(S)-ML-SA1 Activates Autophagy via TRPML1-TFEB Pathway.

Autophagic flux plays a crucial role in various diseases. Recently, the lysosomal ion channel TRPML1 has emerged as a promising target in lysosomal storage diseases, such as mucolipidosis. The discovery of mucolipin synthetic agonist-1 (ML-SA1) has expanded our understanding of TRPML1's function and its potential therapeutic uses. However, ML-SA1 is a racemate with limited cellular potency and poor water solubility. In this study, we synthetized rac-ML-SA1, separated the enantiomers by chiral liquid chromatography and determined their absolute configuration by vibrational circular dichroism (VCD). In addition, we focused on investigating the impact of each enantiomer of ML-SA1 on the TRPML1-TFEB axis. Our findings revealed that (S)-ML-SA1 acts as an agonist for TRPML1 at the lysosomal membrane. This activation prompts transcription factor EB (TFEB) to translocate from the cytosol to the nucleus in a dose-dependent manner within live cells. Consequently, this signaling pathway enhances the expression of coordinated lysosomal expression and regulation (CLEAR) genes and activates autophagic flux. Our study presents evidence for the potential use of (S)-ML-SA1 in the development of new therapies for lysosomal storage diseases that target TRPML1.

Role of TRPA1 in the pharmacological effect triggered by the topical application of trans-anethole in mice.

PURPOSE: This study investigated the pharmacological effects of topical trans-anethole, a natural compound found in anise, star anise, and fennel essential oils, and its relationship with the transient receptor potential of ankyrin 1 (TRPA1). METHODS: The effects of topical anethole were assessed by eye wiping, nociceptive behaviour, and ear oedema in mice. Histological evaluations were performed on the ears of the animals topically treated with anethole. RESULTS: Anethole caused less eye irritation than capsaicin (a TRPV1 agonist) and allyl isothiocyanate (a TRPA1 agonist). Anethole (250 and 500 nmol/20 µL/paw) promoted neurogenic nociception in the paw (20.89 ± 3.53 s and 47.56 ± 8.46 s, respectively) compared with the vehicle (0.88 ± 0.38 s). HC030031 (56.1 nmol/20 µL/paw), a TRPA1 antagonist, abolished this nociceptive response. Anethole (4, 10, and 20 µmol/20 µL/ear) induced ear oedema (30.25 ± 4.78 µm, 78.00 ± 3.74 µm, and 127.50 ± 27.19 µm, respectively) compared with the vehicle (5.00 ± 0.5 µm). HC030031 (56.1 nmol/20 µL/ear) inhibited the oedema induced by anethole (10 µmol/20 µL/ear). Ears pre-treated with anethole or allyl isothiocyanate on the first day and re-exposed to these compounds on the third day showed a reduction in oedema (68.16 ± 6.04% and 38.81 ± 8.98.9%, respectively). Cross-desensitisation between anethole and allyl isothiocyanate was observed. Histological analysis confirmed the beneficial effects of anethol. CONCLUSION: As repeated topical applications of anethole induce the desensitisation of TRPA1, we suggest its clinical application as a topical formulation for treating skin diseases or managing pain associated with this receptor. Anethole may also have advantages over capsaicin and allyl isothiocyanate because of its low pungency and pleasant aroma.

TRPC5 channel participates in myocardial injury in chronic intermittent hypoxia.

OBJECTIVE: The purpose of this study is to develop an animal model of Chronic Intermittent Hypoxia (CIH) and investigate the role of the TRPC5 channel in cardiac damage in OSAHS rats. METHODS: Twelve male Sprague Dawley rats were randomly divided into the CIH group and the Normoxic Control (NC) group. Changes in structure, function, and pathology of heart tissue were observed through echocardiography, transmission electron microscopy, HE-staining, and TUNEL staining. RESULTS: The Interventricular Septum thickness at diastole (IVSd) and End-Diastolic Volume (EDV) of rats in the CIH group significantly increased, whereas the LV ejection fraction and LV fraction shortening significantly decreased. TEM showed that the myofilaments in the CIH group were loosely arranged, the sarcomere length varied, the cell matrix dissolved, the mitochondrial cristae were partly flocculent, the mitochondrial outer membrane dissolved and disappeared, and some mitochondria were swollen and vacuolated. The histopathological examination showed that the cardiomyocytes in the CIH group were swollen with granular degeneration, some of the myocardial fibers were broken and disorganized, and most of the nuclei were vacuolar and hypochromic. CONCLUSION: CIH promoted oxidative stress, the influx of Ca2+, and the activation of the CaN/NFATc signaling pathway, which led to pathological changes in the morphology and ultrastructure of cardiomyocytes, the increase of myocardial apoptosis, and the decrease of myocardial contractility. These changes may be associated with the upregulation of TRPC5.

NCS-1 protein regulates TRPA1 channel through the PI3K pathway in breast cancer and neuronal cells.

The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.

Modulation of TRPV1 on Odontoblast-like Cells Using Capsazepine-Loaded Nanogels.

The modulation of TRPV1 emerges as a promising strategy for dental pain management. This study aimed to assess TRPV1 modulation in a human odontoblast-like cell model using Capsazepine (CZP) loaded in a nanogel delivery system. Gelatin nanogels, synthesized via the emulsification-gelation technique, were characterized and loaded with the TRPV1 antagonist, CZP. HPLC determined a remarkable 67.5 ± 0.04% CZP loading efficiency, with 71.7% of nanogels falling within the 300-950 nm size range, as evidenced by light microscopy. Moreover, CZP-loaded nanogels had a low cytotoxicity. An FTIR analysis showed no adverse chemical interactions, ensuring stability and active release. When examining biological responses, TRPV1 expression and channel activity were assessed in odontoblast-like cells. On the fifth day post-treatment, cells treated with CZP-loaded nanogels exhibited an increased TRPV1 expression and a reduction in calcium fluxes after agonist stimulus (F/F0 ratio 1.18 ± 0.18), resembling the response in free CZP-treated cells (1.28 ± 0.15). A two-way analysis of variance and the Tukey's test were used to determine statistical significance (p < 0.05). This delivery system, proven to be economical and straightforward, holds promise for dental pain management and potential local use. Local administration minimizes systemic adverse effects, making it a practical solution for releasing molecules in the oral cavity.

G Protein-Coupled Receptors and Ion Channels Involvement in Cisplatin-Induced Peripheral Neuropathy: A Review of Preclinical Studies.

Cisplatin is a platinum-based chemotherapy drug widely used to treat various solid tumours. Although it is effective in anti-cancer therapy, many patients develop peripheral neuropathy during and after cisplatin treatment. Peripheral neuropathy results from lesions or diseases in the peripheral somatosensory nervous system and is a significant cause of debilitation and suffering in patients. In recent years, preclinical studies have been conducted to elucidate the mechanisms involved in chemotherapy-induced peripheral neuropathic pain, as well as to promote new therapeutic targets since current treatments are ineffective and are associated with adverse effects. G-protein coupled receptors and ion channels play a significant role in pain processing and may represent promising targets for improving the management of cisplatin-induced neuropathic pain. This review describes the role of G protein-coupled receptors and ion channels in cisplatin-induced pain, analysing preclinical experimental studies that investigated the role of each receptor subtype in the modulation of cisplatin-induced pain.

TRPC5 channel participates in myocardial injury in chronic intermittent hypoxia

Abstract Objective The purpose of this study is to develop an animal model of Chronic Intermittent Hypoxia (CIH) and investigate the role of the TRPC5 channel in cardiac damage in OSAHS rats. Methods Twelve male Sprague Dawley rats were randomly divided into the CIH group and the Normoxic Control (NC) group. Changes in structure, function, and pathology of heart tissue were observed through echocardiography, transmission electron microscopy, HE-staining, and TUNEL staining. Results The Interventricular Septum thickness at diastole (IVSd) and End-Diastolic Volume (EDV) of rats in the CIH group significantly increased, whereas the LV ejection fraction and LV fraction shortening significantly decreased. TEM showed that the myofilaments in the CIH group were loosely arranged, the sarcomere length varied, the cell matrix dissolved, the mitochondrial cristae were partly flocculent, the mitochondrial outer membrane dissolved and disappeared, and some mitochondria were swollen and vacuolated. The histopathological examination showed that the cardiomyocytes in the CIH group were swollen with granular degeneration, some of the myocardial fibers were broken and disorganized, and most of the nuclei were vacuolar and hypochromic. Conclusion CIH promoted oxidative stress, the influx of Ca2+, and the activation of the CaN/NFATc signaling pathway, which led to pathological changes in the morphology and ultrastructure of cardiomyocytes, the increase of myocardial apoptosis, and the decrease of myocardial contractility. These changes may be associated with the upregulation of TRPC5.

Scorpion Peptides and Ion Channels: An Insightful Review of Mechanisms and Drug Development.

The Buthidae family of scorpions consists of arthropods with significant medical relevance, as their venom contains a diverse range of biomolecules, including neurotoxins that selectively target ion channels in cell membranes. These ion channels play a crucial role in regulating physiological processes, and any disturbance in their activity can result in channelopathies, which can lead to various diseases such as autoimmune, cardiovascular, immunological, neurological, and neoplastic conditions. Given the importance of ion channels, scorpion peptides represent a valuable resource for developing drugs with targeted specificity for these channels. This review provides a comprehensive overview of the structure and classification of ion channels, the action of scorpion toxins on these channels, and potential avenues for future research. Overall, this review highlights the significance of scorpion venom as a promising source for discovering novel drugs with therapeutic potential for treating channelopathies.

A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

The transient receptor potential melastatin 2: a new therapeutical target for Parkinson's disease?

The transient receptor potential melastatin 2 is a calcium-permeable cation channel member of the TRP family. Also known as an oxidative stress-activated channel, the transient receptor potential melastatin 2 gating mechanism is dependent on reactive oxygen species. In pathological conditions, transient receptor potential melastatin 2 is overactivated, leading to a Ca2+ influx that alters cell homeostasis and promotes cell death. The role of transient receptor potential melastatin 2 in neurodegenerative diseases, including Alzheimer's disease and ischemia, has already been described and reviewed. However, data on transient receptor potential melastatin 2 involvement in Parkinson's disease pathology has emerged only in recent years and the issue lacks review studies that focus specifically on this topic. The present review aims to elucidate the role of the transient receptor potential melastatin 2 channel in Parkinson's disease by reviewing, summarizing, and discussing the in vitro, in vivo, and human studies published until August 2022. Here we describe fourteen studies that evaluated the transient receptor potential melastatin 2 channel in Parkinson's disease. The Parkinson's disease model used, transient receptor potential melastatin 2 antagonist and genetic approaches, and the main outcomes reported were discussed. The studies described transient receptor potential melastatin 2 activation and enhanced expression in different Parkinson's disease models. They also evidenced protective and restorative effects when using transient receptor potential melastatin 2 antagonists, knockout, or silencing. This review provides a literature overview and suggests where there is a need for more research. As a perspective point, this review shows evidence that supports transient receptor potential melastatin 2 as a pharmacological target for Parkinson's disease in the future.

Capsazepine antagonizes TRPV1 activation induced by thermal and osmotic stimuli in human odontoblast-like cells.

Objectives: Dental pain, which is the main reason for patients consulting dentists, is classified as a public health concern. The study of cellular and molecular mechanisms contributing to pain is a fundamental element for developing new analgesics. By using a selective antagonist in an in vitro model, this study aimed to establish the role of TRPV-1 in human odontoblast-like cells (OLCs) as a therapeutic target for dental pain mediated by noxious thermal and osmotic stimuli. Methods: OLCs were differentiated from dental pulp mesenchymal cells and TRPV1 expression was evaluated. Activation of TRPV-1 was determined by evaluating changes in calcium concentration after stimulation with mannitol and xylitol hyperosmotic solutions or DMEM heated at 45 °C, using the fluorescent calcium probe Fluo-4 AM. In addition, changes in fluorescence (F/F0) due to calcium flux were evaluated using fluorometry and flow cytometry. Simultaneously, the cells were co-stimulated with the selective antagonist capsazepine (CZP). Results: OLCs expressed DSPP and DMP-1, confirming their cellular phenotype. TRPV1 was expressed, and its activation by different stimuli produced an increase in cytosolic Ca2+ which was reduced by the antagonist. Both methods used to evaluate TRPV1 activation through the measurement of calcium probe fluorescence showed similar patterns. Conclusions: These results suggest that TRPV-1 modulation using an antagonist can be implemented as a pharmacological strategy for managing dental pain mediated by hyperosmotic and thermal stimuli.

Paclitaxel Regulates TRPA1 Function and Expression Through PKA and PKC.

Paclitaxel (PTX) is a frequently used anticancer drug that causes peripheral neuropathy. Transient receptor potential ankyrin 1 (TRPA1), a plasma membrane calcium channel, has been associated with PTX toxicity and with other chemotherapy agents such as oxaliplatin and vincristine. However, the effect of PTX on the functional expression and calcium currents of TRPA1 has not been determined. The present study shows the effect of PTX on TRPA1 activity in a neuronal cell line (SH-SY5Y). The effect of PTX on the expression of TRPA1 was assessed through quantitative PCR and Western blot analyses to determine the relative mRNA and protein expression levels. To assess the effect on calcium flux and currents, cells were exposed to PTX; simultaneously, a specific agonist and antagonist of TRPA1 were added to evaluate the differential response in exposed versus control cells. To assess the effect of PKA, PKC and PI3K on PTX-induced TRPA1 increased activity, selective inhibitors were added to these previous experiments. PTX increased the mRNA and protein expression of TRPA1 as well as the TRPA1-mediated Ca2+ currents and intracellular Ca2+ concentrations. This effect was dependent on AITC (a selective specific agonist) and was abolished with HC-030031 (a selective specific antagonist). The inhibition of PKA and PKC reduced the effect of PTX on the functional expression of TRPA1, whereas the inhibition of PI3K had no effects. PTX-induced neuropathy involves TRPA1 activity through an increase in functional expression and is regulated by PKA and PKC signaling. These findings support the role of the TRPA1 channel in the mechanisms altered by PTX, which can be involved in the process that lead to chemotherapy-induced neuropathy.

Disruption of Atrial Rhythmicity by the Air Pollutant 1,2-Naphthoquinone: Role of Beta-Adrenergic and Sensory Receptors.

The combustion of fossil fuels contributes to air pollution (AP), which was linked to about 8.79 million global deaths in 2018, mainly due to respiratory and cardiovascular-related effects. Among these, particulate air pollution (PM2.5) stands out as a major risk factor for heart health, especially during vulnerable phases. Our prior study showed that premature exposure to 1,2-naphthoquinone (1,2-NQ), a chemical found in diesel exhaust particles (DEP), exacerbated asthma in adulthood. Moreover, increased concentration of 1,2-NQ contributed to airway inflammation triggered by PM2.5, employing neurogenic pathways related to the up-regulation of transient receptor potential vanilloid 1 (TRPV1). However, the potential impact of early-life exposure to 1,2-naphthoquinone (1,2-NQ) on atrial fibrillation (AF) has not yet been investigated. This study aims to investigate how inhaling 1,2-NQ in early life affects the autonomic adrenergic system and the role played by TRPV1 in these heart disturbances. C57Bl/6 neonate male mice were exposed to 1,2-NQ (100 nM) or its vehicle at 6, 8, and 10 days of life. Early exposure to 1,2-NQ impairs adrenergic responses in the right atria without markedly affecting cholinergic responses. ECG analysis revealed altered rhythmicity in young mice, suggesting increased sympathetic nervous system activity. Furthermore, 1,2-NQ affected ß1-adrenergic receptor agonist-mediated positive chronotropism, which was prevented by metoprolol, a ß1 receptor blocker. Capsazepine, a TRPV1 blocker but not a TRPC5 blocker, reversed 1,2-NQ-induced cardiac changes. In conclusion, neonate mice exposure to AP 1,2-NQ results in an elevated risk of developing cardiac adrenergic dysfunction, potentially leading to atrial arrhythmia at a young age.

Scorpion peptides and ion channels: an insightful review of mechanisms and drug development

The Buthidae family of scorpions consists of arthropods with significant medical relevance, as their venom contains a diverse range of biomolecules, including neurotoxins that selectively target ion channels in cell membranes. These ion channels play a crucial role in regulating physiological processes, and any disturbance in their activity can result in channelopathies, which can lead to various diseases such as autoimmune, cardiovascular, immunological, neurological, and neoplastic conditions. Given the importance of ion channels, scorpion peptides represent a valuable resource for developing drugs with targeted specificity for these channels. This review provides a comprehensive overview of the structure and classification of ion channels, the action of scorpion toxins on these channels, and potential avenues for future research. Overall, this review highlights the significance of scorpion venom as a promising source for discovering novel drugs with therapeutic potential for treating channelopathies.

A journey from molecule to physiology and in silico tools for drug discovery targeting the transient receptor potential vanilloid type 1 (TRPV1) channel.

The heat and capsaicin receptor TRPV1 channel is widely expressed in nerve terminals of dorsal root ganglia (DRGs) and trigeminal ganglia innervating the body and face, respectively, as well as in other tissues and organs including central nervous system. The TRPV1 channel is a versatile receptor that detects harmful heat, pain, and various internal and external ligands. Hence, it operates as a polymodal sensory channel. Many pathological conditions including neuroinflammation, cancer, psychiatric disorders, and pathological pain, are linked to the abnormal functioning of the TRPV1 in peripheral tissues. Intense biomedical research is underway to discover compounds that can modulate the channel and provide pain relief. The molecular mechanisms underlying temperature sensing remain largely unknown, although they are closely linked to pain transduction. Prolonged exposure to capsaicin generates analgesia, hence numerous capsaicin analogs have been developed to discover efficient analgesics for pain relief. The emergence of in silico tools offered significant techniques for molecular modeling and machine learning algorithms to indentify druggable sites in the channel and for repositioning of current drugs aimed at TRPV1. Here we recapitulate the physiological and pathophysiological functions of the TRPV1 channel, including structural models obtained through cryo-EM, pharmacological compounds tested on TRPV1, and the in silico tools for drug discovery and repositioning.

Endocannabinoids Modulate Production of Osteoclastogenic Factors by Stem Cells of the Apical Papilla In Vitro.

INTRODUCTION: Many mediators are produced during pulp inflammation and necrosis, including endocannabinoids (ECbs), which might affect the function of stem cells of the apical papilla (SCAP), cells of paramount importance for root formation, and regenerative endodontic treatment. The aim of this study was to evaluate the production of osteoclastogenesis-related mediators by SCAP modulated by ECbs and lipopolysaccharide (LPS) in vitro. METHODS: SCAP were cultured and treated with ECb anandamide (AEA), 2-arachidonoylglycerol, or N-arachidonoylaminophenol. All groups were incubated in the presence of a vehicle or LPS and the antagonist of transient receptor potential cation channel subfamily V member 1, capsazepine. After 24 hours, the culture medium supernatants were collected for further quantification of tumor necrosis factor alpha, CCL2, macrophage colony-stimulating factor, osteoprotegerin, and receptor activator of nuclear factor kappa B ligand. RESULTS: Small amounts of tumor necrosis factor alpha and receptor activator of nuclear factor kappa B ligand were detected in SCAP supernatants, and none of the experimental conditions altered their production. A down-regulation in constitutive CCL2 production was observed in the AEA group compared with that in the LPS group. The production of macrophage colony-stimulating factor was significantly increased in all groups treated with AEA compared with the control and LPS-treated groups. Osteoprotegerin was significantly increased by AEA alone and by 2-arachidonoylglycerol and N-arachidonoylaminophenol in the presence of LPS and capsazepine. CONCLUSIONS: AEA modulates some of the osteoclastogenic factors produced by SCAP in a bone resorption protective fashion.

The controversial role of the vagus nerve in mediating ghrelin's actions: gut feelings and beyond.

Ghrelin is a stomach-derived peptide hormone that acts via the growth hormone secretagogue receptor (GHSR) and displays a plethora of neuroendocrine, metabolic, autonomic and behavioral actions. It has been proposed that some actions of ghrelin are exerted via the vagus nerve, which provides a bidirectional communication between the central nervous system and peripheral systems. The vagus nerve comprises sensory fibers, which originate from neurons of the nodose and jugular ganglia, and motor fibers, which originate from neurons of the medulla. Many anatomical studies have mapped GHSR expression in vagal sensory or motor neurons. Also, numerous functional studies investigated the role of the vagus nerve mediating specific actions of ghrelin. Here, we critically review the topic and discuss the available evidence supporting, or not, a role for the vagus nerve mediating some specific actions of ghrelin. We conclude that studies using rats have provided the most congruent evidence indicating that the vagus nerve mediates some actions of ghrelin on the digestive and cardiovascular systems, whereas studies in mice resulted in conflicting observations. Even considering exclusively studies performed in rats, the putative role of the vagus nerve in mediating the orexigenic and growth hormone (GH) secretagogue properties of ghrelin remains debated. In humans, studies are still insufficient to draw definitive conclusions regarding the role of the vagus nerve mediating most of the actions of ghrelin. Thus, the extent to which the vagus nerve mediates ghrelin actions, particularly in humans, is still uncertain and likely one of the most intriguing unsolved aspects of the field.

Inhibition of TRPM2 by AG490 Is Neuroprotective in a Parkinson's Disease Animal Model.

Parkinson's disease (PD) is characterized by motor impairment and dopaminergic neuronal loss. There is no cure for the disease, and treatments have several limitations. The transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel, has been reported to be upregulated in neuronal death. However, there are no in vivo studies evaluating TRPM2's role and neuroprotective effects in PD. Here, we test the hypothesis that TRPM2 is upregulated in the 6-hydroxydopamine (6-OHDA) mouse model of PD and that its inhibition, by the AG490, is neuroprotective. For that, AG490 or vehicle were intraperitoneally administered into C57BL/6 mice. Mice then received 6-OHDA into the right striatum. Motor behavior assessments were evaluated 6, 13, and 20 days after surgery using the cylinder and apomorphine-induced rotational testes, and 7, 14, and 21 days after surgery using rotarod test. Brain samples of substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry and immunoblotting on days 7 and 21. We showed that TRPM2 protein expression was upregulated in 6-OHDA-treated animals. In addition, AG490 prevented dopaminergic neuron loss, microglial activation, and astrocyte reactivity in 6-OHDA-treated animals. The compound improved motor behaviors and Akt/GSK-3ß/caspase-3 signaling. We conclude that TRPM2 inhibition by AG490 is neuroprotective in the 6-OHDA model and that the TRPM2 channel may represent a potential therapeutic target for PD.

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

Endocannabinoids Regulate Stem Cells of the Apical Papilla via a Cannabinoid Receptor and TRPV1-Independent Mechanism.

INTRODUCTION: Endogenous cannabinoids (endocannabinoids [eCBs]) have been shown to have a multitude of functions including neurotransmission and immune modulatory effects. This study aimed to evaluate if stem cells of the apical papilla (SCAP) express the receptors and enzymes of the endocannabinoid system (ECS) and whether eCBs regulate their proliferation and mineralization potential. METHODS: Gene expression of the main components of the ECS and transient receptor potential vanilloid 1 (TRPV1) was evaluated in SCAP cultures. SCAP were treated with 2 concentrations of eCBs and/or capsazepine, a TRPV1 antagonist. SCAP viability was evaluated after 1, 4, and 7 days. Osteogenic differentiation was assessed after 14 days, and the gene expression of mineralization markers was assessed after 7 days. RESULTS: The enzymes of ECS and TRPV1 but not the cannabinoid receptors (cannabinoid receptors 1 and 2) were expressed in SCAP. Anandamide, 2-arachidonoylglycerol, and N-arachidonoylphenolamine (AM-404) reduced SCAP viability in all experimental periods at the highest concentration compared with the group with no treatment. Anandamide and AM-404 did not inhibit SCAP differentiation potential, but 2-arachidonoylglycerol at the highest concentration did. SCAP treated with AM-404 presented a down-regulation in gene expression of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSPP) compared with the proliferation medium group but not with control group. CONCLUSIONS: SCAP expressed the genes of the main components of ECS and TRPV1, and eCBs can affect SCAP viability, mineralization, and gene expression.