The TRPM8 channel as a potential therapeutic target for bladder hypersensitive disorders.

In the lower urinary tract, transient receptor potential (TRP) channels are primarily involved in physiological function, especially in cellular sensors responding to chemical and physical stimuli. Among TRP channels, TRP melastatin 8 (TRPM8) channels, responding to cold temperature and/or chemical agents, such as menthol or icilin, are mainly expressed in the nerve endings of the primary afferent neurons and in the cell bodies of dorsal root ganglia innervating the urinary bladder (via Aδ- and C-fibers); this suggests that TRPM8 channels primarily contribute to bladder sensory (afferent) function. Storage symptoms of overactive bladder, benign prostatic hyperplasia, and interstitial cystitis are commonly related to sensory function (bladder hypersensitivity); thus, TRPM8 channels may also contribute to the pathophysiology of bladder hypersensitivity. Indeed, it has been reported in a pharmacological investigation using rodents that TRPM8 channels contribute to the pathophysiological bladder afferent hypersensitivity of mechanosensitive C-fibers. Similar findings have also been reported in humans. Therefore, a TRPM8 antagonist would be a promising therapeutic target for bladder hypersensitive disorders, including urinary urgency or nociceptive pain. In this review article, the functional role of the TRPM8 channel in the lower urinary tract and the potential of its antagonist for the treatment of bladder disorders was described.

Role of TRPM2-CnA-Drp1 pathway in propofol-induced reduction of renal injury induced by hepatic ischemia-reperfusion in mice / 中华麻醉学杂志

Objective:To evaluate the role of transient receptor potential melastatin 2 (TRPM2)-calcineurin A (CnA)-dynamin-related protein 1 (Drp1) pathway in propofol-induced reduction of renal injury induced by hepatic ischemia-reperfusion (I/R) in mice.Methods:Twenty-four SPF male C57BL6 mice, aged 8 weeks, weighing 20-23 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (group S), hepatic I/R group (group IR), propofol group (group P) and TRPM2 agonist (ADPR) combined with propofol group (AP group). Hepatic I/R injury was induced by occluding the portal vein and hepatic artery supplying the left and middle lobes of the liver for 60 min followed by reperfusion in anesthetized rats.In group P, 0.2 ml normal saline was injected intraperitoneally at 1 h before establishing the model and 1% propofol 30 mg/kg was injected intraperitoneally at 30 min before establishing the model.In group AP, ADPR 10 mg/kg (in 0.2 ml of normal saline) was injected intraperitoneally at 1 h before establishing the model, and 1% propofol 30 mg/kg was injected intraperitoneally at 30 min before establishing the model.The equal volume of normal saline was given intraperitoneally at 1 h and at 30 min before establishing the model in group S and group IR.Blood samples were taken from the eyeballs for determination of the levels of serum urea nitrogen (BUN), creatinine (Cr), aminotransferase (ALT) and aspartate aminotransferase (AST) at 6 h of reperfusion.The animals were then sacrificed and the kidney tissues were taken, the ultrastructure of myocardial mitochondria was observed using transmission electron microscopy, the average diameter of mitochondria was calculated, and the expression of TRPM2, CnA, phospho-Drp1 Ser637 (p-Drp1 Ser637) and cleaved caspase-3 was detected (by Western blot). Results:Compared with group S, the concentrations of serum BUN and Cr were significantly increased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was up-regulated, the expression of p-Drp1 ser637 was down-regulated, and the average diameter of mitochondria was shortened in IR, P and AP groups ( P<0.05). Compared with group IR, the concentrations of serum BUN and Cr were significantly decreased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was down-regulated, the expression of p-Drp1 Ser637 was up-regulated, the average diameter of mitochondria was prolonged ( P<0.05), mitochondrial injury was attenuated, and no significant change was found in the serum ALT and AST concentrations in group P, and no significant change was found in concentrations of BUN and Cr in serum in group AP ( P>0.05). Compared with group P, concentrations of BUN and Cr in serum was significantly increased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was up-regulated, the expression of p-Drp1 Ser637 in kidney tissues was down-regulated, and the average diameter of mitochondria was shortened ( P<0.05), and mitochondrial injury was accentuated in group AP. Conclusion:The mechanism of propofol-induced reduction of renal injury induced by hepatic I/R is related to inhibiting the expression of TRPM2 in kidney tissues, decreasing the level of intracellular CnA and inhibiting dephosphorylation of Drp1 Ser637 in mice.

RQ-00434739, a novel TRPM8 antagonist, inhibits prostaglandin E2-induced hyperactivity of the primary bladder afferent nerves in rats.

AIMS: To examine the effects of RQ-00434739, a novel selective TRPM8 antagonist, on deep body temperature (DBT) and normal bladder sensory function and overactivity and its associated facilitation of mechanosensitive primary bladder single-unit afferent activities (SAAs) induced by intravesical l-menthol or prostaglandin E2 (PGE2) instillation in rats. MAIN METHODS: The effect of RQ-00434739 on DBT was evaluated using intravenous administration of RQ-00434739 (1 mg/kg) or its vehicle under urethane anaesthesia. Cystometry (CMG) was performed on conscious and freely moving rats. SAAs were measured from the left L6 dorsal root under urethane anaesthesia, and the fibers were grouped as Aδ- or C-fiber based on their conduction velocity. For both CMG and SAA measurements, after baseline recording with saline instillation, further recording was performed with intravesical l-menthol (6 mM) or PGE2 (60 µM) instillation after pretreatment with intravenous RQ-00434739 (1 mg/kg) or its vehicle. KEY FINDINGS: RQ-00434739 did not significantly affect DBT. In CMG measurements, RQ-00434739 administration increased mean voided volume. Both l-menthol and PGE2 instillation decreased mean voided volume following vehicle pretreatment, whereas such effects were not observed following RQ-00434739 pretreatment. In SAA measurements, either l-menthol or PGE2 instillations increased SAAs of C-fibers, but not SAAs of Aδ-fibers, in the presence of vehicle. RQ-00434739 pretreatment significantly inhibited the l-menthol- and PGE2-induced activation of C-fiber SAAs. SIGNIFICANCE: The present results demonstrate that blockade of TRPM8 channels can inhibit the pathological activation of mechanosensitive C-fibers and suggest that RQ-00434739 may be a promising therapeutic drug candidate for bladder hypersensitive disorders without affecting DBT.

Effects of Salivary Mg on Head and Neck Carcinoma via TRPM7.

Magnesium (Mg) has been known to play vital roles in regulating growth and various metabolic processes. In recent years, the association between Mg and tumorigenesis has raised more and more attention. However, the effects of Mg on the progression of head and neck carcinoma (HNC), as well as the mechanism behind it, remain undefined. In this study, the roles of Mg in tumorigenic activities were tested in CAL27 and FaDu cells as well as in a xenograft tumor model in nude mice. We demonstrated that a moderate increase in extracellular Mg contributed to the proliferation, migration, and invasion of 2 HNC cell lines, while the addition of Mg in drinking water promoted the growth of xenograft tumors in mice without altering their serum Mg levels. Moreover, TRPM7, a major Mg transporter, was shown to be essential for the tumorigenic activities of HNC and the Mg-induced promotive effects on HNC cells and was further shown to be associated with the activation of AKT/mTOR (mammalian target of rapamycin) signaling. In a preliminary clinical study, we determined the Mg ion concentrations in the stimulated saliva from 72 patients with nasopharynx carcinoma and 12 healthy individuals. Our data revealed that the salivary Mg levels of subjects with nasopharynx carcinoma were significantly higher than those of the healthy controls. This is correlated with our finding showing TRPM7 to be overexpressed in tumor tissues harvested from 9 patients with HNC. Therefore, we can conclude that salivary Mg level, within a certain range, could act as a risk factor for the progression of HNC, which involves the activation of AKT/mTOR signaling pathways through the TRPM7 channel. The control of salivary Mg level and the intervention of TRPM7 should not be ignored during the study of HNC.

KPR-2579, a novel TRPM8 antagonist, inhibits acetic acid-induced bladder afferent hyperactivity in rats.

AIMS: Transient receptor potential melastatin 8 (TRPM8) is proposed to be a promising therapeutic target for hypersensitive bladder disorders. We examined the effects of KPR-2579, a novel selective TRPM8 antagonist, on body temperature and on mechanosensitive bladder single-unit afferent activities (SAAs) provoked by intravesical acetic acid (AA) instillation in rats. METHODS: Female Sprague-Dawley rats were used. Effects of cumulative intravenous (i.v.) administrations of KPR-2579 (0.03-1 mg/kg) on deep body temperature were investigated (N = 18). In separate animals, effects of bolus administration of KPR-2579 (0.03 or 0.3 mg/kg, i.v.) on bladder hyperactivity induced by intravesical instillation of 0.1% AA were investigated using cystometry (N = 57) in a conscious free-moving condition or urethane-anesthetized condition, and SAA measurements (N = 41) were performed in a urethane-anesthetized condition. RESULTS: KPR-2579 at any doses tested did not affect body temperature. In cystometry measurements, a high dose (0.3 mg/kg) of KPR-2579 counteracted the shortened intercontraction interval provoked by AA instillation. In SAA measurements, 48 single afferent fibers (n = 24 in each fiber) were isolated. AA instillations significantly increased the SAAs of C fibers, but not of Aδ fibers, in the presence of KPR-2579's vehicle and a low dose (0.03 mg/kg) of KPR-2579. Pretreatment with a high dose (0.3 mg/kg) of KPR-2579 significantly inhibited the AA-induced activation of C-fiber SAAs. CONCLUSION: The present results suggest that TRPM8 channels play a role in the AA-induced pathological activation of mechanosensitive bladder C fibers in rats. KRP-2579 may be a promising drug for hypersensitive bladder disorders.

Role of GFRα3 in expression and membrane trafficking of TRPM8 in dorsal root ganglion during cold hyperalgesia in rats with neuropathic pain / 中华麻醉学杂志

Objective To evaluate the role of glial cell line-derived neurotrophic factor family re-ceptor alpha3(GFRα3)in the expression and membrane trafficking of transient receptor potential melasta-tin 8(TRPM8)in the dorsal root ganglion(DRG)during cold hyperalgesia in rats with neuropathic pain (NP). Methods Thirty-two healthy adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups(n=8 each) using a random number table: sham operation plus GFRα3 dsRNA group(Sham+dsRNA group), sham operation plus GFRα3 siRNA group(group Sham+siRNA), NP plus GFRα3 dsRNA group(group NP+dsRNA)and NP plus GFRα3 siRNA group(group NP+siRNA). NP was produced by chronic constriction injury to the sciatic nerve. At 10-30 days after operation, GFRα3 dsRNA 10 μg∕20 μl was intrathecally injected once a day for 4 consecutive days in Sham+dsRNA and NP+dsRNA groups, and 10 μg∕20 μl GFRα3 siRNA, of which the sense strand was modified with 2′-O-methyl and 5′-cholesterol, was intrathe-cally injected once a day for 4 consecutive days in Sham+siRNA and NP+siRNA groups. The number of paw lifts on the cold plate, mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency (TWL)were measured on 1 day before operation and 10, 11, 12, 13(before intrathecal injection)and 14 days after operation. The rats were sacrificed after the last behavioral testing, and ipsilateral DRGs of the lumbar segment(L4-6)were dissected for detection of the expression of GFRα3 and TRPM8 in total and membrane proteins by Western blot, and the ratio of TRPM8 expression in the membrane protein to that in the total protein(m∕t ratio)was calculated. Results Compared with group Sham+dsRNA, the number of paw lifts on the cold plate was significantly increased, the MWT was decreased, and TWL was shortened after operation in NP+dsRNA and NP+siRNA groups, the expression of GFRα3 and TRPM8 in total and membrane proteins was significantly up-regulated, and m∕t ratio was increased in group NP+dsRNA, and the expression of GFRα3 in DRGs was significantly down-regulated(P<001), and no significant change was found in the expression of TRPM8 in total and membrane proteins or m∕t ratio in group NP+siRNA(P>005). Compared with group NP+dsRNA, the number of paw lifts on the cold plate was significantly de-creased, the expression of GFRα3 and TRPM8 in total and membrane proteins was down-regulated, m∕t ra-tio was decreased(P<001), and no significant change was found in MWT or TWL in group NP+siRNA (P>005). Conclusion GFRα3 in DRGs can up-regulate the expression of TRPM8 and enhance the membrane trafficking of TRPM8, which may be involved in the maintenance mechanism of cold hyperalgesia in rats with NP.

Relationship between cold hyperalgesia and trafficking of TRPM8 to cell membrane in dorsal root ganglion of rats with neuropathic pain / 中华麻醉学杂志

Objective To evaluate the relationship between cold hyperalgesia and trafficking of transient receptor potential melastatin 8 (TRPM8) to cell membrane in the dorsal root ganglion (DRG) of rats with neuropathic pain (NP).Methods Ninety-six healthy male Sprague-Dawley rats,aged 10-12 weeks,weighing 250-280 g,were divided into sham operation group (S group,n=48) and NP group (n =48) using a random number table.NP was produced by chronic constriction injury to the sciatic nerve.The number of paw lifts on the cold plate and mechanical paw withdrawal threshold (MWT) were measured on 1 day before operation and 1,4,7,10 and 14 days after operation.Rats were sacrificed after behavioral testing,and ipsilateral DRGs of the lumbar segment (L46) were dissected tor detection of the expression of TRPM8 in total and membrane proteins by Western blot,and the ratio of TRPM8 expression in the membrane protein to that in the total protein (m/t ratio) was calculated.Results Compared with group S,the number of paw lifts on the cold plate was significantly increased,the MWT was decreased,the expression of TRPM8 in total and membrane proteins was up-regulated,and m/t ratio was increased on postoperative days 4,7,10 and 14 in group NP (P＜0.05 or 0.01).In group NP,the number of paw lifts on the cold plate was gradually increased with the prolongation of time after operation and reached the peak on postoperative day 10,maintaining at the peak until postoperative day 14;the MWT was gradually decreased and reached the lowest level on postoperative day 10,maintaining at the lowest level until postoperative day 14;the expression of TRPM8 in total and membrane proteins and m/t ratio were gradually increased with the prolongation of time after operation and reached the peak on postoperative day 10,maintaining at the peak level until postoperative day 14 (P＜0.01).Conclusion The mechanism underlying the development of cold hyperalgesia is related to enhanced trafficking of TRPM8 to cell membrane in DRGs of rats with NP.

Itch sensation through transient receptor potential channels: a systematic review and relevance to manual therapy.

OBJECTIVE: Patients may present with a complaint of "itchiness" or an "odd sensation" that can be relieved by manual therapy treatment options, which demonstrates the relevance of transient receptor potential (TRP) channels. There are studies that identify the role of various TRP channels as modulators of the itch sensation; however, discrepancies in the literature exist with respect to the overall neural pathway of the itch sensation, musculoskeletal implications, and decisive therapeutic implications. The purpose of this study was to review the literature and rate the quality of published articles regarding the role of TRP channels in the itch sensation. METHODS: A systematic search of relevant literature that was published in English by a peer-reviewed journal between January 2000 and June 2012 was performed in PubMed. Studies that met the predetermined inclusion criteria regarding the relationship between TRP channels and itch were identified and then evaluated for methodological quality by the Downs and Black Quality Index score system and were summarized. RESULTS: Nine studies were identified that met the inclusion criteria, all of which had fair methodological quality from the perspective of the modified Downs and Black Quality Index. TRPA1, TRPM8, and TRPV1-4 were indicated as key channels responsible for the transmission of the itch sensation. TRPV1 channels convey histamine-dependent itch, and TRPA1 channels convey histamine-independent itch. Temperature, nerve growth factor, and substance-P were also described as important itch modulators. There are similarities between the neural pathways responsible for itch, pain, and temperature, which explain the ability of noxious temperature to suppress the desire to scratch. Although transcutaneous electrical nerve stimulation, innocuous vibration, and cutaneous field stimulation have demonstrated relatively weak attenuation of itch, the use of topical capsaicin, noxious heat, and noxious cold have been demonstrated as effective therapies. CONCLUSIONS: The findings of this review show that studies have assessed the function of TRP channels and itch, rather than identifying the relationship between itch and effective noninvasive treatment options. Therefore, TRP channels could serve as important, complex clinical targets for manual therapists.

Alteration of TRPM8 in Dorsal Root Ganglion in Rat Model of Neuropathic Pain / 天津医药

Objective: To investigate the changes of TRPM8 expression in dorsal root ganglion(DRG) in the rat model of chronic constriction injury(CCI) of the sciatic nerve. Methods: Seventy-two male SD rats weighing 250~280 g were randomly divided into 2 groups (n = 36 each): group I (CCI) and group II (sham operation). The threshold of cold hyperalgesia, heat hyperalgesia and mechanical hyperalgesia were measured before operation (baseline) and at 1, 4, 7, 10 and 14 d after operation. Six rats were killed at each time point in each group. The L5 DRGs ipsilateral to nerve injury were dissected out for determination of transient receptor potential melastatin 8(TRPM8) by immunohistochemical assay. Results: The thresholds of cold, thermal and mechanical stimuli started to decrease at 4 d after CCI in operation group and maintained at a relatively low level until the end of experiment. The cold and thermal hyperalgesia peaked at 10 d after operation and mechanical hyperalgesia at 14 d. Immunohistochemical assay demonstrated that expression of TRPM8 were increased in L5DRG on the operated side significantly at 4 d after CCI and reached the peak at 10 d and was maintained at a high level until the end of experiment. Conclusion: The upregulation of TRPM8 in DRG involved in the mechanism of neuropathic pain.

Study on the effects of acidosis on transient receptor potential channel M7 (TRPM7) in mouse cardiac fibroblast / 中华老年医学杂志

Objective To investigate the effects of acidosis on the current change of transient receptor potential channel M7 (TRPM7) and collagen production in mouse cardiac fibroblast (MCFs), and to explore the pathophysiological function of TRPM7 on the cardiac fibrosis. Methods (1) The model of MCFs was established and isolated. (2) MCFs was subcuhured. (3) Patch clamp technique was used to observe the current characteristics of TRPM7 in low PH solutions. (4) The influence of acidic condition on Ca2+ influx in MCFs was recorded by calcium fluorescent indicators. Results (1) There was a high level expression of TRPM7 in MCFs and the electrophysiological characteristics of TRPM7-like (TRPM7L) was similar to that of TRPM7. (2) Ca2+ influx was increased in acidic condition, and the F340/F380 ratio was increased from 1.0 to 4. 6 at pH 4.0 compared with pH 7.0 (t=2.72, P<0.01). Conclusions (1) TRPM7 is the molecular basis of the native TRPM7L in MCFs and TRPM7L plays an important role in Ca2+ influx. (2) The pathophysiological function of MCFs is influenced by regulation of Ca2+ influx mediated by TRPM7L in the condition of acidosis.

Changes and significance of transient receptor potential melastatin 7 after myocardial infarction in the mouse / 中华老年医学杂志

Objective To investigate the changes of TRPM 7-like current in mouse cardiac fibroblast(CFs) after myocardial infarction and the effect of myocardial ischemia on the TRPM 7 expression and current. Methods (1) The model of myocardial infarction was made and CFs were isolated;(2) CFs were cultured and infected by TRPM 7 siRNA;(3) The effects of myocardial ischemia on TRPM 7 current were recorded by whole cell patch clamp technique;(4) The influences of myocardial isehemia on Ca2+ influx in CFs were recorded by Ca2+ fluorescence imaging. (5) The effects of ischemia on total collagen content of CFs were studied. Results (1) Ca2+ inward current of CFs was increased after myocardial infarction [(16.2±1.7) vs. (7.4±0.7) pA/pF, P<0.053];(2) TRPM 7 current was reduced by 90% after siRNA infection;(3) The total collagen content of CFs after ischemia was approximately 2.3-fold higher than basic value. Conclusions Ca2+ influx in CFs plays an important role in the pathophysiological process of myocardial fibrosis.