Role of transient receptor potential channels in melanocytes and their related diseases / 中华皮肤科杂志

Transient receptor potential (TRP) channels are a special type of non-selective cationic channel family located on the cell membrane or organelle membrane, and notably expressed in melanocytes. This review summarizes recent research progress in biological functions of TRP channels in melanocytes and their involvement in the pathological process of pigmented skin diseases and melanoma, so as to provide a new theoretical basis for the prevention and treatment of melanin-related diseases.

Research advances in the mechanism of action of TRPV4 ion channel protein in liver diseases / 临床肝胆病杂志

As a member of the transient receptor potential ion channel family, transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel and is widely distributed in a variety of tissues and organs. In recent years, more and more studies have shown that TRPV4 channel proteins are closely associated with liver diseases such as liver fibrosis, liver cancer, and polycystic liver disease. This article analyzes the articles on TRPV4 and liver disease and summarizes the exact signaling pathways and possible potential mechanism between TRPV4 and liver disease, so as to provide new ideas for clinical application and further studies.

Comparative study of cold hyperalgesia and mechanical allodynia in two animal models of neuropathic pain: different etiologies and distinct pathophysiological mechanisms

Abstract Neuropathic pain (NP) affects more than 8% of the global population. The proposed action of the transient receptor potential ankyrin 1 (TRPA1) as a mechanosensor and the characterization of the transient receptor potential melastatin 8 (TRPM8) as a cold thermosensor raises the question of whether these receptors are implicated in NP. Our study aimed to evaluate the involvement of TRPA1 and TRPM8 in cold and mechanical signal transduction to obtain a comparative view in rat models of streptozotocin-induced diabetes (STZ) and chronic constriction injury of the sciatic nerve (CCI). The electronic von Frey test showed that STZ rats presented mechanical allodynia that was first evidenced on the 14th day after diabetes confirmation, and four days after CCI. This phenomenon was reduced by the intraplantar (ipl) administration of a TRPA1 receptor antagonist (HC-030031; 40 µL/300 µg/paw) in both NP models. Only CCI rats displayed cold hyperalgesia based on the cold plate test. The pharmacological blocking of TRPA1 through the injection of the antagonist attenuated cold hyperalgesia in this NP model. STZ animals showed a reduction in the number of flinches induced by the intraplantar injection of mustard oil (MO; TRPA1 agonist; 0.1%/50 µL/paw), or intraplantar injection of menthol (MT; TRPM8 agonist; 0.5% and 1%/50 µL/paw). The response induced by the ipl administration of MT (1%/50 µL/paw) was significantly different between the CCI and SHAM groups. Together, these data suggest a different pattern in nociceptive behavior associated with different models of NP, suggesting a variant involvement of TRPA1 and TRPM8 in both conditions

The Effects of Lung Ischaemia/Reperfusion on TRPM Gene Expression

ABSTRACT Objective: Transient receptor potential melastatin (TRPM) are integral membrane proteins that have broad range of cellular functions. Roles of TRPM2, TRPM3, TRPM4 and TRPM7 among these channels are very important, and their roles in lung ischaemia/reperfusion injury have not been evaluated yet. The aim of this study is to investigate the contribution of these genes in lung ischaemia/reperfusion injury and evaluate histopathology of tissues. Methods: A total of 40 Wistar albino rats were enrolled for the study. Ischaemia was performed by the application of an atramvatic clamp to pulmonary artery. Gene expressions were determined by the semi-quantitative reverse transcription-polymerase chain reaction method. Histopatholical evaluations were held by a standard haematoxyline-eosin staining. Results: The major histopathological tissue damage was observed in ischaemia performed groups, and expression of TRPM channels was found to be obviously downregulated. Substantial changes were determined between TRPM2, TRPM3, TRPM4 and TRPM7 and lung ischaemia/reperfusion injury. In particular, expression of TRPM2 and TRPM7 was reversibly downregulated in ischaemia. Yet, the expression of TRPM3 and TRPM4 was irreversibly down-regulated after ischaemia. Conclusion: Consequently, these results indicate that TRPM family of cation channels may have significant roles in the lung ischaemia/reperfusion injury.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Correlation between transient receptor potential canonical channel with heart and kidney injure of rat model of obstructive sleep apnea hypopnea syndrome / 浙江大学学报·医学版

OBJECTIVE@#To investigate the expression of transient receptor potential canonical channels (TRPCs) in the heart and kidney of rat model of obstructive sleep apnea hypopnea syndrome (OSAHS).@*METHODS@#Eighteen male SD rats were randomly assigned to intermittent hypoxia (IH) group (=9 ) and control group (=9). In IH group, rats were placed in a chamber and exposed to intermittent hypoxia for 8h (10AM-6PM) daily. The expression of TRPC-related mRNA and protein in the heart and kidney tissue were detected by qRT-PCR and Western blotting, respectively.@*RESULTS@#The mRNA expressions of TRPC3/TRPC4/TRPC5 in heart tissues of IH group were increased significantly compared with the control group (all >0.05); while there were no significant differences in the mRNA expressions of TRPC1/TRPC3/TRPC4/TRPC5/TRPC6/TRPC7 in kidney tissue between two groups (all 0.05).@*CONCLUSIONS@#The IH rat model shows that TRPC5 channel is likely to be involved in the OSAHS induced pathophysiological changes in the myocardium and may become a target to prevent OSAHS related cardiac damage.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.

The role of calmodulin in regulating calcium-permeable PKD2L1 channel activity

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when coexpressed with CaM and CaMΔN. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ΔEF-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

Effect of transient receptor potential channel m4 on cerebral artery myogenic tone following subarachnoid hemorrhage in rats / 中国脑血管病杂志

Objective: To investigate the effect of transient receptor potential channel M4 (TRPM4) on cerebral artery myogenic tone following subarachnoid hemorrhage (SAH) in rats. Methods: Sixty cleangrade healthy SD rats of both sexes were randomly divided into SAH group (n = 30) and sham operation group (n =30) according to the random number table. A SAH model was induced by injecting autologous rat tail artery blood into the suprasellar cistern of rats with stereotactic apparatus. 0.2 ml autologous arterial blood was injected into the rats of the SAH group and 0.2 ml isotonic saline was injected into the rats of the sham operation group. Five days after inducing the SAH model,the cerebral arteries of rats were separated, and the diameter of cerebral arteries was observed by the pressurized myography, and the myogenic tone was calculated. The cerebral arteries of each experimental group were observed by time-sequence method for artificial cerebrospinal fluid(aCSF), containing 0. 03 mmol/L TRPM4 specific blocker 9-phenanthrol (9- Phe) aCSF and containing 0. 1 mmol/L diltiazem in the calcium-free aCSF in the arterial diameters, and according to this, the myogenic tone was calculated. Results: After SAH, when the cerebral artery was located in aCSF,the cerebral artery diameter of the SAH group was significantly smaller than that of the sham operation group (45.9 ±6. 6 u.m vs. 74. 9 ±5. 1 u,m,i = 5. 08,P <0. 05). Cerebral artery myogenic tone was significantly higher than that of the sham operation group (58. 7 ±2. 4% vs. 41. 2 ±3. 4%;t - 2.11,P < 0. 05). After adding 9-Phe to aCSF, the diastole of cerebral artery diameters of the SAH group and sham operation group was more obvious than that before adding (98. 4 ± 6. 1 and 95. 1 ±5.5 u,m, respectively). The difference was statistically significant (both P<0.05). The decreased myogenic tone in the SAH group was significantly higher than that in the sham operation group (42. 4 ±4. 4% vs. 15. 3 ± 3. 2%;t =2. 83,P < 0. 05). Conclusion: After SAH,TRPM4 mediates the increased myogenic lone of cerebral artery in rats.

Effects of honokiol on activation of transient receptor potential channel in rat spinal dorsal root ganglion cells and pruritus in mouse models / 中华皮肤科杂志

Objective To evaluate the effect of honokiol on the activation of transient receptor potential (TRP) channel in rat spinal dorsal root ganglion cells and pruritus in mouse models.Methods Healthy male ICR mice aged 4-6 weeks were used to establish histamine-induced and acetone/ether/water (AEW)-induced itching models separately.Totally,mice were randomly divided into 7 groups (histamineinduced model experiment) or 6 groups (AEW-induced model experiment):normal control group and model group both gavaged with sodium chloride physiological solution,solvent group gavaged with sodium carboxymethylcellulose solution,chlorphenamine group (only set up in the histamine-induced model experiment) gavaged with chlorphenamine,50,25 and 12.5 mg/kg honokiol groups gavaged with 50,25 and 12.5 mg/kg honokiol respectively.In the histamine-induced model experiment,the mice were all injected with histamine except the normal control group injected with sodium chloride physiological solution 24 hours after the gavage treatment,while the mice in the AEW-induced model experiment were all topically treated with AEW except the normal control group topically treated with sodium chloride physiological solution for 4 days,followed by gavage with different drugs.The anti-itch effect of each treatment was evaluated by counting the scratching frequency within 30 minutes.Rat spinal dorsal root ganglion (DRG)cells were isolated and subjected to a primary culture.Then,the DRG cells were divided into 6 groups:capsaicin or allyl isothiocyanate (AITC)-induced model group pre-incubated with Hank's balanced salt solution (HBSS),500 μmol/L capsazepine or 10 μmol/L HSC030031 group pre-incubated with capsazepine or HSC030031,solvent group pre-incubated with dimethyl sulfoxide (DMSO),3 honokiol groups preincubated with 7.81,15.63 and 31.25 mg/L honokiol respectively,and Ca2+ fluorescence imaging system was used to observe changes of Ca2+ influx in these cells after capsaicin or AITC stimulation.Statistical analysis was carried out with SPSS 20.0 software by using one-way analysis of variance and Dunnett-t test.Results In the histamine-induced mouse models,the scratching frequency was significantly lower in the 50 and 25 mg/kg honokiol groups than in the model group (21.88 and 21.14 vs.63.70,t =3.48,3.49 respectively,both P =0.003),while no significant difference in the scratching frequency was observed between the 12.5 mg/kg honokiol group and the model group (t =2.01,P =0.062).After the treatment with 50 mg/kg honokiol in the AEW-induced mouse models,the scratching frequency significantly decreased compared with the model group (61.4 vs.101.17,t =0.45,P =0.009),while there were no significant differences among the 25,12.5 mg/kg honokiol groups and the model group (all P ＞ 0.05).Compared with the capsaicin or AITC-induced model group,the increase of Ca2+ fluorescence signal in the DRG cells was significantly inhibited in the 31.25 mg/L honokiol group:at the 45th second,the rate of relative fluorescence intensity change (AF/F0) was 1.11 in the model group,but-0.11 in the 31.25 mg/L honokiol group in the capsaicin-induced model experiment,and 0.56 in the model group,but 0.00 in the 31.25 mg/L honokiol group in the AITC-induced model experiment.Conclusion Honokiol shows an inhibitory effect on mouse models of pruritus induced by histaminergic or non-histaminergic factors,likely by inhibiting Ca2+ influx through activated TRPV 1 and TRPA 1 channels in the DRG cells.

Prediction of itching diagnostic marker through RNA sequencing of contact hypersensitivity and skin scratching stimulation mice models

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a , Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment.

Skin Barrier and Calcium

Epidermal barrier formation and the maintenance of barrier homeostasis are essential to protect us from the external environments and organisms. Moreover, impaired keratinocytes differentiation and dysfunctional skin barrier can be the primary causes or aggravating factors for many inflammatory skin diseases including atopic dermatitis and psoriasis. Therefore, understanding the regulation mechanisms of keratinocytes differentiation and skin barrier homeostasis is important to understand many skin diseases and establish an effective treatment strategy. Calcium ions (Ca²⁺) and their concentration gradient in the epidermis are essential in regulating many skin functions, including keratinocyte differentiation, skin barrier formation, and permeability barrier homeostasis. Recent studies have suggested that the intracellular Ca²⁺ stores such as the endoplasmic reticulum (ER) are the major components that form the epidermal calcium gradient and the ER calcium homeostasis is crucial for regulating keratinocytes differentiation, intercellular junction formation, antimicrobial barrier, and permeability barrier homeostasis. Thus, both Ca²⁺ release from intracellular stores, such as the ER and Ca²⁺ influx mechanisms are important in skin barrier. In addition, growing evidences identified the functional existence and the role of many types of calcium channels which mediate calcium flux in keratinocytes. In this review, the origin of epidermal calcium gradient and their role in the formation and regulation of skin barrier are focused. We also focus on the role of ER calcium homeostasis in skin barrier. Furthermore, the distribution and role of epidermal calcium channels, including transient receptor potential channels, store-operated calcium entry channel Orai1, and voltage-gated calcium channels in skin barrier are discussed.

Role of transient receptor potential channel 8 in development and maintenance of neuropathic pain in rats: the relationship with nuclear factor kappa B in dorsal root ganglion / 中华麻醉学杂志

Objective To evaluate the role of transient receptor potential channel 8 (TRPM8) in the development and maintenance of neuropathic pain and the relationship with nuclear factor kappa B (NF-κB) in the dorsal root ganglion of rats.Methods Thirty-six pathogen-free healthy adult male SpragueDawley rats,weighing 180-200 g,aged 6-8 weeks,were divided into 3 groups (n=12 each) using a random number table:sham opeation group (group S),neuropathic pain group (group NP) and TRPM8 bocker BCTC group (group BCTC).Neuropathic pain was induced by ligation of the right sciatic nerve in anesthetized rats.BCTC 20 nmol was intrathecally injected once a day on preoperative day 1 and postoperative days 1,3,7 and 10 in group BCTC.The thermal,mechanical and cold pain thresholds were measured on preoperative day 1 and postoperative days 1,3,7,10 and 14.The dorsal root ganglions of the lumbar segment (L4-6) were removed on postoperative days 7 and 14 for determination of the expression of TRPM8 and NF-κB p65 by Western blot.Results Compared with group S,the thermal and mechanical thresholds were significantly decreased on postoperative days 1-14,and the cold pain threshold was decreased on postoperative days 3-14 in NP and BCTC groups,the expression of TRPM8 and NF-κB p65 in dorsal root ganglions was up-regulated on postoperative days 7 and 14 in group NP (P＜0.05),and no significant change was found in the expression of TRPM8 and NF-κB p65 in dorsal root ganglions in group BCTC (P＞0.05).Compared with group NP,the thermal pain threshold was significantly decreased and the cold pain threshold was increased on postoperative days 3-14,the expression of TRPM8 and NF-κB p65 in dorsal root ganglions was up-regulated on postoperative days 7 and 14,and no significant change was found in the mechanical pain threshold in group BCTC (P＞0.05).Conclusion NF-κB activation after opening of TR-PM8 in dorsal root ganglion neurons is involved in the development and maintenance of neuropathie pain in rats.

Effect of TRPC6 after astrocytes activation in traumatic brain injury / 重庆医学

Objective To explore the role of astrocytes transient receptor potential channel 6(TRPC6) in rat brain injury (TBI).Methods Thirty-nine male Sprague-Dawley(SD) rats were divided into the sham operation group,injury group and deferoxamine(DFX) group(n=13).According to the previous model construction scheme established by our research group Feeney method,the rat brain impact injury model was established.The Morris water maze test was performed and the defected brain volume was measured.The immunofluorescence was adopted to detect the co-expression of TRPC6 and GFAP.Then Western blot was performed.Results The defected brain volume after TBI in the DFX group was significantly decreased compared with the injury group [(115.35 ± 13.70)mm3 vs.(209.99 ± 16.70)mm3]] (P＜0.05).The Morris water maze test found that the platform search strategy and search time in the DFX group were(3.13 ± 0.35) and(36.15 ± 26.63)s,which were significantly improve d compared with (2.13±0.64) and(110±47.34)s in the injury group(P＜0.05).The immunofluorescence found that GFAP in the DFX group was highly expressed,moreover the co-expression with TRPC6 was increased.Western blot found that TRPC6 in the DFX group was significantly down-regulated(P＜0.01).Conclusion In rat TBI early stage,strocytes are activated after DFX treatment and TRPC6 is highly expressed,playing a neuroprotective role.

Effect of ultraviolet radiation on expression of transient receptor potential ankyrin 1 in HaCaT cells / 中华皮肤科杂志

Objective To investigate the photoregulation of transient receptor potential ankyrin 1 (TRPA1) in HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 225-mJ/cm2 UVA radiation groups and 25-mJ/cm2 UVB radiation groups.HaCaT cells in the UVA radiation groups were further classified into 6 groups:blank control group 1 receiving no treatment,retinal group 1 treated with 12 μmol/L retinal alone,UVA group treated with 225 mJ/cm2 UVA radiation alone,retinal + UVA group (UVA-TRPA1 control group),retinal + UVA + 500 μmol/L cinnamaldehyde group (UVA-TRPA1 agonist group) and retinal + UVA + 1 mmol/L camphor group (UVA-TRPA1 antagonist group).Additionally,HaCaT cells in the UVB radiation groups were also further classified into 6 groups:blank control group 2 receiving no treatment,retinal group 2 treated with 12 μmol/L retinal alone,UVB group treated with 25-mJ/cm2 UVB radiation,retinal + UVB group (UVB-TRPA1 control group),retinal + UVB + 500 μmol/L cinnamaldehyde group (UVB-TRPA1 agonist group) and retinal + UVB + 1 mmol/L camphor group (UVB-TRPA1 antagonist group).Real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of TRPA 1 respectively.Flow cytometry was conducted to investigate changes of calcium influx in HaCaT cells in the above groups.Results qPCR and Western blot analysis showed that TRPA1 mRNA and protein were expressed in HaCaT cells.The fluorescence intensity of calcium influx significantly differed among the blank control group 1,retinal group 1,UVA group and retinal + UVA group (155.06 ± 7.62,148.37 ± 18.77,166.92 ± 3.71 and 331.333 ± 40.563;F =44.509,P ＜ 0.01),as well as among the blank control group 2,retinal group 2,UVB group and retinal + UVB group (150.20 ± 1.73,171.66 ± 56.23,147.56 ± 6.60 and 250.44 ± 9.13;F =85.261,P ＜ 0.01).Additionally,retinal + UVA/UVB groups showed significantly higher fluorescence intensity of calcium influx compared with the blank control groups (q =18.442,6.052,P ＜ 0.01).The TRPA1 agonist cinnamaldehyde and its antagonist camphor could regulate the UVA-and UVB-induced calcium influx (P ＜ 0.001).Compared with the blank control group 1 and 2 respectively,the fluorescence intensity of retinal-dependent calcium influx was significantly higher in the UVA/UVB-TRPA1 agonist group (q =14.934,32.770,P ＜ 0.001),and significantly lower in the UVA/UVB-TRPA1 antagonist group (q =7.986,14.596,P ＜ 0.001).Conclusion TRPA1 is expressed in HaCaT cells,and UVA or UVB can regulate the calcium influx in HaCaT cells by adjusting the activity of TRPA 1.

Effect of ultraviolet radiation on expression of transient receptor potential ankyrin 1 in HaCaT cells / 中华皮肤科杂志

Objective To investigate the photoregulation of transient receptor potential ankyrin 1 (TRPA1) in HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 225-mJ/cm2 UVA radiation groups and 25-mJ/cm2 UVB radiation groups.HaCaT cells in the UVA radiation groups were further classified into 6 groups:blank control group 1 receiving no treatment,retinal group 1 treated with 12 μmol/L retinal alone,UVA group treated with 225 mJ/cm2 UVA radiation alone,retinal + UVA group (UVA-TRPA1 control group),retinal + UVA + 500 μmol/L cinnamaldehyde group (UVA-TRPA1 agonist group) and retinal + UVA + 1 mmol/L camphor group (UVA-TRPA1 antagonist group).Additionally,HaCaT cells in the UVB radiation groups were also further classified into 6 groups:blank control group 2 receiving no treatment,retinal group 2 treated with 12 μmol/L retinal alone,UVB group treated with 25-mJ/cm2 UVB radiation,retinal + UVB group (UVB-TRPA1 control group),retinal + UVB + 500 μmol/L cinnamaldehyde group (UVB-TRPA1 agonist group) and retinal + UVB + 1 mmol/L camphor group (UVB-TRPA1 antagonist group).Real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of TRPA 1 respectively.Flow cytometry was conducted to investigate changes of calcium influx in HaCaT cells in the above groups.Results qPCR and Western blot analysis showed that TRPA1 mRNA and protein were expressed in HaCaT cells.The fluorescence intensity of calcium influx significantly differed among the blank control group 1,retinal group 1,UVA group and retinal + UVA group (155.06 ± 7.62,148.37 ± 18.77,166.92 ± 3.71 and 331.333 ± 40.563;F =44.509,P ＜ 0.01),as well as among the blank control group 2,retinal group 2,UVB group and retinal + UVB group (150.20 ± 1.73,171.66 ± 56.23,147.56 ± 6.60 and 250.44 ± 9.13;F =85.261,P ＜ 0.01).Additionally,retinal + UVA/UVB groups showed significantly higher fluorescence intensity of calcium influx compared with the blank control groups (q =18.442,6.052,P ＜ 0.01).The TRPA1 agonist cinnamaldehyde and its antagonist camphor could regulate the UVA-and UVB-induced calcium influx (P ＜ 0.001).Compared with the blank control group 1 and 2 respectively,the fluorescence intensity of retinal-dependent calcium influx was significantly higher in the UVA/UVB-TRPA1 agonist group (q =14.934,32.770,P ＜ 0.001),and significantly lower in the UVA/UVB-TRPA1 antagonist group (q =7.986,14.596,P ＜ 0.001).Conclusion TRPA1 is expressed in HaCaT cells,and UVA or UVB can regulate the calcium influx in HaCaT cells by adjusting the activity of TRPA 1.

Canales iónicos Receptores de Potencial Transitorio y su papel protagónico en la terapia analgésica / Transient receptor potential ion channels and their leading role in analgesic therapy

En la búsqueda de nuevas opciones terapéuticas para el tratamiento del dolor, se ha llegado al descubrimiento de canales iónicos que actúan como receptores y están presentes en neuronas nociceptoras aferentes primarias. Entre estos receptores, se encuentran los canales iónicos receptores de potencial transitorio que regulan las vías involucradas en el dolor y la nocicepción. Se realizó una revisión actualizada de los principales canales iónicos receptores de potencial transitorio implicados en la fisiopatología del dolor. Se hace una reseña histórica del descubrimiento de estas moléculas y sus estudios avanzados. A continuación se revisan las diferentes familias de estos canales con su clasificación, nomenclatura, estructura y funciones celulares. También se hace un recuento de la relación de estos canales con la analgesia, así como el mecanismo de acción de algunos analgésicos que actúan sobre ellos. Finalmente, se detallan importantes consideraciones a tomar en cuenta, que pudieran influir sobre la utilización de estos medicamentos en la clínica. Por tal motivo, el trabajo procura ser una revisión que abarque el rol de los canales TRP como nuevas dianas farmacológicas en el tratamiento del dolor.

In the search for new therapeutic options for pain treatment, new ion channels have been discovered, which act as receptors and are present in primary afferent nociceptor neurons. Some of them are the transient receptor potential ion channels that regulate the pathways involved in pain and nociception. An updated review of the main transient potential receptors ion channels involved in pain physiopathology and a historical review of the discovery of these molecules and advanced studies on them were also made. Then the different families of these channels with their classification, nomenclature, structure and cell functions were also examined. An account of the relationships of these channels with analgesia as well as the mechanism of action of some analgesics acting upon them was also presented. Finally, important considerations were given, which should be taken into account since they might influence on the clinical use of these drugs. For these reasons, the paper intends to be a review covering the role of the transient receptor potential channels as new pharmacological targets in the pain treatment.

Expression of TRPV1 and Electrophysiological Characteristics of Colon-specific Dorsal Root Ganglion Neurons in Rat Model of Visceral Hypersensitivity / 胃肠病学

Background:Visceral hypersensitivity is considered to be one of the major pathophysiological mechanisms of irritable bowel syndrome. Aims:To investigate the expression of TRPV1 and electrophysiological characteristics of colon-specific dorsal root ganglion( DRG)neurons in rat model of visceral hypersensitivity. Methods:Twenty 10-day-old rats were randomly divided into two groups. In model group,visceral hypersensitivity was induced by colorectal administration of acetic acid;while in control group the same amount of saline was administered. Colon-specific DRG neurons were labeled retrogradely by injection of DiI,a fluorochrome,into the colon wall. Expression of TRPV1 in DRG neurons was detected by immunofluorescence and the electrophysiological characteristics of DRG neurons was detected by using patch-clamp technique. Results:In model group,the expression rate of TRPV1 in colon-specific DRG neurons was significantly higher than that in controls(46. 1% vs. 36. 6% ,P <0. 01),the average rheobase was significantly decreased[(57. 80 ±1. 32)pA vs.(73. 45 ± 4. 51)pA,P < 0. 05],while the frequency of action potentials(APs)in response to doubling rheobase stimulation was significantly increased[(8. 20 ± 1. 10)Hz vs. (4. 54 ± 0. 66)Hz,P < 0. 05]. Score of abdominal withdrawal reflex(AWR)under a 60 mm Hg colorectal distention was positively correlated with the expression rate of TRPV1 and the frequency of APs in response to doubling rheobase stimulation(r = 0. 87 and r = 0. 73,P < 0. 01),but was negatively correlated with the rheobase(r = - 0. 81,P < 0. 01)in model group. Conclusions:Increased expression of TRPV1 and excitability in colon-specific DRG neurons might be a crucial step in formation of visceral hypersensitivity.

Effect of subarachnoid hemorrhage on transient receptor potential melastatin 4 channel activity / 中国脑血管病杂志

Objective To study the effect of spontaneous subarachnoid hemorrhage (SAH)on transient receptor potential melastatin 4 (TRPM4)channel activity. Methods Seventeen SD rats of clean grade were selected. They were randomly divided into either a SAH (n = 10)or a sham operation group (n = 7) according to the random number table. At day 5 after SAH modeling,the cerebral arteries were harvested and the cerebral arterial smooth muscle cells were isolated using enzymatic digestion method. Western blot was used to detect TRPM4 expression and translocation rate. Patch-clamp techniques were used to study the maximum current intensity of the TRPM4 single channel in cerebral arterial smooth muscle cells. Results The fluorescent-stained TRPM4 were observed in cerebral arterial smooth muscle cells in the 2 groups of rats. The relative quantities of TRPM4 in the total protein of the sham operation group and the SAH group were 24 ± 3% and 32 ± 4% respectively. There was significant difference between the 2 groups (t = 4. 47,P 0. 05). Conclusion SAH has the induced effect for TRPM4 activity.

Effect of TRPM8 on proliferation and apoptosis of human bladder transitional cell carcinoma cell T24 / 中国医师杂志

Objective To investigate the effect of down-regulation of transient receptor potential melastatin 8 (TRPM8) on the proliferation and apoptosis of human bladder transitional cell carcinoma cells T24.Methods shRNA targeting TRPM8 was designed and synthesized, and then transfected into the T24 cells via lipofectamine 2000 mediation.The proliferation and apoptosis of T24 cells were detected with methyl thiazolyl tetrazolium (MTT) assay and flow cytometry.Expression of extracellular regulated kinase (ERK), cyclin D1, and Bcl-2 were detected with Western blot.Results TRPM8-targeted shRNA downregulated TRPM8 expression of T24 cells.MTT assay showed a significant acceleration of the proliferation of shRNA interference group compared to blank and control groups (P ＜0.01).Compared to control group, cell apoptosis rate was significantly higher in shRNA interference group (P ＜ 0.01).In addition, the expressions of PI3K, cyclin D1, and Bcl-2 were decreased in shRNA interference group.Conclusions Down-regulation of TRPM8 can induce inhibition of proliferation and promotion of cell apoptosis in human bladder transitional cell carcinoma cells T24 via regulating PI3K.It might be regarded as a novel target for clinical diagnosis and gene therapy of bladder cancer.