocannabinoids. Here, we investigated whether CBG protects against colon tumourigenesis. Cell growth was evaluated in colorectal cancer (CRC) cells using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and 3-amino-7-dimethylamino-2-methylphenazine hydrochloride assays; apoptosis was examined by histology and by assessing caspase 3/7 activity; reactive oxygen species (ROS) production by a fluorescent probe; CB receptors, TRP and CCAAT/enhancer-binding protein homologous protein (CHOP) messenger RNA (mRNA) expression were quantified by reverse transcription-polymerase chain reaction; small hairpin RNA-vector silencing of TRPM8 was performed by electroporation. The in vivo antineoplastic effect of CBG was assessed using mouse models of colon cancer. CRC cells expressed TRPM8, CB1, CB2, 5-HT1A receptors, TRPA1, TRPV1 and TRPV2 mRNA. CBG promoted apoptosis, stimulated ROS production, upregulated CHOP mRNA and reduced cell growth in CRC cells. CBG effect on cell growth was independent from TRPA1, TRPV1 and TRPV2 channels activation, was further increased by a CB2 receptor antagonist, and mimicked by other TRPM8 channel blockers but not by a 5-HT1A antagonist. Furthermore, the effect of CBG on cell growth and on CHOP mRNA expression was reduced in TRPM8 silenced cells. In vivo, CBG inhibited the growth of xenograft tumours as well as chemically induced colon carcinogenesis. CBG hampers colon cancer progression in vivo and selectively inhibits the growth of CRC cells, an effect shared by other TRPM8 antagonists. CBG should be considered translationally in CRC prevention and cure.

Human TRPM8 and TRPA1 pain channels, including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

TRPM8 (transient receptor potential M8) and TRPA1 (transient receptor potential A1) are cold-temperature-sensitive nociceptors expressed in sensory neurons but their behaviour in neuronal cells is poorly understood. Therefore DNA expression constructs containing human TRPM8 or TRPA1 cDNAs were transfected into HEK (human embryonic kidney cells)-293 or SH-SY5Y neuroblastoma cells and G418 resistant clones analysed for effects of agonists and antagonists on intracellular Ca2+ levels. Approximately 51% of HEK-293 and 12% of SH-SY5Y cell clones expressed the transfected TRP channel. TRPM8 and TRPA1 assays were inhibited by probenecid, indicating the need to avoid this agent in TRP channel studies. A double-residue mutation in ICL-1 (intracellular loop-1) of TRPM8 (SV762,763EL, mimicking serine phosphorylation) or one in the C-terminal tail region (FK1045,1046AG, a lysine knockout) retained sensitivity to agonists (WS 12, menthol) and antagonist {AMTB [N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide]}. SNP (single nucleotide polymorphism) variants in TRPA1 ICL-1 (R797T, S804N) and TRPA1 fusion protein containing C-terminal (His)10 retained sensitivity to agonists (cinnamaldehyde, allyl-isothiocyanate, carvacrol, eugenol) and antagonists (HC-030031, A967079). One SNP variant, 797T, possessed increased sensitivity to agonists. TRPA1 became repressed in SH-SY5Y clones but was rapidly rescued by Src-family inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Conversely, TRPM8 in SH-SY5Y cells was inhibited by PP2. Further studies utilizing SH-SY5Y may identify structural features of TRPA1 and TRPM8 involved in conferring differential post-translational regulation.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

TRPM8 and Nav1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

BackgroundFamilial amyloidotic polyneuropathy (FAP) is a peripheral neuropathy caused by the extracellular accumulation and deposition of insoluble transthyretin (TTR) aggregates. However the molecular mechanism that underlies TTR toxicity in peripheral nerves is unclear. Previous studies have suggested that amyloidogenic proteins can aggregate into oligomers which disrupt intracellular calcium homeostasis by increasing the permeability of the plasma membrane to extracellular calcium. The aim of the present study was to examine the effect of TTR on calcium influx in dorsal root ganglion neurons.ResultsLevels of intracellular cytosolic calcium were monitored in dorsal root ganglion (DRG) neurons isolated from embryonic rats using the calcium-sensitive fluorescent indicator Fluo4. An amyloidogenic mutant form of TTR, L55P, induced calcium influx into the growth cones of DRG neurons, whereas wild-type TTR had no significant effect. Atomic force microscopy and dynamic light scattering studies confirmed that the L55P TTR contained oligomeric species of TTR. The effect of L55P TTR was decreased by blockers of voltage-gated calcium channels (VGCC), as well as by blockers of Nav1.8 voltage-gated sodium channels and transient receptor potential M8 (TRPM8) channels. siRNA knockdown of TRPM8 channels using three different TRPM8 siRNAs strongly inhibited calcium influx in DRG growth cones.ConclusionsThese data suggest that activation of TRPM8 channels triggers the activation of Nav1.8 channels which leads to calcium influx through VGCC. We suggest that TTR-induced calcium influx into DRG neurons may contribute to the pathophysiology of FAP. Furthermore, we speculate that similar mechanisms may mediate the toxic effects of other amyloidogenic proteins such as the β-amyloid protein of Alzheimer's disease.

AFM imaging reveals the tetrameric structure of the TRPM8 channel

Several members of the transient receptor potential (TRP) channel superfamily have been shown to assemble as tetramers. Here we have determined the subunit stoichiometry of the transient receptor potential M8 (TRPM8) channel using atomic force microscopy (AFM). TRPM8 channels were isolated from transfected cells, and complexes were formed between the channels and antibodies against a V5 epitope tag present on each subunit. The complexes were then subjected to AFM imaging. A frequency distribution of the molecular volumes of antibody decorated channels had a peak at 1305 nm 3, close to the expected size of a TRPM8 tetramer. The frequency distribution of angles between pairs of bound antibodies had two peaks, at 93° and 172°, confirming that the channel assembles as a tetramer. We suggest that this assembly pattern is common to all members of the TRP channel superfamily.

Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells

We investigated the effects of transient receptor potential M8 (TRPM8) channel on the proliferation and motility of androgen-independent prostate cancer PC-3 cells. After being permanently transfected with an empty vector and cDNA encoding the TRPM8 protein, cells were analysed for cell cycle distribution and motility using flow cytometry and scratch assay. Immunocytochemistry and Ca2+ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells. Cell cycle distribution and scratch assay analysis revealed that TRPM8 induced cell cycle arrest at the G0/G1 stage (P < 0.05) and facilitated the cell apoptosis induced by starvation (P < 0.05). Furthermore, TRPM8 inhibited the migration of PC-3-TRPM8 cells (P < 0.01) through the inactivation of focal-adhesion kinase. It appears that TRPM8 was not essential for the survival of PC-3 cells; however, the overexpression of TRPM8 had negative effects on the proliferation and migration of PC-3 cells. Thus, TRPM8 and its agonists may serve as important targets for the treatment of prostate cancer.

Plasticity in intact Aδ- and C-fibers contributes to cold hypersensitivity in neuropathic rats

Cold hypersensitivity is a common sensory abnormality accompanying peripheral neuropathies and is difficult to treat. Progress has been made in understanding peripheral mechanisms underlying neuropathic pain but little is known concerning peripheral mechanisms of cold hypersensitivity. The aim of this study was to analyze the contribution of uninjured primary afferents to the cold hypersensitivity that develops in neuropathic rats. Rats with a lumbar 5 (L5) and L6 spinal nerve ligation (SNL, Chung model) but not sham, developed mechanical allodynia, evidenced by decreased paw withdrawal thresholds and increased magnitude of response to von Frey stimulation. Cold hypersensitivity also developed in SNL but not sham rats, evidenced by enhanced nociceptive behaviors induced by placement on a cold plate (6 °C) or application of icilin (a transient receptor potential M8 (TRPM8)/transient receptor potential A1 (TRPA1) receptor agonist) to nerve-injured hind paws. Single fiber recordings demonstrated that the mean conduction velocities of intact L4 cutaneous Aδ- and C-fibers were not different between naive and SNL rats; however, mechanical thresholds of the Aδ- but not the C-fibers were significantly decreased in SNL compared with naive. There was a higher prevalence of C-mechanoheat-cold (CMHC) fibers in SNL compared with naive, but the overall percentage of cold-sensitive C-fibers was not significantly increased compared with naive. This was in contrast to the numerous changes in Aδ-fibers: the percentage of L4 cold sensitive Aδ-, but not C-fibers, was significantly increased, the percentage of L4 icilin-sensitive Aδ-, but not C-fibers, was significantly increased, the icilin-induced activity of L4 Aδ-, but not C-fibers, was significantly increased. Icilin-induced activity was blocked by the TRPA1 antagonist Ruthenium Red. The results indicate plasticity in both Aδ- and C-uninjured fibers, but Aδ fibers appear to provide a major contribution to cold hypersensitivity in neuropathic rats.

Ca 2+-dependent PKC activation mediates menthol-induced desensitization of transient receptor potential M8

In 1950, Hensel and Zotterman reported cooling-induced desensitization of cold receptors by extracellular discharge recordings of cold fibers. Since then, however, its intracellular mechanism has remained unresolved. We studied menthol-induced desensitization of cold/menthol receptors (TRPM8, transient receptor potential M8) expressed in HEK cells. TRPM8 desensitization depended on extracellular Ca 2+ ions, indicating that Ca 2+ influx-induced [Ca 2+] i elevation caused the desensitization. We studied whether Ca 2+-dependent kinase, PKC, mediated TRPM8 desensitization. PMA, a PKC activator, desensitized TRPM8. Inhibitor of Ca 2+-dependent PKC isozymes specifically abolished PMA-induced TRPM8 desensitization. PMA similarly desensitized wild type TRPM8 and mutant TRPM8, in which serine or threonine residues in some putative PKC phosphorylation sites were replaced by alanine. PMA treatment did not induce internalization of TRPM8. As the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated TRPM8 causes Ca 2+-dependent PKC isozymes to desensitize TRPM8 itself.