TRPM8 Activation Research Articles

Protective effects of menthol against olanzapine-induced metabolic alterations in female mice

AimMetabolic comorbidities such as obesity type 2 diabetes, insulin resistance, glucose intolerance, dyslipidemia are the major contributors for lower life expectancy and reduced patient compliance during antipsychotic therapy in patients with severe mental illnesses such as schizophrenia, bipolar disorder, and depression. TRPM8 activation by menthol is also reported to alleviate high fat diet-induced obesity in mice. Additionally, this TRPM8 activation leads to increase in gene expression of thermogenic genes in white adipocytes and dietary menthol was found to increase browning of WAT along with improved glucose utilization. Therefore, we aimed to evaluate the plausible role of TRPM8 channels in olanzapine-induced metabolic alterations in female balb/c mice. Methods6 weeks olanzapine (6 mg kg−1, per oral) model was used in female balb/c mice. Pharmacological manipulation of TRPM8 channel was done using menthol and N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB), the agonist and antagonist respectively. Key resultsMenthol co-treatment for six weeks prevented olanzapine-induced metabolic alterations such as weight gain, increased food intake, decreased energy expenditure, adiposity, liver lipid accumulation, systemic inflammation and insulin resistance. Although no significant change in TRPM8 mRNA expression was found in the hypothalamus, however, some of the protective effects of menthol were absent in presence of AMTB indicating possible involvement of TRPM8 channels. ConclusionOur results suggest possible therapeutic implications of menthol in the management of antipsychotic-induced weight gain and other metabolic alterations.

Conservation of the cooling agent binding pocket within the TRPM subfamily.

Transient Receptor Potential (TRP) channels are a large and diverse family of tetrameric cation selective channels that are activated by many different types of stimuli, including noxious heat or cold, organic ligands such as vanilloids or cooling agents, or intracellular Ca 2+ . Structures available for all subtypes of TRP channels reveal that the transmembrane domains are closely related despite their unique sensitivity to activating stimuli. Here we use computational and electrophysiological approaches to explore the conservation of the cooling agent binding pocket identified within the S1-S4 domain of the Melastatin subfamily member TRPM8, the mammalian sensor of noxious cold, with other TRPM channel subtypes. We find that a subset of TRPM channels, including TRPM2, TRPM4 and TRPM5, contain pockets very similar to the cooling agent binding pocket in TRPM8. We then show how the cooling agent icilin modulates activation of TRPM4 to intracellular Ca 2+ , enhancing the sensitivity of the channel to Ca 2+ and diminishing outward-rectification to promote opening at negative voltages. Mutations known to promote or diminish activation of TRPM8 by cooling agents similarly alter activation of TRPM4 by icilin, suggesting that icilin binds to the cooling agent binding pocket to promote opening of the channel. These findings demonstrate that TRPM4 and TRPM8 channels share related ligand binding pockets that are allosterically coupled to opening of the pore.

Neuregulin 1 mitigated prolactin deficiency through enhancing TRPM8 signaling under the influence of melatonin in senescent pituitary lactotrophs

The age-related alterations in pituitary function, including changes in prolactin (PRL) production contributes to the systemic susceptibility to age-related diseases. Our previous research has shown the involvement of Nrg1 in regulating the expression and secretion of PRL. However, the precise role of Nrg1 in mitigating the senescence of pituitary lactotrophs and the underlying mechanisms are yet to be comprehended. Here, data from the GEPIA database was used to evaluate the association between transient receptor potential cation channel subfamily M member 8 (TRPM8) and PRL in normal human pituitary tissues, followed by immunofluorescence verification using a human pituitary tissue microarray. TRPM8 levels showed a significant positive association with PRL expression in normal human pituitary tissues, and both TRPM8 and PRL levels declined during aging, suggesting that TRPM8 may regulate pituitary aging by affecting PRL production. It was also found that treatment with exogenous neuregulin 1 (Nrg1) markedly delayed the senescence of GH3 cells (rat lactotroph cell line) generated by D-galactose (D-gal). In addition, melatonin reduced the levels of senescence-related markers in senescent pituitary cells by promoting Nrg1 / ErbB4 signaling, stimulating PRL expression and secretion. Further investigation showed that Nrg1 attenuated senescence in pituitary cells by increasing TRPM8 expression. Downregulation of TRPM8 activation eliminated Nrg1-mediated amelioration of pituitary cell senescence. These findings demonstrate the critical function of Nrg1 / ErbB signaling in delaying pituitary lactotroph cell senescence and enhancing PRL production via promoting TRPM8 expression under the modulation of melatonin.

#788 Mitigation of tubular apoptosis and cystogenesis in ADPKD: the impact of TRPM8 through restriction of mitochondrial function

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD), characterized by numerous fluid-filled cysts and a progressive decline in renal function, is a prevalent hereditary kidney disorder. Approximately 70% of individuals affected by ADPKD may develop end-stage renal disease, typically manifesting around the median age of 58 years. Despite its prevalence, effective clinical treatments for ADPKD remain elusive. Transient receptor potential melastatin member 8 (TRPM8), serving as a sensor for cold temperatures and activated by the cooling agent menthol, orchestrates cellular Ca2+ signaling by facilitating Ca2+ entry into the cytosol. However, the role of TRPM8 in ADPKD remains undefined. Method The WT 9-12, an epithelial cell line isolated from the proximal tubule of a patient with ADPKD, was utilized for in vitro experiments. Eight-week-old wild-type (WT), Pkd1 knockdown (pkd1kd), and Trpm8, Pkd1 double mutant (T-/-P) mice were used for experiments. Renal Trpm8 was activated by administration of a 0.5% menthol diet for 7 months in mice. The cyst index was measured by H&E stain and calculated using Image J. Apoptosis was evaluated by TUNEL assay (In Situ Cell Death Detection Kit, TMR red). Protein and mRNA expression of mice kidneys was detected by Western blotting and qRT-PCR, respectively. Blood urea nitrogen (BUN) was measured by HITACHI 7600 analyzer. Results The activation of TRPM8 by menthol treatment resulted in an elevation of intracellular Ca2+ levels, subsequently impeding proliferation in WT9-12 cells. Consistently, Trpm8 activation suppressed kidney weight, kidney weight-to-body weight ratio, BUN, fibrosis, and cystogenesis in Pkd1kd mice, while no such effects were observed in WT and T-/-P mice. This suggests a protective role of Trpm8 in ADPKD. Mechanistically, Trpm8 activation mitigated tubular apoptosis in the kidneys of Pkd1kd mice, as evidenced by reduced TUNEL-positive cells and downregulated Bax and cleaved caspase-3, with no such effects observed in WT and T-/-P mice. Intriguingly, the proliferation-associated signaling pathway, ERK, was also suppressed by Trpm8. Proteomic analysis of mice kidneys revealed that Trpm8 regulates mitochondrial dysfunction, EIF2 and Sirtuin signaling pathways. The result was verified in WT9-12 cells showing reduced mitochondrial respiration, particularly impacting oxidative phosphorylation by TRPM8. Conclusion Trpm8 inhibits tubular apoptosis and cyst formation through the restriction of mitochondrial function, subsequently retarding ADPKD development in mice. These findings illuminate a novel and promising strategy for intervening in ADPKD.

Chief Complaint: Nasal Congestion

Nasal obstruction is the subjective perception and objective state of insufficient airflow through the nose. Nasal congestion, on the other hand, describes a state of not just inadequate airflow or obstructive phenomena but also pressure and mucus related states to the patient. Nasal receptors belonging to the transient receptor potential (TRP) protein family mediate the sense of nasal patency via the trigeminal nerve. TRPM8 responds to temperatures around 8–22 °C, and is stimulated by menthol and other cooling agents. The radiant effects of airflow create heat loss to activate these receptors and humans perceive this as nasal patency rather than the direct detection of airflow. The thermovascular state of the mucosa, in conditions such as rhinitis, influence TRPM8 activation. Nasal endoscopy can show signs of rhinitis and should be considered an essential part of the workup of nasal congestion. Efforts to relieve nasal congestion need to manage the mucosal state and surgery needs to ensures that the nasal cavity mucosa is exposed to the cooling effects of airflow rather than simply creating a passage to the nasopharynx.

Thermosensitive TRP Channels Are Functionally Expressed and Influence the Lipogenesis in Human Meibomian Gland Cells.

While the involvement of thermosensitive transient receptor potential channels (TRPs) in dry eye disease (DED) has been known for years, their expression in the meibomian gland (MG) has never been investigated. This study aims to show their expression and involvement in the lipogenesis of the MG, providing a possible new drug target in the treatment of DED. Our RT-PCR, Western blot and immunofluorescence analysis showed the expression of TRPV1, TRPV3, TRPV4 and TRPM8 in the MG at the gene and the protein level. RT-PCR also showed gene expression of TRPV2 but not TRPA1. Calcium imaging and planar patch-clamping performed on an immortalized human meibomian gland epithelial cell line (hMGECs) demonstrated increasing whole-cell currents after the application of capsaicin (TRPV1) or icilin (TRPM8). Decreasing whole-cell currents could be registered after the application of AMG9810 (TRPV1) or AMTB (TRPM8). Oil red O staining on hMGECs showed an increase in lipid expression after TRPV1 activation and a decrease after TRPM8 activation. We conclude that thermo-TRPs are expressed at the gene and the protein level in MGs. Moreover, TRPV1 and TRPM8's functional expression and their contribution to their lipid expression could be demonstrated. Therefore, TRPs are potential drug targets and their clinical relevance in the therapy of meibomian gland dysfunction requires further investigation.

Borneol exerts its antipruritic effects by inhibiting TRPA1 and activating TRPM8

Ethnopharmacological relevanceBorneol is a long-established traditional Chinese medicine that has been found to be effective in treating pain and itchy skin. However, whether borneol has a therapeutic effect on chronic itch and its related mechanisms remain unclear. Aim of the studyTo investigate the antipruritic effect of borneol and its molecular mechanism. Materials and methodsDrugBAN framework and molecular docking were applied to predict the targets of borneol, and the calcium imaging or patch-clamp recording analysis were used to detect the effects of borneol on TRPA1, TRPM8 or TRPV3 channels in HEK293T cells. In addition, various mouse models of acute itch and chronic itch were established to evaluate the antipruritic effects of borneol on C57BL/6J mice. Then, the borneol-induced pruritic relief was further investigated in Trpa1−/−, Trpm8−/−, or Trpa1−/−/Trpm8−/− mice. The effects of borneol on the activation of TRPM8 and the inhibition of TRPA1 were also measured in dorsal root ganglia neurons of wild-type (WT), Trpm8−/− and Trpv1−/− mice. Lastly, a randomized, double-blind study of adult patients was conducted to evaluate the clinical antipruritic effect of borneol. ResultsTRPA1, TRPV3 and TRPM8 are the potential targets of borneol according to the results of DrugBAN algorithm and molecular docking. Calcium imaging and patch-clamp recording analysis demonstrated that borneol activates TRPM8 channel-induced cell excitability and inhibits TRPA1 channel-mediated cell excitability in transfected HEK293T cells. Animal behavior analysis showed that borneol can significantly reduce acute and chronic itch behavior in C57BL/6J mice, but this effect was eliminated in Trpa1−/−, Trpm8−/− mice, or at least in Trpa1−/−/Trpm8−/− mice. Borneol elicits TRPM8 channel induced [Ca2+]i responses but inhibits AITC or SADBE-induced activation of TRPA1 channels in dorsal root ganglia neurons of WT and Trpv1−/− mice, respectively. Furthermore, the clinical results indicated that borneol could reduce itching symptoms in patients and its efficacy is similar to that of menthol. ConclusionBorneol has therapeutic effects on multiple pruritus models in mice and patients with chronic itch, and the mechanism may be through inhibiting TRPA1 and activating TRPM8.

Cooling‐promoted myogenic differentiation of murine bone marrow mesenchymal stem cells through TRPM8 activation in vitro

AbstractTRPM8 agonist has been reported to promote osteogenic differentiation of mesenchymal stem cells (MSCs), therefore we evaluated whether cooling‐induced activation of TRPM8 promotes myogenic differentiation of MSCs. We used 5‐azacytidine as a myogenic differentiation inducer in murine bone marrow‐derived MSCs. Addition of menthol, a TRPM8 agonist, to the differentiation induction medium significantly, increased the percentage of MyoD‐positive cells, a specific marker of myogenic differentiation. We performed intracellular Ca2+ imaging experiments using fura‐2 to confirm TRPM8 activation by cooling stimulation. The results confirmed that intracellular Ca2+ concentration ([Ca2+]i) increases due to TRPM8 activation, and TRPM8 antagonist inhibits increase in [Ca2+]i at medium temperatures below 19°C. We also examined the effect of cooling exposure time on myogenic differentiation of MSCs using an external cooling stimulus set at 17°C. The results showed that 60 min of cooling had an acceleratory effect on differentiation (2.18 ± 0.27 times). We observed that the TRPM8 antagonist counteracted the differentiation‐promoting effect of the cooling. These results suggest that TRPM8 might modulate the multiple differentiation pathways of MSCs, and that cooling is an effective way of activating TRPM8, which regulates MSCs differentiation in vitro.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Modulation of TRPM8 alters the phagocytic activity of microglia and induces changes in sub-cellular organelle functions

In this work, we investigated the presence and function of TRPM8, a non-selective and cold-sensitive Ca2+-permeable ion channel in the primary microglia cell as well as in microglia cell line BV2. We demonstrate that primary microglia as well as BV2 express TRPM8 endogenously. Both pharmacological activation or inhibition of TRPM8 causes enhanced uptake of bacterial particles at early time points of infection. In BV2, TRPM8 activation and/or LPS-signaling alters its surface expression and cytosolic ROS production. TRPM8 modulation in the absence and presence of LPS causes differential regulation of cytosolic pH and lysosomal pH. Notably, TRPM8 modulation also alters the correlation between lysosomal pH and cytosolic pH depending on TRPM8 modulation and the presence or absence of LPS. Collectively our data suggest that TRPM8 is involved in the regulation of subcellular organelle, i.e. mitochondrial and lysosomal functions. Data also suggest that primarily TRPM8 activation, but often deviation from endogenous TRPM8 function is linked with better innate immune function mediated by microglial cells. We suggest that TRPM8-mediated regulations of sub-cellular organelle functions are more context-dependent manner. Such understanding is relevant in the context of microglial cell functions and innate immunity.

Abstract P229: Beyond Cooling Sensation: Topical Penile Administration Of The Trpm8 Agonist Improves Hypertension Associated-erectile Dysfunction

Background and Purpose: Erectile dysfunction (ED) is an indicator for futurecardiovascular events. The search for new targets is crucial due to the low efficacy ofPDE5 inhibitors in patients with endothelial dysfunction. Menthol, a TRPM8 agonist, iswidely used in preparations to improve sensation during sexual intercourse, although nodata are reported for the improvement of ED. So, we hypothesize that chronic topicalactivation of TRPM8 improves hypertension associated-ED. Experimental Approach: Adult (9-12 weeks) male spontaneously hypertensive rats (SHR) and Wistar rats wereused for protein expression analysis and TRPM8 agonist curves in isolated CC. We alsotreated rats topically on the penis for 30 days with carbopol 940 (hydrogel, vehicle) ormenthol (carbopol 940 + menthol 0.1%). After treatment, we measured the mean bloodpressure (MBP), CC and iliac (IL) artery contractility. Key Results: Both strains of rathad similar TRPM8 expression in CC by western blotting and immunofluorescence.TRPM8 activation by cooling compounds, icilin (10 -8 –10 -4 M) or menthol (10 -7 –10 -3 M),relaxed CC strips pre-contracted by phenylephrine (PE, 10 -6 or 10 -5 M), but with reducedeffect in SHR. Interestingly, the topical penile treatment with menthol decreased the MBPof the SHR (151.8±6.8 mmHg, n=6) compared to the SHR vehicle group (174.6±2.6mmHg, n=5; p<0.05). In addition, in CC, the concentration-response curves to PE andthromboxane agonists were shifted to the left after menthol treatment. Interestingly, nochange in acetylcholine and sodium nitroprusside (SNP) relaxation were observed aftertopical penile treatment with menthol. The concentration-response curves to PE and SNPwere shifted to the left in IL arterial rings. This suggests topical menthol administrationinduced systemic vascular influence leading to a response like the normotensive Wistar. Conclusion: TRPM8 channels are expressed and functional in the CC, with decreasedsensitivity in SHR. Also, our data suggest that topical administration of menthol improvesthe reactivity of vascular tissue, demonstrating the potential of TRPM8 activation fortherapeutic benefits in hypertension associated-ED.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging.TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found.In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

Molecular determinants of TRPM8 function: key clues for a cool modulation.

Cold thermoreceptor neurons detect temperature drops with highly sensitive molecular machinery concentrated in their peripheral free nerve endings. The main molecular entity responsible for cold transduction in these neurons is the thermo-TRP channel TRPM8. Cold, cooling compounds such as menthol, voltage, and osmolality rises activate this polymodal ion channel. Dysregulation of TRPM8 activity underlies several physiopathological conditions, including painful cold hypersensitivity in response to axonal damage, migraine, dry-eye disease, overactive bladder, and several forms of cancer. Although TRPM8 could be an attractive target for treating these highly prevalent diseases, there is still a need for potent and specific modulators potentially suitable for future clinical trials. This goal requires a complete understanding of the molecular determinants underlying TRPM8 activation by chemical and physical agonists, inhibition by antagonists, and the modulatory mechanisms behind its function to guide future and more successful treatment strategies. This review recapitulates information obtained from different mutagenesis approaches that have allowed the identification of specific amino acids in the cavity comprised of the S1-S4 and TRP domains that determine modulation by chemical ligands. In addition, we summarize different studies revealing specific regions within the N- and C-terminus and the transmembrane domain that contribute to cold-dependent TRPM8 gating. We also highlight the latest milestone in the field: cryo-electron microscopy structures of TRPM8, which have provided a better comprehension of the 21years of extensive research in this ion channel, shedding light on the molecular bases underlying its modulation, and promoting the future rational design of novel drugs to selectively regulate abnormal TRPM8 activity under pathophysiological conditions.

Topical borneol relieves nonhistaminergic pruritus via targeting TRPA1 and TRPM8 channels in peripheral nerve terminals of mice

Borneol has been used successfully for the treatment of itchy skin in traditional Chinese medicine. However, the antipruritic effect of borneol has rarely been studied, and the mechanism is unclear. Here, we showed that topical application of borneol on skin substantially suppressed pruritogen chloroquine- and compound 48/80-induced itching in mice. The potential targets of borneol, including transient receptor potential cation channel subfamily V member 3 (TRPV3), transient receptor potential cation channel subfamily A member 1 (TRPA1), transient receptor potential cation channel subfamily M member 8 (TRPM8), and gamma-aminobutyric acid type A (GABAA) receptor were pharmacologically inhibited or genetically knocked out one by one in mouse. Itching behavior studies demonstrated that the antipruritic effect of borneol is largely independent of TRPV3 and GABAA receptor, and TRPA1 and TRPM8 channels are responsible for a major portion of the effect of borneol on chloroquine-induced nonhistaminergic itching. Borneol activates TRPM8 and inhibits TRPA1 in sensory neurons of mice. Topical co-application of TRPA1 antagonist and TRPM8 agonist mimicked the effect of borneol on chloroquine-induced itching. Intrathecal injection of a group II metabotropic glutamate receptor antagonist partially attenuated the effect of borneol and completely abolished the effect of TRPM8 agonist on chloroquine-induced itching, suggesting that a spinal glutamatergic mechanism is involved. In contrast, the effect of borneol on compound 48/80-induced histaminergic itching occurs through TRPA1-and TRPM8-independent mechanisms. Our work demonstrates that borneol is an effective topical itch reliever, and TRPA1 inhibition and TRPM8 activation in peripheral nerve terminals account for its antipruritic effect.

(224) Icilin, a TRPM8 Agonist, Induces Relaxation and Reduces Sensitivity to Phenylephrine in Pudendal Artery from Control and Diabetic Mice

Abstract Introduction Diabetes mellitus (DM) is a chronic metabolic disease with epidemiological relevance worldwide. One of the most common and most underestimated complications of diabetes is erectile dysfunction (ED). It is estimated that approximately 35-90% of diabetic men present ED as an early complication. Previous studies have shown that TRPM8 activation triggers relaxation of pudendal artery with increased sensitivity in the hypertensive rats. However, despite the correlation between ED and diabetes, research on the role of TRPM8 channels in the vasculature responsible for sexual function in diabetic condition still needs to be elucidated. Objective In this way, the aim of this study was to investigate TRPM8 channels in arteries vital to erection of the penis. We hypothesized that TRPM8 activation would induce relaxation and reduce sensitivity to alpha-adrenergic stimulation in pudendal arteries from diabetic mice. Methods Male control and db/db mice were euthanized, and the internal pudendal arteries were mounted on DMT wire myographs for the measurement of isometric force. Results Cumulative administration of icilin (10-8 to 10-4M), a TRPM8 agonist, induced endothelium-dependent and endothelium-independent relaxation in pre-contracted rings with phenylephrine (Phe,1μM). Relaxant effect induced by icilin was similar between pudendal arteries from db/db (Emax: 56.31 +/− 1.90%, n=5) and control (Emax:46.57 +/− 4.45%, n=5) mice. Diabetes increased the contractility of the pudendal artery in response to phenylephrine in concentration of 3x10-6M (99.40 +/− 4.65%, n=4) compared to control (85.44 +/− 2.36%, n=4). Interestingly, pre-incubation with icilin (10-4M) decreased the potency of the contraction induced by phenylephrine (10-9 to 3x10-5M) compared to untreated rings in both diabetic (pD2: 6.39 +/− 0.07 vs 6.67 +/− 0.05, n=4) and control (pD2: 6.43 +/− 0.09 vs 6.82 +/− 0.05, n=4) animals. In the pudendal artery, diabetes decreased the sensitivity to acetylcholine compared to control (pD2: 7.17 +/− 0.06 vs 7.42 +/− 0.03, n=4). However, the relaxation induced by acetylcholine (10-9 to 3x10-5M) was not changed by pre-incubation with icilin. Conclusions Icilin is able to promote relaxation with the same magnitude in pudendal arteries from diabetic and control mice. Increased responses to phenylephrine in pudendal artery may contribute to the development of ED. In addition, pre-incubation with icilin reduced sensitivity to phenylephrine, suggesting that the icilin could be a helpful treatment for ED. Disclosure No

TRPM8 channel activation induces relaxation in pudendal artery and corpus cavernosum: Can TRPM8 channel be a target to improve diabetic erectile dysfunction?

Erectile dysfunction (ED) is one of the most common and underestimated complications of diabetes. Previous studies have shown that TRPM8 activation triggers relaxation of internal pudendal arteries (IPA) with increased sensitivity in hypertensive rats. Despite the correlation between ED and diabetes, research on the role of TRPM8 channels in the IPA and corpus cavernosum (CC) in diabetes still needs to be elucidated. We hypothesized that preserved TRPM8 channel function in diabetic mice could be a therapeutic target for the treatment of ED. Male lean and db/db mice were euthanized, and the IPA were mounted on DMT wire myographs and CC were mounted on AVS organ bath for the measurement of isometric force. Concentration-response curves to icilin or menthol (TRPM8 agonists) were obtained. The CC TRPM8 channels expression was evaluated by western blot. Second harmonic generation was used to evaluate collagen deposition in IPA. Data are expressed as mean ± S.E.M of 3-5 mice. Non-linear regression curve was performed, and the maximum response (Emax) was analyzed using Student’s t test (p<0.05). Similar TRPM8 channel expression was observed in the CC from lean and db/db mice. Menthol-induced relaxation of the CC was reduced in diabetic mice (Emax: 105.00 ± 5.00%) compared to lean mice (Emax: 139.44 ± 15.91%) . Relaxant effect induced by icilin and menthol was similar between IPA from db/db (Emax icilin: 95.56 ± 0.86% and Emax menthol: 93.88 ± 2.33%) and lean (Emax icilin: 97.05 ± 0.87% and Emax menthol: 101.10 ± 1.75%) mice. Interestingly, pre-incubation with icilin (10-4M) for 30 minutes decreased the potency of phenylephrine-induced contraction compared to untreated IPA rings in both db/db and lean mice. In the IPA, diabetes decreased the sensitivity to acetylcholine compared to lean. However, the relaxation induced by acetylcholine was not changed by pre-incubation with icilin. Diabetic mice (db/db) IPA tended to have a decrease in arterial collagen content compared to lean mice. Although menthol-induced relaxation of the CC was reduced in diabetic mice, icilin and menthol were able to promote similar relaxation in IPA from diabetic and lean mice. In this way, TRPM8 function was preserved in IPA from diabetic mice. Icilin reduced sensitivity to phenylephrine in IPA from both mice. These data suggest that activation of TRPM8 could be a therapeutic target for the treatment of ED. SMSNA, NIH (RO1DK132948) and FAPESB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP-RyR-BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

High-throughput investigation of temperature- and ligand-activation of the TRPM8 channel.

The TRPM8 channel functions as the main cold sensor in sensory nerves, where decreases in temperature cause opening of the cation-conducting channel pore. Activation of TRPM8 in neurons is modulated by voltage, lipids, G-proteins, protein kinases, cytosolic calcium and natural compounds such as menthol. These mechanisms tune neural excitability and are likely important for how sensory stimuli are detected, integrated, and perceived as cold, warmth, pain, or analgesia. The molecular mechanisms of activation and regulation of the TRPM8 channel by chemical and physical stimuli are largely unknown, including the mechanism of cold-activation. Here we set out to systematically identify which residues in the transmembrane domain of the TRPM8 channel are determinant for its sensitivity to menthol or cold. We generated a library containing all possible single-residue substitutions and deletions in the transmembrane domain of the channel. This library was benchmarked using patch-clamp electrophysiology and we have developed a high-throughput fluorescence-based assay of ion channel activity. Finally, we will use Sort-seq methods to link genotypes to phenotypes for each of the variants in our libraries. Through this method, it will be possible to link individual residue changes to functional outcomes in TRPM8.