RESUMO
The transient receptor potential cation channel, subfamily M, members 6 and 7 (TRPM6 and TRPM7) are homologous membrane proteins encompassing cation channel units fused to cytosolic serine/threonine-protein kinase domains. Clinical studies and experiments with animal disease models suggested that selective inhibition of TRPM6 and TRPM7 currents might be beneficial for subjects with immune and cardiovascular disorders, tumours and other pathologies, but the suitable pharmacological toolkit remains underdeveloped. The present study identified small synthetic molecules acting specifically on the channel moieties of TRPM6 and TRPM7. Using electrophysiological analysis in conjunction with Ca2+ imaging, we show that iloperidone and ifenprodil inhibit the channel activity of recombinant TRPM6 with IC50 values of 0.73 and 3.33 µM, respectively, without an impact on the TRPM7 channel. We also found that VER155008 suppresses the TRPM7 channel with an IC50 value of 0.11 µM but does not affect TRPM6. Finally, the effects of iloperidone and VER155008 were found to be suitable for blocking native endogenous TRPM6 and TRPM7 in a collection of mouse and human cell models. Hence, the identification of iloperidone, ifenprodil, and VER155008 allows for the first time to selectively manipulate TRPM6 and TRPM7 currents.
Assuntos
Canais de Cátion TRPM , Animais , Humanos , Isoxazóis/farmacologia , Magnésio/metabolismo , Camundongos , Piperidinas/farmacologia , Proteínas Serina-Treonina Quinases , Nucleosídeos de Purina/farmacologia , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating multi-systemic chronic condition of unknown aetiology classified as an immune dysfunction syndrome and neurological disorder. The discovery of the widely expressed Transient Receptor Potential Melastatin 3 (TRPM3) as a nociceptor channel substantially targeted by certain opioid receptors, and its implication in calcium (Ca2+)-dependent Natural Killer (NK) cell immune functions has raised the possibility that TRPM3 may be pharmacologically targeted to treat characteristic symptoms of ME/CFS. Naltrexone hydrochloride (NTX) acts as an antagonist to the mu (µ)-opioid receptor thus negating its inhibitory function on TRPM3. Based on the benefits reported by patients on their symptoms, low dose NTX (LDN, 3.0-5.0â mg/day) treatment seems to offer some potential benefit suggesting that its effect may be targeted towards the pathomechanism of ME/CFS. As there is no literature confirming the efficacy of LDN for ME/CFS patients in vitro, this study investigates the potential therapeutic effect of LDN in ME/CFS patients. TRPM3 ion channel activity was measured after modulation with Pregnenolone sulfate (PregS) and ononetin in NK cells on 9 ME/CFS patients taking LDN and 9 age- and sex-matched healthy controls using whole-cell patch-clamp technique. We report that ME/CFS patients taking LDN have restored TRPM3-like ionic currents in NK cells. Small ionic currents with a typical TRPM3-like outward rectification were measured after application of PregS, a TRPM3-agonist, in NK cells from patients taking LDN. Additionally, PregS-evoked ionic currents through TRPM3 were significantly modulated by ononetin, a TRPM3-antagonist, in NK cells from ME/CFS patients taking LDN. These data support the hypothesis that LDN may have potential as a treatment for ME/CFS by characterising the underlying regulatory mechanisms of LDN treatment involving TRPM3 and opioid receptors in NK cells. Finally, this study may serve for the repurpose of marketed drugs, as well as support the approval of prospective randomized clinical studies on the role and dose of NTX in treating ME/CFS patients.
Assuntos
Síndrome de Fadiga Crônica/tratamento farmacológico , Células Matadoras Naturais/efeitos dos fármacos , Naltrexona/administração & dosagem , Antagonistas de Entorpecentes/administração & dosagem , Receptores Opioides mu/antagonistas & inibidores , Canais de Cátion TRPM/efeitos dos fármacos , Adulto , Sinalização do Cálcio/efeitos dos fármacos , Estudos de Casos e Controles , Reposicionamento de Medicamentos , Síndrome de Fadiga Crônica/imunologia , Síndrome de Fadiga Crônica/metabolismo , Síndrome de Fadiga Crônica/fisiopatologia , Feminino , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Pessoa de Meia-Idade , Naltrexona/efeitos adversos , Antagonistas de Entorpecentes/efeitos adversos , Receptores Opioides mu/metabolismo , Canais de Cátion TRPM/metabolismo , Resultado do TratamentoRESUMO
Oxaliplatin, a platinum-based chemotherapeutic drug, which is used as first-line treatment for some types of colorectal carcinoma, causes peripheral neuropathic pain in patients. In addition, an acute peripheral pain syndrome develop in almost 90% of patients immediately after oxaliplatin treatment, which is poorly understood mechanistically but correlates with incidence and severity of the later-occurring neuropathy. Here we investigated the effects of acute oxaliplatin treatment in a murine model, showing that male and female mice develop mechanical hypersensitivity 24 h after oxaliplatin treatment. Interestingly, we found that the levels of several lipids were significantly altered in nervous tissue during oxaliplatin-induced acute pain. Specifically, the linoleic acid metabolite 9,10-EpOME (epoxide of linoleic acid) as well as the lysophospholipids lysophosphatidylcholine (LPC) 18:1 and LPC 16:0 were significantly increased 24 h after oxaliplatin treatment in sciatic nerve, DRGs, or spinal cord tissue as revealed by untargeted and targeted lipidomics. In contrast, inflammatory markers including cytokines and chemokines, ROS markers, and growth factors are unchanged in the respective nervous system tissues. Importantly, LPC 18:1 and LPC 16:0 can induce Ca2+ transients in primary sensory neurons, and we identify LPC 18:1 as a previously unknown endogenous activator of the ligand-gated calcium channels transient receptor potential V1 and M8 (transient receptor potential vanilloid 1 and transient receptor potential melastatin 8) in primary sensory neurons using both pharmacological inhibition and genetic knockout. Additionally, a peripheral LPC 18:1 injection was sufficient to induce mechanical hypersensitivity in naive mice. Hence, targeting signaling lipid pathways may ameliorate oxaliplatin-induced acute peripheral pain and the subsequent long-lasting neuropathy.SIGNIFICANCE STATEMENT The first-line cytostatic drug oxaliplatin can cause acute peripheral pain and chronic neuropathic pain. The former is causally connected with the chronic neuropathic pain, but its mechanisms are poorly understood. Here, we performed a broad unbiased analysis of cytokines, chemokines, growth factors, and â¼200 lipids in nervous system tissues 24 h after oxaliplatin treatment, which revealed a crucial role of lysophospholipids lysophosphatidylcholine (LPC) 18:1, LPC 16:0, and 9,10-EpOME in oxaliplatin-induced acute pain. We demonstrate for the first time that LPC 18:1 contributes to the activation of the ion channels transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 in sensory neurons and causes mechanical hypersensitivity after peripheral injection in vivo These findings suggest that the LPC-mediated lipid signaling is involved in oxaliplatin-induced acute peripheral pain.
Assuntos
Antineoplásicos , Lisofosfolipídeos , Oxaliplatina , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/fisiopatologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Hiperalgesia/induzido quimicamente , Ácido Linoleico , Lipidômica , Lisofosfatidilcolinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor/induzido quimicamente , Dor/psicologia , Doenças do Sistema Nervoso Periférico/psicologia , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPV/efeitos dos fármacosRESUMO
Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL® cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL® cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Mentol/efeitos adversos , Doenças Respiratórias/induzido quimicamente , Aerossóis , Antioxidantes/metabolismo , Cálcio/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Canais de Cátion TRPM/biossíntese , Canais de Cátion TRPM/efeitos dos fármacosRESUMO
After almost 50 years of praziquantel (PZQ) research, Park and Marchant (Trends Parasitol 36:182-194, 2020) described the Ca++-permeable transient receptor potential (TRP) channel Sm.TRPMPZQ in Schistosoma mansoni as target of PZQ. Here we describe the deadly cascade in schistosomes which is induced by the (R)-PZQ enantiomer that includes contemporaneous stereoselective activation of Sm.TRPMPZQ-mediated Ca++ influx, disturbed Ca++ homeostasis, Ca++-dependent spastic paralysis, and Ca++- and PZQ-dependent disruption of parasitic teguments. Under normal conditions, there is a reversible balance between bilayer, isotropic, and HII phases in biological membranes (Jouhet 2013). In vitro, we could observe an irreversible but not stereoselective transition to the HII phase in liposomes consisting of phosphatidylethanolamine (PE) and phosphatidylserine (PS), two naturally occurring phospholipids in schistosomes, by the concerted action of Ca++ and PZQ (Harder 2013). HII structures are a prerequisite for induction of fusion processes (Jouhet 2013), which, indeed, become visible as blebs, vacuolation processes, and large balloon-like surface exudates in a large variety of PZQ-sensitive parasitic flukes and cestodes after PZQ treatment. These tegument damages are irreversible. As homologs of Sm.TRPMPZQ are also present in the other trematodes S. japonicum, S. haematobium, or Clonorchis sinensis and cestodes Taenia solium, Echinococcus multilocularis, or Hymenolepis microstoma (Park and Marchant, Trends Parasitol 36:182-194, 2020), it is suggested that a similar deadly cascade will be operating generally in PZQ-sensitive parasites.
Assuntos
Cestoides/efeitos dos fármacos , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Trematódeos/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Praziquantel/uso terapêutico , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologia , Canais de Cátion TRPM/efeitos dos fármacosRESUMO
Parkinson's disease (PD) is one of most common neurodegenerative diseases. Environmental stressors such as oxidative stress (OS), calcium ion influx, apoptosis, and inflammation mechanisms are linked to activated microglia in patients with PD. The OS-dependent activated transient receptor potential melastatin 2 (TRPM2) channel is modulated in several neurons by glutathione (GSH). However, the cellular and molecular effects of GSH alteration on TRPM2 activation, OS, apoptosis, and inflammation in the microglia remain elusive. The microglia of TRPM2 wild-type (TRPM2-WT) and knockout (TRPM2-KO) mice were divided into control, PD model (MPP), L-buthionine sulfoximine (BSO), MPP + BSO and MPP + BSO + GSH groups. MPP-induced increases in apoptosis, death, OS, lipid peroxidation, PARP1, caspase-3 and caspase-9, inflammatory cytokines (IL-1ß, TNF-α, IL-6), and intracellular free Zn2+ and Ca2+ levels in the microglia of TRPM2-WT mice were further increased by the BSO treatment, although they were diminished by the GSH treatment. Their levels were further reduced by PARP1 inhibitors (PJ34 and DPQ) and TRPM2 blockers (ACA and 2-APB). However, the effects of MPP and BSO were not observed in the microglia of TRPM2-KO mice. Taken together, our data demonstrate that maintaining GSH homeostasis is not only important for quenching OS in the microglia of patients with PD but also equally critical to modulating TRPM2, thus suppressing inflammatory responses elicited by environmental stressors.
Assuntos
Glutationa/metabolismo , Microglia/efeitos dos fármacos , Neurotoxinas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Animais , Apoptose/efeitos dos fármacos , Butionina Sulfoximina/farmacologia , Camundongos Knockout , Microglia/metabolismo , Neurônios/metabolismo , Canais de Cátion TRPM/efeitos dos fármacosRESUMO
Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in maintaining cell survival following oxidant injury. Here, we show that TRPM2 is highly expressed in acute myeloid leukemia (AML). The role of TRPM2 in AML was studied following depletion with CRISPR/Cas9 technology in U937 cells. In in vitro experiments and in xenografts, depletion of TRPM2 in AML inhibited leukemia proliferation, and doxorubicin sensitivity was increased. Mitochondrial function including oxygen consumption rate and ATP production was reduced, impairing cellular bioenergetics. Mitochondrial membrane potential and mitochondrial calcium uptake were significantly decreased in depleted cells. Mitochondrial reactive oxygen species (ROS) were significantly increased, and Nrf2 was decreased, reducing the antioxidant response. In TRPM2-depleted cells, ULK1, Atg7, and Atg5 protein levels were decreased, leading to autophagy inhibition. Consistently, ATF4 and CREB, two master transcription factors for autophagosome biogenesis, were reduced in TRPM2-depleted cells. In addition, Atg13 and FIP200, which are known to stabilize ULK1 protein, were decreased. Reconstitution with TRPM2 fully restored proliferation, viability, and autophagy; ATF4 and CREB fully restored proliferation and viability but only partially restored autophagy. TRPM2 expression reduced the elevated ROS found in depleted cells. These data show that TRPM2 has an important role in AML proliferation and survival through regulation of key transcription factors and target genes involved in mitochondrial function, bioenergetics, the antioxidant response, and autophagy. Targeting TRPM2 may represent a novel therapeutic approach to inhibit myeloid leukemia growth and enhance susceptibility to chemotherapeutic agents through multiple pathways.
Assuntos
Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Canais de Cátion TRPM/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Glibenclamide protects against ischemic injury in both preclinical and clinical studies, presumably by blocking the de novo assembled sulfonylurea receptor 1-transient receptor potential M4 (Sur1-Trpm4) channel induced by ischemia. However, glibenclamide may cause unexpected serious hypoglycemia. Here, we tested whether glimepiride, another sulfonylurea with better safety, has comparable efficacy with glibenclamide and whether gene deletion of Trpm4 (Trpm4-/-) exerts similar effect. Wild-type (WT) mice subjected to temporary middle cerebral artery occlusion (tMCAO) were randomized to receive glibenclamide (an initial dose of 10⯵g/kg and additional doses of 1.2⯵g every 8â¯h), three different doses of glimepiride (10⯵g/kg, 100⯵g/kg and 1â¯mg/kg) or vehicle after ischemia, while tMCAO-treated Trpm4-/- mice were randomized to receive vehicle or glimepiride. Neurological function, infarct volume, edema formation, the integrity of blood-brain barrier and inflammatory reaction were evaluated at 24â¯h after ischemia. In tMCAO-treated WT mice, 10⯵g/kg and 100⯵g/kg glimepiride had comparable efficacy with glibenclamide in improving longa score and grip test score, reducing infarct volume, mitigating brain edema, lessening extravasation of Evans blue dye and IgG, restoring tight junction protein expression as well as suppressing inflammatory cytokines. Compared with WT mice, Trpm4-/- mice showed less neurological deficit, smaller cerebral infarction, lighter brain edema and more integrity of blood-brain barrier. As expected, glimepiride did not provide additional neuroprotection compared with vehicle in the tMCAO-treated Trpm4-/- mice. Glimepiride shows comparable efficacy with glibenclamide in alleviating brain injury after ischemic stroke in mice, possibly via targeting the Sur1-Trpm4 channel.
Assuntos
Edema Encefálico/fisiopatologia , Encéfalo/efeitos dos fármacos , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Infarto da Artéria Cerebral Média/fisiopatologia , Inflamação/fisiopatologia , AVC Isquêmico/fisiopatologia , Compostos de Sulfonilureia/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Claudina-5/efeitos dos fármacos , Claudina-5/genética , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Perfilação da Expressão Gênica , Hipoglicemia/induzido quimicamente , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/genética , AVC Isquêmico/genética , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Camundongos , Camundongos Knockout , Fármacos Neuroprotetores , Ocludina/efeitos dos fármacos , Ocludina/genética , Distribuição Aleatória , Receptores de Sulfonilureias/efeitos dos fármacos , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismo , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
N-(p-amylcinnamoyl) anthranilic acid (ACA) is a blocker of transient receptor potential melastatin-2 (TRPM2) which is a non-selective, Ca2+-permeable and oxidative stress sensor cation channel. Intracerebroventricular (ICV) streptozotocin (STZ) induction successfully generates spatial memory deficits in rats. The purpose of this study was to investigate effects of ACA on a rat model of STZ-induced learning and memory deficits. A total of 60 Wistar rats randomly divided into six groups; (1) control, (2) sham-operated, (3) ICV-STZ administered, (4) ICV-STZ + memantine (5 mg/kg i.p.), (5) ICV-STZ + ACA (25 mg/kg i.p.) and (6) a combination therapy group, ICV-STZ + ACA (25 mg/kg) + memantine (5 mg/kg). Effects of the drugs on spatial memory deficits were appraised in Morris water maze (MWM) apparatus. Anxiety-like behavior of the rats were also assessed by using both the elevated plus maze (EPM) and open field maze (OFM) apparatuses. Western blot analysis of hippocampal tissues revealed TRPM2-L channel protein expression levels. Serum levels of tumor necrosis factor alpha (TNF-α) and malondialdehyde (MDA) were detected by enzyme-linked immunosorbent assay (ELISA) kits. Memantine treatment ameliorated the spatial memory deficits induced, as evidenced by the MWM tests. However, ACA treatment did not provide any improvement, instead positive effects of memantine were attenuated by ACA treatment. Western blot analysis in hippocampal tissues showed that TRPM2-L protein expression was markedly suppressed in ICV-STZ administered group. The ACA treatment reversed that suppression. Surprisingly, the memantine treatment resulted in overexpression of TRPM2-L, to a certain extent. Examination of the rats in EPM and OFM apparatuses, as a display of anxiety-like behavior, did not reveal any marked difference among groups. Serum levels of TNF-α and MDA also did not vary significantly among groups, as well. Conclusively, our findings showed for the first time that TRPM2-L protein expression was significantly suppressed in the ICV-STZ induced memory deficit model. Even though ACA reversed this suppression, no improvement in spatial memory was observed following ACA treatment.
Assuntos
Memantina , Transtornos da Memória , ortoaminobenzoatos , Animais , Aprendizagem em Labirinto , Memantina/farmacologia , Transtornos da Memória/tratamento farmacológico , Estresse Oxidativo , Distribuição Aleatória , Ratos , Ratos Wistar , Estreptozocina , Canais de Cátion TRPM/efeitos dos fármacos , ortoaminobenzoatos/farmacologiaRESUMO
The anthelmintic praziquantel (±PZQ) serves as a highly effective antischistosomal therapy. ±PZQ causes a rapid paralysis of adult schistosome worms and deleterious effects on the worm tegument. In addition to these activities against the parasite, ±PZQ also modulates host vascular tone in blood vessels where the adult worms reside. In resting mesenteric arteries ±PZQ causes a constriction of basal tone, an effect mediated by (R)-PZQ activation of endogenous serotoninergic G protein coupled receptors (GPCRs). Here, we demonstrate a novel vasodilatory action of ±PZQ in mesenteric vessels that are precontracted by high potassium-evoked depolarization, an effect previously reported to be associated with agonists of the transient receptor potential melastatin 8 channel (TRPM8). Pharmacological profiling a panel of 17 human TRPs demonstrated ±PZQ activity against a subset of human TRP channels. Several host TRP channels (hTRPA1, hTRPC3, hTRPC7) were activated by both (R)-PZQ and (S)-PZQ over a micromolar range whereas hTRPM8 showed stereoselective activation by (S)-PZQ. The relaxant effect of ±PZQ in mesenteric arteries was caused by (S)-PZQ, and mimicked by TRPM8 agonists. However, persistence of both (S)-PZQ and TRPM8 agonist evoked vessel relaxation in TRPM8 knockout tissue suggested that canonical TRPM8 does not mediate this (S)-PZQ effect. We conclude that (S)-PZQ is vasoactive over the micromolar range in mesenteric arteries although the molecular mediators of this effect remain to be identified. These data expand our knowledge of the polypharmacology and host vascular efficacy of this clinically important anthelmintic.
Assuntos
Anti-Helmínticos/farmacologia , Praziquantel/farmacologia , Canais de Cátion TRPM/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Feminino , Células HEK293 , Humanos , Artérias Mesentéricas/efeitos dos fármacos , Camundongos , Estereoisomerismo , Canais de Cátion TRPM/metabolismoRESUMO
AIMS: To investigate the localization of phosphodiesterase 5 (PDE5) and the molecular mechanism underlying the effect of the PDE5 inhibitor tadalafil in signal transduction in the bladder urothelium. METHODS: PDE5 expression in rat bladder tissues and cultured primary rat bladder urothelial cells was evaluated using immunochemistry and western blot assays. Ca2+ influx in cells exposed to isotonic solution, hypotonic solution, a selective transient receptor potential vanilloid 2 (TRPV2) channel agonist (cannabidiol), a selective TRPV4 channel agonist (GSK1016790A), a TRP cation channel melastatin 7 (TRPM7) channel agonist (PIP2), or a purinergic receptor agonist (ATP) in the presence or absence of 10 µM tadalafil was evaluated using calcium imaging techniques. We also evaluated stretch-induced changes in ATP concentration in the mouse bladder in the presence or absence of 100 µM tadalafil. RESULTS: Immunochemistry and western blot analyses demonstrated that PDE5 is abundantly expressed in the bladder urothelium and in primary rat urothelial cells. Ca2+ influx induced by hypotonic stimulation, GSK1016790A, or cannabidiol was significantly inhibited by tadalafil, whereas ATP-induced Ca2+ influx was unaffected by tadalafil. PIP2 did not induce Ca2+ influx. ATP release in tadalafil-pretreated bladders significantly decreased compared to control bladders. CONCLUSIONS: Tadalafil attenuates Ca2+ influx via TRPV4 and TRPV2, and inhibits ATP release in the bladder urothelium. These findings indicate that tadalafil functions as an inhibitor of urothelial signal transduction.
Assuntos
Cálcio/metabolismo , Tono Muscular/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Canais de Cátion TRPV/efeitos dos fármacos , Tadalafila/farmacologia , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Soluções Hipotônicas , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPV/agonistas , Bexiga Urinária/citologia , Urotélio/citologiaRESUMO
OBJECTIVES: We aimed to reveal the role of 5-fluorouracil (5-FU) and Leucovorin (LV) along with transient receptor potential protein melastatin 2 (TRPM2) channels in breast and colon cancer cells during the treatment process. BACKGROUND: 5-FU and LV are widely used in breast and colon cancers for chemotherapy. It has been reported that the expression of TRPM2 channels increased intensively in cancer cells. METHODS: Breast (MCF7) and colon (Caco-2) cells were cultured and divided into seven main groups. The cells in the group were incubated with 5-FU and LV for 24 hrs and then incubated with Antranilic acid. The effects of medicines were investigated on all molecular pathways of apoptosis. RESULTS: It was found that 5FU and LCV, administered separately and together on breast cancer cell culture and colon cancer cell culture increased the intracellular calcium levels by stimulation of TRPM2 channels in both cancer cells. CONCLUSION: As the result of our study, it has been shown that apoptotic effects of 5FU and LV on both colon and breast cancer cells were directly related to TRPM2 channels and that TRPM2 channels played an important role in the whole molecular pathway of apoptosis leading to increased intracellular Ca2+ (Ca2+) levels and increased mitochondrial depolarisation (Fig. 6, Ref. 43).
Assuntos
Neoplasias da Mama , Neoplasias do Colo , Fluoruracila , Leucovorina , Canais de Cátion TRPM , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Células CACO-2 , Cálcio , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Fluoruracila/farmacologia , Humanos , Leucovorina/farmacologia , Estresse Oxidativo , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPVRESUMO
Oxytocin (OT) is a critical molecule for social recognition that mediates social and emotional behaviors. OT is released during stress and acts as an anxiolytic factor. To know the precise molecular mechanisms underlying OT release into the brain during stress is important. It has been reported that intracellular concentrations of free calcium in the hypothalamic neurons are elevated by simultaneous stimulation of cyclic ADP-ribose (cADPR) and heat. We have reported in vitro and in vivo data that supports the idea that release of OT in the brain of male mice is regulated by cADPR and fever in relation to stress conditions. 1) Significantly higher levels of OT release were observed in hypothalamus cultures isolated from subordinate mice in group-housed males compared to dominant males after cage-switch stress; 2) OT concentrations in micro-perfusates at the paraventricular nucleus upon perfusion stimulation with cADPR were enhanced in subordinate mice compared to dominant mice; 3) The OT concentration in the cerebrospinal fluid (CSF) was higher in endotoxin-shock mice with fever compared to controls with no body temperature increase; and 4) In mice exposed to new environmental stress, the CSF OT level transiently increased 5 min after exposure, while the rectal temperature increased from 36.6 °C to 37.8 °C from 5 to 15 min after exposure. In this review, we examine whether or not cADPR and hyperthermia co-regulate hypothalamic OT secretion during social stress through the elevation of intracellular free Ca2+ concentrations involved in CD38-dependent Ca2+ mobilization and TRPM2-dependent Ca2+ influx. Finally, we propose that the interaction between CD38 and TRPM2 seems to be a new mechanism for stress-induced release of OT, which may result in anxiolytic effects for temporal recovery from social impairments in children with autism spectrum disorder during hyperthermia.
Assuntos
Febre/tratamento farmacológico , Hipotálamo/efeitos dos fármacos , Ocitocina/metabolismo , Canais de Cátion TRPM/efeitos dos fármacos , ADP-Ribosil Ciclase 1/efeitos dos fármacos , Animais , Humanos , Hipotálamo/metabolismo , Ocitocina/farmacologiaRESUMO
Spices in food and beverages and compounds in tobacco smoke interact with sensory irritant receptors of the transient receptor potential (TRP) cation channel family. TRPV1 (vanilloid type 1), TRPA1 (ankyrin 1) and TRPM8 (melastatin 8) not only elicit action potential signaling through trigeminal nerves, eventually evoking pungent or cooling sensations, but by their calcium conductance they also stimulate the release of calcitonin gene-related peptide (CGRP). This is measured as an index of neuronal activation to elucidate the chemo- and thermosensory transduction in the isolated mouse buccal mucosa of wild types and pertinent knockouts. We found that the lipophilic capsaicin, mustard oil and menthol effectively get access to the nerve endings below the multilayered squamous epithelium, while cigarette smoke and its gaseous phase were weakly effective releasing CGRP. The hydrophilic nicotine was ineffective unless applied unprotonated in alkaline (pH9) solution, activating TRPA1 and TRPV1. Also, mustard oil activated both these irritant receptors in millimolar but only TRPA1 in micromolar concentrations; in combination (1 mm) with heat (45 °C), it showed supraadditive, that is heat sensitizing, effects in TRPV1 and TRPA1 knockouts, suggesting action on an unknown heat-activated channel and mustard oil receptor. Menthol caused little CGRP release by itself, but in subliminal concentration (2 mm), it enabled a robust cold response that was absent in TRPM8-/- but retained in TRPA1-/- and strongly reduced by TRPM8 inhibitors. In conclusion, all three relevant irritant receptors are functionally expressed in the oral mucosa and play their specific roles in inducing neurogenic inflammation and sensitization to heat and cold.
Assuntos
Capsaicina/farmacologia , Mucosa Bucal/efeitos dos fármacos , Canal de Cátion TRPA1/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPV/efeitos dos fármacos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Camundongos , Células Receptoras Sensoriais/metabolismo , Canais de Potencial de Receptor Transitório/efeitos dos fármacosRESUMO
After spinal cord injury (SCI), blood-spinal cord barrier (BSCB) disruption and progressive hemorrhage lead to secondary injury, subsequent apoptosis and/or necrosis of neurons and glia, causing permanent neurological deficits. Growing evidence indicates that mithramycin A (MA), an anti-cancer drug, has neuroprotective effects in ischemic brain injury and Huntington's disease (HD). However, the precise mechanism underlying its protective effects is largely unknown. Here, we examined the effect of MA on BSCB breakdown and hemorrhage as well as subsequent inflammation after SCI. After moderate spinal cord contusion injury at T9, MA (150 µg/kg) was immediately injected intraperitoneally (i.p.) and further injected once a day for 5 days. Our data show that MA attenuated BSCB disruption and hemorrhage, and inhibited the infiltration of neutrophils and macrophages after SCI. Consistent with these findings, the expression of inflammatory mediators was significantly alleviated by MA. MA also inhibited the expression and activation of matrix metalloprotease-9 (MMP-9) after injury, which is known to disrupt BSCB and the degradation of tight junction (TJ) proteins. In addition, the expression of sulfonylurea receptor 1 (SUR1) and transient receptor potential melastatin 4 (TRPM4), which are known to mediate hemorrhage at an early stage after SCI, was significantly blocked by MA treatment. Finally, MA inhibited apoptotic cell death and improved functional recovery after injury. Thus, our results demonstrated that MA improves functional recovery by attenuating BSCB disruption and hemorrhage through the downregulation of SUR1/TRPM4 and MMP-9 after SCI.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Plicamicina/análogos & derivados , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/patologia , Animais , Hemorragia/patologia , Masculino , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Plicamicina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Sulfonilureias/biossíntese , Receptores de Sulfonilureias/efeitos dos fármacos , Canais de Cátion TRPM/biossíntese , Canais de Cátion TRPM/efeitos dos fármacosRESUMO
PCB19, a 2,2',6-trichlorinated biphenyl, is one of many non-dioxin-like polychlorinated biphenyls (NDL-PCBs), which are ubiquitous pollutants. NDL-PCBs affect cytosolic Ca2+ signaling by promoting Ca2+ release from ryanodine receptor-sensitive Ca2+ pools and inhibiting store-operated Ca2+ entry (SOCE) from the extracellular space. However, NDL-PCB-mediated SOCE inhibition has only been demonstrated in PC12 cells, in which SOCE is thought to be mainly mediated by TRPC family channels. Here, we investigated the effect of PCB19 on SOCE using human embryonic kidney 293 (HEK293) cells, human leukemia T cell line Jurkat-T cells and human promyelocytoma HL-60 cells which are the cell lines that are previously demonstrated to mediate the most common form of SOCE solely by the intrinsic Orai channels. PCB19 reduced thapsigargin-induced Ca2+ influx after Ca2+ pool depletion in HEK293 cells. SOCEs in HEK293, Jurkat T, HL-60 and PC12 cells showed distinct sensitivities to SOCE inhibitors such as Gd3+ and ML-9; however, PCB19 also showed a common effect of inhibiting SOCEs in all cell lines. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current but not the TRPM7 current. These results imply that PCB19 inhibits not only TRPC-mediated SOCE as in PC12 cells but also Orai-mediated SOCE as in many other cells including HEK293, Jurkat T and HL-60 cells.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proteína ORAI1/efeitos dos fármacos , Bifenilos Policlorados/farmacologia , Animais , Azepinas/farmacologia , Gadolínio/farmacologia , Células HEK293 , Células HL-60 , Humanos , Células Jurkat , Células PC12 , Ratos , Canais de Cátion TRPM/efeitos dos fármacos , Tapsigargina/antagonistas & inibidores , Tapsigargina/farmacologiaRESUMO
Anti-epidermal growth factor receptor (EGFR) drugs such as erlotinib and gefitinib cause a side effect of hypomagnesemia, but chemotherapy to treat this has not yet been developed. The transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of Mg2+ in the renal tubule. We reported previously that the expression of TRPM6 is up-regulated by epidermal growth factor (EGF) in renal tubular epithelial NRK-52E and HEK293 cells. EGF-induced elevation of TRPM6 expression was inhibited by erlotinib, gefitinib, and lapatinib. We found that tumor necrosis factor-α (TNF-α) increases TRPM6 expression in the presence of erlotinib. Therefore, we investigated what molecules are involved in the up-regulation of TRPM6 expression by TNF-α. EGF increased the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2), which were inhibited by erlotinib. TNF-α did not change p-ERK1/2 levels, but increased the phosphorylation and nuclear localization of nuclear factor-κB (NF-κB), which were blocked by the NF-κB inhibitors BAY 11-7082 and pyrrolidinedithiocarbamate ammonium. Similarly, luciferase reporter activity of human TRPM6 was increased by TNF-α, which was blocked by NF-κB inhibitors, and was inhibited by a mutation in the κB-binding site in the proximal region of the TRPM6 promoter. A chromatin immunoprecipitation assay revealed that NF-κB binds to the κB-binding site, which was blocked by NF-κB inhibitors. In the presence of erlotinib, TNF-α increased Mg2+ influx, which was blocked by NF-κB inhibitors. These results suggest that TNF-α reverses the reduction in Mg2+ reabsorption caused by anti-EGFR drugs. J. Cell. Physiol. 232: 2841-2850, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/toxicidade , Túbulos Renais/efeitos dos fármacos , Magnésio/metabolismo , Inibidores de Proteínas Quinases/toxicidade , Reabsorção Renal/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Sítios de Ligação , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gefitinibe , Células HEK293 , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/fisiopatologia , Lapatinib , NF-kappa B/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Quinazolinas/toxicidade , Ratos , Canais de Cátion TRPM/metabolismo , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 µM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 µM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).
Assuntos
Antipruriginosos/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio , Mentol/farmacologia , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Anilidas/farmacologia , Animais , Artérias , Canais de Cálcio Tipo L/metabolismo , Imuno-Histoquímica , Mentol/análogos & derivados , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Pirimidinonas/farmacologia , Ratos , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/metabolismo , Cauda , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
BACKGROUND: Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD). Low magnesium levels are associated with VC, and recent in vitro studies confirm a protective role of magnesium, which is mediated by its entry into the VSMCs through the Transient Receptor Potential Melastatin 7 (TRPM7) channel. The role of Angiotensin II (Ang II) on VC is still unclear. As Ang II is able to stimulate TRPM7 activity, we hypothesize that it might prevent VC. Thus, the aim of this study was to dissect the direct effect of Ang II on VC. MATERIALS AND METHODS: We worked with a model of high phosphate (HP)-induced calcification in human aortic smooth muscle cells, which resembles the CKD-related VC. RESULTS: Addition of Ang II to cells growing in HP decreased calcification, which was associated with the upregulation of the osteogenic factors BMP2, Runx2/Cbfa1, Osterix and ALP. A reduction of magnesium entry into the HP-calcifying cells was found. The treatment with Ang II avoided this reduction, which was reversed by the cotreatment with the TRPM7-inhibitor 2-APB. The protective effect of Ang II was related to AT1R-induced ERK1/2 MAPKinase activation. HP-induced calcification was also associated with the upregulation of the canonical Wnt/beta-catenin pathway, while its downregulation was related to attenuation of calcification by Ang II. CONCLUSION: As hypothesized, Ang II prevented phosphate-induced calcification in VSMCs, which appears mediated by the increase of magnesium influx and by the activation of the ERK1/2 and the inhibition of the canonical Wnt/beta-catenin signalling pathways.
Assuntos
Angiotensina II/farmacologia , Magnésio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Calcificação Vascular/metabolismo , Vasoconstritores/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Compostos de Boro/farmacologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição Sp7 , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Defects in primary cilia lead to a variety of human diseases. One of these, polycystic kidney disease, can be caused by defects in a Ca²âº-gated ion channel (TRPP2) found on the cilium. Other ciliary functions also contribute to cystogenesis, and defects in apical Ca²âº homeostasis have been implicated. By recording directly from the native cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin, we have identified a second Ca²âº-gated channel in the ciliary membrane: the transient receptor potential cation channel, subfamily M, member 4 (TRPM4). In excised primary cilia, TRPM4 was found to have a low sensitivity to Ca²âº, with an EC50 of 646 µM at +100 mV. It was inhibited by MgATP and by 9-phenanthrol. The channel was not permeable to Ca²âº or Clâ» and had a permeability ratio PK/PNa of 1.42. Reducing the expression of Trpm4 mRNA with short hairpin (sh) RNA reduced the TRPM4 current by 87% and shortened primary cilia by 43%. When phospholipase C was inhibited, the sensitivity to cytoplasmic Ca²âº greatly increased (EC50 = 26 µM at +100 mV), which is consistent with previous reports that phosphatidylinositol 4,5-bisphosphate (PIP2) modulates the channel. MgATP did not restore the channel to a preinactivation state, suggesting that the enzyme or substrate necessary for making PIP2 is not abundant in primary cilia of mIMCD-3 cells. The function of TRPM4 in renal primary cilia is not yet known, but it is likely to influence the apical Ca²âº dynamics of the cell, perhaps in tandem with TRPP2.