Transient Receptor Potential Melastatin Research Articles

Separation of Oral Cooling and Warming Requires TRPM8.

Cooling sensations arise inside the mouth during ingestive and homeostasis behaviors. Oral presence of cooling temperature engages the cold and menthol receptor TRPM8 (transient receptor potential melastatin 8) on trigeminal afferents. Yet, how TRPM8 influences brain and behavioral responses to oral temperature is undefined. Here we used in vivo neurophysiology to record action potentials stimulated by cooling and warming of oral tissues from trigeminal nucleus caudalis neurons in female and male wild-type and TRPM8 gene deficient mice. Using these lines, we also measured orobehavioral licking responses to cool and warm water in a novel, temperature-controlled fluid choice test. Capture of antidromic electrophysiological responses to thalamic stimulation identified that wild-type central trigeminal neurons showed diverse responses to oral cooling. Some neurons displayed relatively strong excitation to cold <10°C (COLD neurons) while others responded to only a segment of mild cool temperatures below 30°C (COOL neurons). Notably, TRPM8 deficient mice retained COLD-type but lacked COOL cells. This deficit impaired population responses to mild cooling temperatures below 30°C and allowed warmth-like (≥35°C) neural activity to pervade the normally innocuous cool temperature range, predicting TRPM8 deficient mice would show anomalously similar orobehavioral responses to warm and cool temperatures. Accordingly, TRPM8 deficient mice avoided both warm (35°C) and mild cool (≤30°C) water and sought colder temperatures in fluid licking tests, whereas control mice avoided warm but were indifferent to mild cool and colder water. Results imply TRPM8 input separates cool from warm temperature sensing and suggest other thermoreceptors also participate in oral cooling sensation.

Identification of Ligands for Ion Channels: TRPM2.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, nonselective cation channel with a widespread distribution throughout the body. It is involved in many pathological and physiological processes, making it a potential therapeutic target for various diseases, including Alzheimer's disease, Parkinson's disease, and cancers. New analytical techniques are beneficial for gaining a deeper understanding of its involvement in disease pathogenesis and for advancing the drug discovery for TRPM2-related diseases. In this work, we present the application of collision-induced affinity selection mass spectrometry (CIAS-MS) for the direct identification of ligands binding to TRPM2. CIAS-MS circumvents the need for high mass detection typically associated with mass spectrometry of large membrane proteins. Instead, it focuses on the detection of small molecules dissociated from the ligand-protein-detergent complexes. This affinity selection approach consolidates all affinity selection steps within the mass spectrometer, resulting in a streamlined process. We showed the direct identification of a known TRPM2 ligand dissociated from the protein-ligand complex. We demonstrated that CIAS-MS can identify binding ligands from complex mixtures of compounds and screened a compound library against TRPM2. We investigated the impact of voltage increments and ligand concentrations on the dissociation behavior of the binding ligand, revealing a dose-dependent relationship.

TRPM2 knockdown attenuates myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice via regulating the MEK/ERK and mTORC1 signaling pathway

Diabetic cardiomyopathy (DCM) is a major complication of diabetes. Transient receptor potential melastatin 2 (TRPM2) activity increases in diabetic oxidative stress state, and it is involved in myocardial damage and repair. We explore the protective effect of TRPM2 knockdown on the progression of DCM. A type 2 diabetes animal model was established in C57BL/6N mice by long-term high-fat diet (HFD) feeding combined with a single injection of 100-mg/kg streptozotocin (STZ). Genetic knockdown of TRPM2 in heart was accomplished by the intravenous injection via the tail vein of adeno-associated virus type 9 carrying TRPM2 shRNA. Neonatal rat ventricular myocytes was exposed to 45 mM of high-glucose (HG) stimulation for 72 h in vitro to mimic the in vivo conditions. Western blot, real-time quantitative PCR (RT-qPCR), immunohistochemistry and fluorescence, electron, CCK-8, and flow cytometry were used to evaluate the phenotype of cardiac inflammation, fibrosis, apoptosis, and autophagy. Mice with HFD/STZ-induced diabetes exhibited systolic and diastolic dysfunction, as demonstrated by increased myocardial apoptosis and autophagy inhibition in the heart. Compared to control group, the protein expression of TRPM2, bax, cleaved caspase-3, and P62 was significantly elevated, and the protein expression of bcl-2 and LC3-II was significantly decreased in the myocardial tissues of the HFD/STZ-induced diabetes group. Knockdown of TRPM2 significantly reversed the HFD/STZ-induced myocardial apoptosis and autophagy inhibition. TRPM2 silencing attenuated HG-induced apoptosis and autophagy inhibition in primary cardiomyocytes via regulating the MEK/ERK mTORC1 signaling pathway. TRPM2 knockdown attenuates hyperglycemia-induced myocardial apoptosis and promotes autophagy in HFD/STZ-induced diabetic mice or HG-stimulated cardiomyocytes via regulating the MEK/ERK and mTORC1 signaling pathway.

In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel

Background Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4. Methods We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence. Results In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment. Conclusion M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.

Targeting Grancalcin Accelerates Wound Healing by Improving Angiogenesis in Diabetes.

Chronic diabetic wounds are a serious complication of diabetes and often result in limb amputations and confer high mortality rates. The proinflammatory secretome in the wound perpetuates defective neovascularization and contributes to dysregulated tissue repair. This study aims to design a gelatin methacrylamide (GelMA) hydrogel to sustained the release of grancalcin-neutralizing antibody (GCA-NAb) and evaluate it as a potential scaffold to promote diabetic wound healing. Results show that the expression of grancalcin(GCA), a protein secreted by bone marrow-derived immune cells, is elevated in the wound sites of individuals and animals with diabetic ulcers. Genetic inhibition of grancalcin expression accelerates vascularization and healing in an animal model. Mechanistic studies show that grancalcin binds to transient receptor potential melastatin 8(TRPM8) and partially inactivates its downstream signaling pathways, thereby impairing angiogenesis in vitro and ex vivo. Systemic or topical administration of a GCA-NAb accelerate wound repair in mice with diabetes. The data suggest that GCA is a potential therapeutic target for the treatment of diabetic ulcers.

A biophysically comprehensive model of urothelial afferent neurons: implications for sensory signalling in urinary bladder.

The urothelium is the innermost layer of the bladder wall; it plays a pivotal role in bladder sensory transduction by responding to chemical and mechanical stimuli. The urothelium also acts as a physical barrier between urine and the outer layers of the bladder wall. There is intricate sensory communication between the layers of the bladder wall and the neurons that supply the bladder, which eventually translates into the regulation of mechanical activity. In response to natural stimuli, urothelial cells release substances such as ATP, nitric oxide (NO), substance P, acetylcholine (ACh), and adenosine. These act on adjacent urothelial cells, myofibroblasts, and urothelial afferent neurons (UAN), controlling the contractile activity of the bladder. There is rising evidence on the importance of urothelial sensory signalling, yet a comprehensive understanding of the functioning of the urothelium-afferent neurons and the factors that govern it remains elusive to date. Until now, the biophysical studies done on UAN have been unable to provide adequate information on the ion channel composition of the neuron, which is paramount to understanding the electrical functioning of the UAN and, by extension, afferent signalling. To this end, we have attempted to model UAN to decipher the ionic mechanisms underlying the excitability of the UAN. In contrast to previous models, our model was built and validated using morphological and biophysical properties consistent with experimental findings for the UAN. The model included all the channels thus far known to be expressed in UAN, including; voltage-gated sodium and potassium channels, N, L, T, P/Q, R-type calcium channels, large-conductance calcium-dependent potassium (BK) channels, small conductance calcium-dependent (SK) channels, Hyperpolarisation activated cation (HCN) channels, transient receptor potential melastatin (TRPM8), transient receptor potential vanilloid (TRPV1) channel, calcium-activated chloride(CaCC) channels, and internal calcium dynamics. Our UAN model a) was constrained as far as possible by experimental data from the literature for the channels and the spiking activity, b) was validated by reproducing the experimental responses to current-clamp and voltage-clamp protocols c) was used as a base for modelling the non-urothelial afferent neurons (NUAN). Using our models, we also gained insights into the variations in ion channels between UAN and NUAN neurons.

Functional conservation of the cooling agent binding site in the transient receptor potential melastatin subfamily

Functional conservation of the cooling agent binding site in the transient receptor potential melastatin subfamily

Expression and function of transient receptor potential melastatin 3 in the spinal afferent innervation of the mouse colon.

Abdominal pain is a cardinal symptom of inflammatory bowel disease (IBD). Transient receptor potential (TRP) channels contribute to abdominal pain in preclinical models of IBD, and TRP melastatin 3 (TRPM3) has recently been implicated in inflammatory bladder and joint pain in rodents. We hypothesized that TRPM3 is involved in colonic sensation and is sensitized during colitis. We used immunohistochemistry, ratiometric Ca2+ imaging, and colonic afferent nerve recordings in mice to evaluate TRPM3 protein expression in colon-projecting dorsal root ganglion (DRG) neurons, as well as functional activity in DRG neurons and colonic afferent nerves. Colitis was induced using dextran sulfate sodium (DSS) in drinking water. TRPM3 protein expression was observed in 76% of colon-projecting DRG neurons and was often colocalized with calcitonin gene-related peptide. The magnitudes of intracellular Ca2+ transients in DRG neurons in response to the TRPM3 agonists CIM-0216 and pregnenolone sulfate sodium were significantly greater in neurons from mice with colitis compared with controls. In addition, the percentage of DRG neurons from mice with colitis that responded to CIM-0216 was significantly increased. CIM-0216 also increased the firing rate of colonic afferent nerves from control and mice with colitis. The TRPM3 inhibitor isosakuranetin inhibited the mechanosensitive response to distension of wide dynamic range afferent nerve units from mice with colitis but had no effect in control mice. Thus, TRPM3 contributes to colonic sensory transduction and may be a potential target for treating pain in IBD.NEW & NOTEWORTHY This is the first study to characterize TRPM3 protein expression and function in colon-projecting DRG neurons. A TRPM3 agonist excited DRG neurons and colonic afferent nerves from healthy mice. TRPM3 agonist responses in DRG neurons were elevated during colitis. Inhibiting TRPM3 reduced the firing of wide dynamic range afferent nerves from mice with colitis but had no effect in control mice.

Joint CB1 and NGF Receptor Activation Suppresses TRPM8 Activation in Etoposide-Resistant Retinoblastoma Cells.

In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca2+ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca2+ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca2+ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca2+ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk.

Effect of TRPM8 on pain symptom and neuronal proliferation in mice with interstitial cystitis/bladder pain syndrome

Objective: To explore the effects between transient receptor potential melastatin 8 (TRPM8) on pain symptom and neuronal proliferation in mice with interstitial cystitis/bladder pain syndrome (IC/BPS). Methods: Female wild-type C57BL/6 mice (8-10 weeks old) were divided into control group, IC/BPS model group, and IC/BPS model+menthol group (6 mice each) by random number table method; TRPM8 knockout mice were randomly divided into TRPM8 knockout group and TRPM8 knockout model group (6 mice each). The IC/BPS model group, the IC/BPS model+menthol group, and the TRPM8 knockout model group were injected subcutaneously with residues 65-84 of murine uroplakin 3A (UPK3A65-84). The IC/BPS model+menthol group continued to be injected with menthol. After successful modeling, the differences in pain thresholds between the groups were assessed by mechanosensitivity. The bladder wall was injected with a cell membrane red fluorescent probe (Dil), and the dorsal root ganglion (DRG) tissues were collected 10 days later. The differences in the protein and mRNA levels of TRPM8 and GAP43 in the groups were detected by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Immunofluorescence was used to detect the distribution of TRPM8 expression with GAP43 or Dil in DRG tissues. The relationship between TRPM8 and pain symptom and its role in neuronal proliferation in IC/BPS mice were analyzed. Results: The models were all constructed successfully. Compared with the control group, the pain thresholds of mice in the IC/BPS model group and IC/BPS model+menthol group were reduced [(8.50±1.22), (5.50±1.05) vs (11.67±2.16), respectively, all P<0.001]. Compared with the control group, the expression of TRPM8 mRNA was elevated in the IC/BPS model group and IC/BPS model+menthol group, while TRPM8 was not expressed in the TRPM8 knockout group [(3.16±0.05), (6.46±0.21), and 0 vs (1.00±0.06), respectively, all P<0.001]. The expression of TRPM8 protein and mRNA in each group had the same trend (P<0.001). Compared with the control group, the expression of GAP43 mRNA in the DRG of the IC/BPS model group and the IC/BPS model+menthol group was increased, whereas the expression of GAP43 mRNA in the TRPM8 knockout model group was decreased (all P<0.001). The trend of GAP43 protein expression was the same as that of mRNA expression (P<0.001). Immunofluorescence results showed an increase in the number of GAP43-positive neurons in the DRG of the IC/BPS model group and the IC/BPS model+menthol group, and a decrease in the TRPM8 knockout group compared with the control group (all P<0.001). Compared with the control group, the number of Dil-positive neurons in the DRG of the IC/BPS model group and the IC/BPS model+menthol group was increased, while the TRPM8 knockout group was decreased (all P<0.001). Conclusion: TRPM8 can exacerbate pain symptom in IC/BPS mice, and the mechanism may be related to the induction of sensory nerve proliferation at the DRG level.

TRPM8 inhibits substance P release from primary sensory neurons via PKA/GSK-3beta to protect colonic epithelium in colitis

Transient receptor potential melastatin 8 (TRPM8) is a cold sensory receptor in primary sensory neurons that regulates various neuronal functions. Substance P (SP) is a pro-inflammatory neuropeptide secreted by the neurons, and it aggravates colitis. However, the regulatory role of TRPM8 in SP release is still unclear. Our study aimed to investigate TRPM8’s role in SP release from primary sensory neurons during colitis and clarify the effect of SP on colonic epithelium. We analyzed inflammatory bowel disease patients’ data from the Gene Expression Omnibus dataset. Dextran sulfate sodium (DSS, 2.5%)-induced colitis in mice, mouse dorsal root ganglion (DRG) neurons, ND7/23 cell line, and mouse or human colonic organoids were used for this experiment. Our study found that TRPM8, TAC1 and WNT3A expression were significantly correlated with the severity of ulcerative colitis in patients and DSS-induced colitis in mice. The TRPM8 agonist (menthol) and the SP receptor antagonist (Aprepitant) can attenuate colitis in mice, but the effects were not additive. Menthol promoted calcium ion influx in mouse DRG neurons and inhibited the combination and phosphorylation of PKAca from the cAMP signaling pathway and GSK-3β from the Wnt/β-catenin signaling pathway, thereby inhibiting the effect of Wnt3a-driven β-catenin on promoting SP release in ND7/23 cells. Long-term stimulation with SP inhibited proliferation and enhanced apoptosis in both mouse and human colonic organoids. Conclusively, TRPM8 inhibits SP release from primary sensory neurons by inhibiting the interaction between PKAca and GSK-3β, thereby inhibiting the role of SP in promoting colonic epithelial apoptosis and relieving colitis.

Inhibition of TRPM8 function by prostacyclin receptor agonists requires coupling to Gq/11 proteins.

The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI2) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors. We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890. Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs. Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation.

Mouse transient receptor potential melastatin 2 (TRPM2) isoform 7 attenuates full-length mouse TRPM2 activity through reductions in its expression by targeting it to ER-associated degradation.

Transient receptor potential melastatin 2 (TRPM2) assembles into tetramers to function as an oxidative stress-sensitive Ca2+ channel at the surface membrane. Limited information is currently available on the 10 protein isoforms of mouse TRPM2 (mTRPM2) identified. This study investigated whether these isoforms function as Ca2+ channels and examined their effects on full-length mTRPM2 activity using the HEK 293 cell exogenous expression system. Only full-length mTRPM2, isoform 1 localized to the surface membrane and was activated by oxidative stress. Isoform 7 was clearly recognized by protein quality control systems and degraded by endoplasmic reticulum-associated degradation after transmembrane proteolysis. In the co-expression system, the activation and expression of full-length mTRPM2 were attenuated by its co-expression with isoform 7, but not with the other isoforms. This decrease in the expression of full-length mTRPM2 was recovered by the proteasomal inhibitor. The present results suggest that isoforms other than isoform 1 did not function as oxidative stress-sensitive channels and also that only isoform 7 attenuated the activation of full-length mTRPM2 by targeting it to endoplasmic reticulum-associated degradation. The present study will provide important information on the functional nature of mTRPM2 isoforms for the elucidation of their roles in physiological and patho-physiological responses in vivo using mouse models.

Corneal acetylcholine regulates sensory nerve activity via nicotinic receptors

PurposeSensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea. MethodsLocalization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [3H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs. ResultsChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [3H] acetylcholine was synthesized from [3H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist. ConclusionsThe corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation.

MiR-133a-3p/TRPM4 axis improves palmitic acid induced vascular endothelial injury.

Background: Vascular endothelial injury is a contributing factor to the development of atherosclerosis and the resulting cardiovascular diseases. One particular factor involved in endothelial cell apoptosis and atherosclerosis is palmitic acid (PA), which is a long-chain saturated fatty acid. In addition, transient receptor potential melastatin 4 (TRPM4), a non-selective cation channel, plays a significant role in endothelial dysfunction caused by various factors related to cardiovascular diseases. Despite this, the specific role and mechanisms of TRPM4 in atherosclerosis have not been fully understood. Methods: The protein and mRNA expressions of TRPM4, apoptosis - and inflammation-related factors were measured after PA treatment. The effect of TRPM4 knockout on the protein and mRNA expression of apoptosis and inflammation-related factors was detected. The changes of intracellular Ca2+, mitochondrial membrane potential, and reactive oxygen species were detected by Fluo-4 AM, JC-1, and DCFH-DA probes, respectively. To confirm the binding of miR-133a-3p to TRPM4, a dual luciferase reporter gene assay was conducted. Finally, the effects of miR-133a-3p and TRPM4 on intracellular Ca2+, mitochondrial membrane potential, and reactive oxygen species were examined. Results: Following PA treatment, the expression of TRPM4 increases, leading to calcium overload in endothelial cells. This calcium influx causes the assemblage of Bcl-2, resulting in the opening of mitochondrial calcium channels and mitochondrial damage, ultimately triggering apoptosis. Throughout this process, the mRNA and protein levels of IL-1β, ICAM-1, and VCAM1 significantly increase. Database screenings and luciferase assays have shown that miR-133a-3p preferentially binds to the 3'UTR region of TRPM4 mRNA, suppressing TRPM4 expression. During PA-induced endothelial injury, miR-133a-3p is significantly decreased, but overexpression of miR-133a-3p can attenuate the progression of endothelial injury. On the other hand, overexpression of TRPM4 counteracts the aforementioned changes. Conclusion: TRPM4 participates in vascular endothelial injury caused by PA. Therefore, targeting TRPM4 or miR-133a-3p may offer a novel pharmacological approach to preventing endothelial injury.

Severe inflammation in C57/BL6 mice leads to prolonged cognitive impairment by initiating the IL-1β/TRPM2 pathway

BackgroundSepsis could lead to chronic cognitive impairment by unclear molecular mechanisms. Transient receptor potential melastatin-2 (TRPM2) is essential against immunity-related activities and inflammation. Our study attempted to decipher the relationship between cognitive impairment caused by severe inflammation and TRPM2 expression levels. MethodsSevere inflammation was induced by intraperitoneally injecting C57/BL6 mice with a high dosage (5 mg kg−1) of Lipopolysaccharide (LPS). Fear conditioning and a Morris water maze test were performed to examine the cognitive abilities of the mice. Moreover, the signaling and expression of pro-inflammatory cytokines and TRPM2 were measured using Western blotting and Reverse transcription-polymerase chain reaction (RT-PCR). Flow cytometry and immunofluorescence staining helped to determine the astrocyte apoptosis rate. ResultsSevere inflammation can lead to long-term cognitive impairment in C57/BL6 mice. The interleukin-1 beta (IL-1β) levels intra-hippocampus were significantly elevated until P14 post-LPS introduction. At both P7 and P14, there is an up-regulation of TRPM2 expression within hippocampus. Administration of recombinant IL-1β to astrocytes results in a significant up-regulation of TRPM2 expression. IL-1β or TRPM2 level knockdown helped counter the cognitive impairment caused by significant inflammation. ConclusionsA continuous increase in IL-1β levels within the hippocampus can lead to cognitive impairment by enhancing TRPM2 levels caused by severe inflammation.

Bidirectional regulation mechanism of TRPM2 channel: role in oxidative stress, inflammation and ischemia-reperfusion injury.

Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that exhibits Ca2+ permeability. The TRPM2 channel is expressed in various tissues and cells and can be activated by multiple factors, including endogenous ligands, Ca2+, reactive oxygen species (ROS) and temperature. This article reviews the multiple roles of the TRPM2 channel in physiological and pathological processes, particularly on oxidative stress, inflammation and ischemia-reperfusion (I/R) injury. In oxidative stress, the excessive influx of Ca2+ caused by the activation of the TRPM2 channel may exacerbate cellular damage. However, under specific conditions, activating the TRPM2 channel can have a protective effect on cells. In inflammation, the activation of the TRPM2 channel may not only promote inflammatory response but also inhibit inflammation by regulating ROS production and bactericidal ability of macrophages and neutrophils. In I/R, the activation of the TRPM2 channel may worsen I/R injury to various organs, including the brain, heart, kidney and liver. However, activating the TRPM2 channel may protect the myocardium from I/R injury by regulating calcium influx and phosphorylating proline-rich tyrosine kinase 2 (Pyk2). A thorough investigation of the bidirectional role and regulatory mechanism of the TRPM2 channel in these physiological and pathological processes will aid in identifying new targets and strategies for treatment of related diseases.

Hypomagnesaemia leading to parathyroid dysfunction, hypocalcaemia, and hypokalaemia as a complication of long-term treatment with a proton pump inhibitor - a literature review.

Proton pump inhibitors (PPIs) are one of the most frequently used medications worldwide. The side effects of this class of drugs have been widely studied. However, their impact on the electrolyte balance is frequently forgotten. Long-term PPI administration can lead to profound electrolyte disturbances, namely hypomagnesaemia as well as, secondary to very low magnesium levels, hypocalcaemia and hypokalaemia. In this paper we comprehensively review the complexity of the mechanisms contributing to electrolyte imbalance following PPI (proton pump inhibitors) by changing the pH in the intestinal lumen, interfering with the active cellular transport of magnesium regulated by the transient receptor potential melastatin cation channels TRPM6 and TRPM7. The accompanying hypomagnesaemia causes unblocking of the renal outer medullary potassium channel (ROMK), which results in increased potassium loss in the ascending limb of the loop of Henle. Hypokalaemia caused by hypomagnesaemia is resistant to potassium supplementation because the loss of this element in urine increases with the supply of potassium. Additionally, within the calcium-sensitive receptor (CASR), dissociation of magnesium from the alpha subunit of G protein caused by hypomagnesaemia increases its activity, leading to inhibition of PTH secretion and hypocalcaemia resistant to calcium supplementation. All this means that in some patients, chronic use of proton pump inhibitors by affecting the absorption of magnesium, may lead to life-threatening electrolyte disorders.

TRPM8 knockdown relieved inflammatory response and cell apoptosis in pneumonia model induced by Streptococcus pneumoniae in vitro.

Streptococcus pneumoniae infection remains a leading cause ofpneumonia-related deaths. Transient receptor potential melastatin 8 (TRPM8) exerts crucial roles in lung diseases. We first dissected the role of TRPM8 in pneumococcalpneumonia and the mechanism related to TRPM8 effects. TRPM8 expression and inflammation cytokines level were determined in 15 paired patients and controls. A549 cells were pretreated with si-TRPM8, followed by infection with S. pneumoniae D39 strain (D39). TRPM8 expressions in D39-treated cells were detected and the effect of TRPM8 inhibition on the viability, apoptosis, and inflammation induced by D39 was evaluated. To explore the mechanism underlying TRPM8 effects, cells in D39+si-TRPM8 group were further treated with MAPK activator (Anisomycin, ANIS). TRPM8 was highly expressed in patients and cell models at mRNA or/and protein levels. Cytokines of TNF-α, IL-1β and IL-6 were intensely upregulated in the serum samples of patients and cells infected with D39 (p<0.05). TRPM8 knockdown attenuated the reduced cell viability and increased cell apoptosis (reflected by the upregulation of Bax and downregulation of Bcl-2) in D39 group (p<0.05). The expression level of inflammation cytokines was lower in D39+si-TRPM8 group than D39 group (p<0.05). The protein levels of NF-κB p-p65 and p-p38 MAPK were intensely accumulated in D39 treated cells, while reduced by TRPM8 inhibition (p<0.05). ANIS addition significantly attenuated the altered cell viability, cell apoptosis and inflammation response in D39+si-TRPM8 group (p<0.05). TRPM8 knockdown relieved D39 infection-caused inflammation and cell apoptosis via NF-κB/MAPK signaling.

Design, synthesis, and biological evaluation of 2-(naphthalen-1-yloxy)-N-phenylacetamide derivatives as TRPM4 inhibitors for the treatment of prostate cancer

Transient receptor potential melastatin 4 (TRPM4) is considered to be a potential target for cancer and other human diseases. Herein, a series of 2-(naphthalen-1-yloxy)-N-phenylacetamide derivatives were designed and synthesized as new TRPM4 inhibitors, aiming to improve cellular potency. One of the most promising compounds, 7d (ZX08903), displayed promising antiproliferative activity against prostate cancer cell lines. 7d also suppressed colony formation and the expression of androgen receptor (AR) protein in prostate cancer cells. Furthermore, 7d can concentration-dependently induce cell apoptosis in prostate cancer cells. Collectively, these findings indicated that compound 7d may serve as a promising lead compound for further anticancer drug development.