RESUMO
Zika virus (ZIKV) infection may lead to severe neurological consequences, including seizures, and early infancy death. However, the involved mechanisms are still largely unknown. TRPC channels play an important role in regulating nervous system excitability and are implicated in seizure development. We investigated whether TRPCs might be involved in the pathogenesis of ZIKV infection. We found that ZIKV infection increases TRPC4 expression in host cells via the interaction between the ZIKV-NS3 protein and CaMKII, enhancing TRPC4-mediated calcium influx. Pharmacological inhibition of CaMKII decreased both pCREB and TRPC4 protein levels, whereas the suppression of either TRPC4 or CaMKII improved the survival rate of ZIKV-infected cells and reduced viral protein production, likely by impeding the replication phase of the viral life cycle. TRPC4 or CaMKII inhibitors also reduced seizures and increased the survival of ZIKV-infected neonatal mice and blocked the spread of ZIKV in brain organoids derived from human-induced pluripotent stem cells. These findings suggest that targeting CaMKII or TRPC4 may offer a promising approach for developing novel anti-ZIKV therapies, capable of preventing ZIKV-associated seizures and death.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Canais de Cátion TRPC , Infecção por Zika virus , Zika virus , Infecção por Zika virus/virologia , Infecção por Zika virus/metabolismo , Animais , Humanos , Zika virus/fisiologia , Zika virus/efeitos dos fármacos , Camundongos , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Células HEK293 , Proteínas Virais/metabolismo , Convulsões/virologia , Convulsões/metabolismo , Convulsões/tratamento farmacológico , Proteases Virais , Serina Endopeptidases , Nucleosídeo-Trifosfatase , RNA Helicases DEAD-boxRESUMO
Transient receptor potential canonical 5 (TRPC5) is a calcium-permeable non-selective cation channel involved in various pathophysiological processes, including renal injury. Recently, GFB-887, an investigational pyridazinone TRPC5 inhibitor, demonstrated significant therapeutic potential in a Phase II clinical trial for focal segmental glomerulosclerosis (FSGS), a rare and severe form of chronic kidney disease (CKD). In the current study, based on the structure of GFB-887, we conducted extensive structural modification to explore novel TRPC5 inhibitors with desirable drug-like properties and robust nephroprotective efficacy. A series of pyridazinone derivatives featuring a novel tetrahydroimidazo[1,2-a]pyrazine scaffold were synthesized and their activities were evaluated in HEK-293 cells stably expressing TRPC5 using a fluorescence-based Ca2+ mobilization assay. Among these compounds, compound 12 is turned out to be a potent TRPC5 inhibitor with apparent affinity comparable to the parent compound GBF-887. Compound 12 is highly selective on TRPC4/5 over TRPC3/6/7 and hERG channels, along with acceptable pharmacokinetic properties and a favorable safety profile. More importantly, in a rat model of hypertension-induced renal injury, oral administration of compound 12 (10 mg/kg, BID) efficaciously reduced mean blood pressure, inhibited proteinuria, and protected podocyte damage. These findings further confirmed the potential of TRPC5 inhibitors on the CKD treatment and provided compound 12 to be a valuable tool for exploring TRPC4/5 pathophysiology.
Assuntos
Hipertensão , Pirazinas , Canais de Cátion TRPC , Animais , Humanos , Ratos , Pirazinas/química , Pirazinas/farmacologia , Pirazinas/síntese química , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/metabolismo , Células HEK293 , Relação Estrutura-Atividade , Masculino , Hipertensão/tratamento farmacológico , Descoberta de Drogas , Estrutura Molecular , Piridazinas/farmacologia , Piridazinas/química , Piridazinas/síntese química , Relação Dose-Resposta a Droga , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/química , Anti-Hipertensivos/síntese química , Ratos Sprague-Dawley , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/síntese químicaRESUMO
Transient receptor potential canonical 3 (TRPC3) channels are important in regulating Ca2+ homeostasis and have been implicated in the pathophysiology of chemically induced seizures. Inherited seizure susceptibility in genetically epilepsy-prone rats (GEPR-3s) has been linked to increased voltage-gated Ca2+ channel currents in the inferior colliculus neurons, which can affect intraneuronal Ca2+ homeostasis. However, whether TRPC3 channels also contribute to inherited seizure susceptibility in GEPR-3s is unclear. This study investigated the effects of JW-65, a potent and selective inhibitor of TRPC3 channels, on acoustically evoked seizure susceptibility in adult male and female GEPR-3s. These seizures consisted of wild running seizures (WRSs) that evolved into generalized tonic-clonic seizures (GTCSs). The results showed that acute administration of low doses of JW-65 significantly decreased by 55-89% the occurrence of WRSs and GTCSs and the seizure severity in both male and female GEPR-3s. This antiseizure effect was accompanied by increased seizure latency and decreased seizure duration. Additionally, female GEPR-3s were more responsive to JW-65's antiseizure effects than males. Moreover, JW-65 treatment for five consecutive days completely suppressed acoustically evoked seizures in male and female GEPR-3s. These findings suggest that inhibiting TRPC3 channels could be a promising antiseizure strategy targeting Ca2+ signaling mechanisms in inherited generalized tonic-clonic epilepsy.
Assuntos
Convulsões , Canais de Cátion TRPC , Animais , Masculino , Feminino , Convulsões/fisiopatologia , Convulsões/genética , Convulsões/induzido quimicamente , Ratos , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Predisposição Genética para Doença , Epilepsia/fisiopatologia , Epilepsia/genética , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológicoRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder associated with mitochondrial dysfunctions and oxidative stress. However, to date, therapeutics targeting these pathological events have not managed to translate from bench to bedside for clinical use. One of the major reasons for the lack of translational success has been the use of classical model systems that do not replicate the disease pathology and progression with the same degree of robustness. Therefore, we employed a more physiologically relevant model involving alpha-synuclein-preformed fibrils (PFF) exposure to SH-SY5Y cells and Sprague Dawley rats. We further explored the possible involvement of transient receptor potential canonical 5 (TRPC5) channels in PD-like pathology induced by these alpha-synuclein-preformed fibrils with emphasis on amelioration of oxidative stress and mitochondrial health. We observed that alpha-synuclein PFF exposure produced neurobehavioural deficits that were positively ameliorated after treatment with the TRPC5 inhibitor clemizole. Furthermore, Clemizole also reduced p-alpha-synuclein and diminished oxidative stress levels which resulted in overall improvements in mitochondrial biogenesis and functions. Finally, the results of the pharmacological modulation were further validated using siRNA-mediated knockdown of TRPC5 channels, which also decreased p-alpha-synuclein expression. Together, the results of this study could be superimposed in the future for exploring the beneficial effects of TRPC5 channel modulation for other neurodegenerative disorders and synucleopathies.
Assuntos
Mitocôndrias , Estresse Oxidativo , Ratos Sprague-Dawley , Canais de Cátion TRPC , alfa-Sinucleína , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Animais , Ratos , Estresse Oxidativo/efeitos dos fármacos , Humanos , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Masculino , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/induzido quimicamente , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/tratamento farmacológicoRESUMO
Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.
Assuntos
Cálcio , Oligonucleotídeos Antissenso , Canais de Cátion TRPC , Humanos , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/antagonistas & inibidores , Cálcio/metabolismo , Células A549 , Animais , Camundongos , Imidazóis/farmacologia , Canal de Cátion TRPC6/metabolismo , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/antagonistas & inibidores , Ácido Egtázico/farmacologia , Ácido Egtázico/análogos & derivados , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Linhagem Celular TumoralRESUMO
In intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mICAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mICAT and Ca2+ signalling in mouse ileal myocytes, and on intestinal motility. Ileal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca2+ imaging using Fura-2. Functional assessment of Pico145's effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. Carbachol (50 µM)-induced mICAT was strongly inhibited by Pico145 starting from 1 pM. The IC50 value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTPγS (200 µM), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca2+ rises were inhibited with IC50 of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only â¼43 % current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. Thus, Pico145 is a potent GI-active small-molecule which completely inhibits mICAT at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.
Assuntos
Receptores Muscarínicos , Canais de Cátion TRPC , Animais , Masculino , Camundongos , Carbacol/farmacologia , Motilidade Gastrointestinal , Miócitos de Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/metabolismoRESUMO
Transient receptor potential canonical (TRPC) channels are a class of non-selective cation channels expressed in a variety of tissues and organ systems where they functionally regulate physiological and pathological processes. TRPC5 has been shown to be a promising target for focal segmental glomerulosclerosis treatment. In this study, we report the synthesis and biological evaluation of a novel series of benzimidazole-based TRPC5 inhibitors. One compound, 8b, is 100-fold more potent than the parent compound, AC1903, in the suppression of TRPC5 channel activity. Interestingly, both AC1903 and 8b also suppressed TRPC4 channel activity with similar potency. Compound 8b also significantly blunts protamine sulfate-induced reorganization of podocyte cytoskeleton, interleukin (IL)-17-induced cell proliferation, and the expression of proinflammatory mediators in human keratinocyte HaCaT cells.
Assuntos
Indazóis , Canais de Cátion TRPC , Humanos , Indazóis/farmacologia , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/metabolismoRESUMO
A deepening understanding of the relationship between transient receptor potential canonical channel 5 (TRPC5) and chronic kidney disease (CKD), has led to the emergence of several types of TRPC5 inhibitors displaying clear therapeutic effect. Herein, we report the synthesis and biological evaluation of a series of pyrroledione TRPC5 inhibitors, culminating in the discovery of compound 16g with subtype selectivity. Compared with GFB-8438, a potent TRPC5 inhibitor (Goldfinch Bio), compound 16g showed improved inhibition of TRPC5 and enhanced protective effect against protamine sulfates (PS)-induced podocyte injury in vitro. In addition, compound 16g did not induce cell death in primary cultured hepatocytes and immortalized podocytes in a preliminary toxicity assessment, indicating its utility as a potent and safe inhibitor for studying the function of TRPC5.
Assuntos
Descoberta de Drogas , Pirróis/farmacologia , Canais de Cátion TRPC/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Protaminas , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Canais de Cátion TRPC/metabolismoRESUMO
Nonsense mutations, which occur in â¼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4-C1, and TRPC5-C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8-18 µM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake.
Assuntos
Aminoglicosídeos , Códon sem Sentido , Indazóis , Canais de Cátion TRPC , Aminoglicosídeos/farmacologia , Códon sem Sentido/efeitos dos fármacos , Humanos , Indazóis/farmacologia , Inibidores da Síntese de Proteínas , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismoRESUMO
The roles of ion channels, miRNAs and, neurotransmitters in the pathophysiology of major depressive disorder (MDD) are not yet fully elucidated. The current study aims to investigate ion channel gene expressions in the brain, the therapeutic efficacies of TRPC1, TRPM4, and CHRNA6 inhibitors, miRNAs specific to these ion channels and, neurotransmitter interactions in a chronic unpredictable mild stress (CUMS) induced MDD rat model. 48 two-month-old male albino Wistar rats were divided into Control, CUMS, Sham, CUMS+Pico145 (TRPC1 inhibitor), CUMS+ 9-Phe (TRPM4 inhibitor), and CUMS+BPiDl (CHRNA6 inhibitor) groups. Seven-week CUMS was used to induce MDD. Inhibitors were administered subacutely on the final of CUMS. Rats were subjected to behavioral tests. Gene expression levels were analyzed using qRT-PCR and neurotransmitter levels using ELISA. CUMS lead to a significant upregulation in the expression of channels in the hippocampus, and channels in the prefrontal cortex. Behavioral experiments determined the antidepressant effects as follows: Pico145 > BPiDl > 9-Phe. Compared to the Control, serotonin and noradrenaline levels remained unchanged, whereas dopamine levels increased. Acetylcholine levels decreased in CUMS and CUMS+Pico145 groups. CUMS significantly altered the expression of 6 miRNAs in the brain. BPiDl upregulated the expression of miR-6334 and Pico145 upregulated the expression of miR-135b-5p and miR-875 in the prefrontal cortex. The interactions of ion channels, miRNAs, and disruptions of neurotransmitter networks can play an important role in the pathophysiology of MDD. Moreover, as shown in this study, ion channel inhibitors have significant potential in the treatment of this disease.
Assuntos
Encéfalo/efeitos dos fármacos , Depressão/tratamento farmacológico , Canais Iônicos/efeitos dos fármacos , MicroRNAs/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Estresse Psicológico/tratamento farmacológico , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPM/antagonistas & inibidores , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Depressão/metabolismo , Modelos Animais de Doenças , Masculino , MicroRNAs/metabolismo , Ratos , Ratos Wistar , Estresse Psicológico/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPM/metabolismoRESUMO
TRPC5 channel is a nonselective cation channel that participates in diverse physiological processes. TRPC5 inhibitors show promise in the treatment of anxiety disorder, depression, and kidney disease. However, the binding sites and inhibitory mechanism of TRPC5 inhibitors remain elusive. Here, we present the cryo-EM structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and HC-070, to the resolution of 2.7 Å. The structures reveal that clemizole binds inside the voltage sensor-like domain of each subunit. In contrast, HC-070 is wedged between adjacent subunits and replaces the glycerol group of a putative diacylglycerol molecule near the extracellular side. Moreover, we found mutations in the inhibitor binding pockets altered the potency of inhibitors. These structures suggest that both clemizole and HC-070 exert the inhibitory functions by stabilizing the ion channel in a nonconductive closed state. These results pave the way for further design and optimization of inhibitors targeting human TRPC5.
Assuntos
Benzimidazóis/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/química , Benzimidazóis/metabolismo , Sítios de Ligação , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Humanos , Modelos Moleculares , Canais de Cátion TRPC/metabolismoRESUMO
TRPC4 ion channel was reported to be regulated by small molecular inhibitors and calmodulin. We discuss these findings in the context of other members of TRPC subfamily modulated by different stimulants.
Assuntos
Calmodulina/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Calmodulina/farmacologia , Humanos , Estrutura Secundária de Proteína , Canais de Cátion TRPC/químicaRESUMO
Regulation of Ca2+ homeostasis is essential for neuronal function and its survival. Recent data suggest that TRPC1 function as the endogenous store-mediated Ca2+ entry channel in dopaminergic cells, and loss of TRPC1 function leads to neurodegeneration; however, its regulation is not fully identified. Here we provide evidence that the sigma 1 receptor contributes to the loss of dopaminergic cells by blocking TRPC1-mediated Ca2+ entry. Importantly, downregulation of sigma 1 receptor expression significantly decreased neurotoxin-induced loss of dopaminergic cells as measured by MTT assays and caspase activity was also inhibited. Importantly, sigma 1 receptor inhibited TRPC1-mediated Ca2+ entry and silencing of sigma 1 receptor significantly restored store-dependent Ca2+ influx. Although co-immunoprecipitation failed to show an interaction between the TRPC1 and sigma 1 receptor, store depletion promoted a decrease in the sigma 1 receptor-STIM1 association. Neurotoxin-induced loss of Ca2+ entry was significantly restored in cells that had decreased sigma 1 receptor expression. Furthermore, TRPC1 or STIM1 silencing inhibited store-mediated Ca2+ entry, which was further increased upon the downregulation of the sigma 1 receptor expression. TRPC1 silencing prevented the increased neuroprotection and caspase activity observed upon the downregulation of sigma 1 receptor. Finally, sigma 1 receptor activation also significantly decreased TRPC1-mediated Ca2+ entry and lead to an increase in neurodegeneration. In contrast, addition of sigma 1 receptor antagonist prevented neurotoxin-induced neurodegeneration and facilitated TRPC1-mediated Ca2+ influx. Together these results suggest that the sigma 1 receptor is involved in the inhibition of TRPC1- mediated Ca2+ entry, which leads to the degeneration in the dopaminergic cells, and prevention of sigma 1 receptor function could protect neuronal cell death as observed in Parkinson's disease.
Assuntos
Cálcio/metabolismo , Morte Celular/fisiologia , Neurônios Dopaminérgicos/metabolismo , Receptores sigma/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Compostos de Boro/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Canais de Cátion TRPC/antagonistas & inibidores , Receptor Sigma-1RESUMO
OBJECTIVES: Various factors could interfere the biological performance of DPSCs during post-thawed process. Yet, little has been known about optimization of the recovery medium for DPSCs. Thus, our study aimed to explore the effects of adding recombinant bFGF on DPSCs after 3-month cryopreservation as well as the underlying mechanisms. MATERIALS AND METHODS: DPSCs were extracted from impacted third molars and purified by MACS. The properties of CD146+ DPSCs (P3) were identified by CCK-8 and flow cytometry. After cryopreservation for 3 months, recovered DPSCs (P4) were immediately supplied with a series of bFGF and analysed cellular proliferation by CCK-8. Then, the optimal dosage of bFGF was determined to further identify apoptosis and TRPC1 channel through Western blot. The succeeding passage (P5) from bFGF pre-treated DPSCs was cultivated in bFGF-free culture medium, cellular proliferation and stemness were verified, and pluripotency was analysed by neurogenic, osteogenic and adipogenic differentiation. RESULTS: It is found that adding 20 ng/mL bFGF in culture medium could significantly promote the proliferation of freshly thawed DPSCs (P4) through suppressing apoptosis, activating ERK pathway and up-regulating TRPC1. Such proliferative superiority could be inherited to the succeeding passage (P5) from bFGF pre-stimulated DPSCs, meanwhile, stemness and pluripotency have not been compromised. CONCLUSIONS: This study illustrated a safe and feasible cell culture technique to rapidly amplify post-thawed DPSCs with robust regenerative potency, which brightening the future of stem cells banking and tissue engineering.
Assuntos
Proliferação de Células/efeitos dos fármacos , Polpa Dentária/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Adipogenia/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Antígeno CD146/metabolismo , Diferenciação Celular/efeitos dos fármacos , Criopreservação , Meios de Cultura/química , Polpa Dentária/citologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Homeobox Nanog/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. Xanthine-based modulators include the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Here we report the structure of a small-molecule-bound TRPC1/4/5 channel-human TRPC5 in complex with the xanthine Pico145-to 3.0 Å. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.
Assuntos
Canais de Cátion TRPC , Xantinas , Humanos , Lipídeos/química , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/química , Canais de Cátion TRPC/metabolismo , Xantinas/química , Xantinas/metabolismoRESUMO
Traumatic brain injury (TBI) can cause physical, cognitive, social, and behavioral changes that can lead to permanent disability or death. After primary brain injury, translocated free zinc can accumulate in neurons and lead to secondary events such as oxidative stress, inflammation, edema, swelling, and cognitive impairment. Under pathological conditions, such as ischemia and TBI, excessive zinc release, and accumulation occurs in neurons. Based on previous research, it hypothesized that calcium as well as zinc would be influx into the TRPC5 channel. Therefore, we hypothesized that the suppression of TRPC5 would prevent neuronal cell death by reducing the influx of zinc and calcium. To test our hypothesis, we used a TBI animal model. After the TBI, we immediately injected NU6027 (1 mg/kg, intraperitoneal), TRPC5 inhibitor, and then sacrificed animals 24 h later. We conducted Fluoro-Jade B (FJB) staining to confirm the presence of degenerating neurons in the hippocampal cornus ammonis 3 (CA3). After the TBI, the degenerating neuronal cell count was decreased in the NU6027-treated group compared with the vehicle-treated group. Our findings suggest that the suppression of TRPC5 can open a new therapeutic window for a reduction of the neuronal death that may occur after TBI.
Assuntos
Lesões Encefálicas Traumáticas/patologia , Morte Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Compostos Nitrosos/farmacologia , Pirimidinas/farmacologia , Animais , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/fisiopatologia , Contagem de Células , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Neurônios/patologia , Neurônios/fisiologia , Compostos Nitrosos/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Pirimidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPC/antagonistas & inibidores , Zinco/metabolismoRESUMO
Increased blood pressure variability (BPV) has been proved to be associated with cardiovascular morbidity and mortality. It is of great significance to elucidate the mechanism of BPV increase. The cation channel transient receptor potential canonical 6 (TRPC6) is involved in a series of cardiovascular disease. Our experiment aimed to explore the role of TRPC6 in the development of BPV increase. Sino-aortic denervation (SAD) operation was applied to establish the model of BPV increase in rats. The BPV was presented as the standard deviation to the mean of systolic or diastolic blood pressure every 1 h during 12 h of the light period. SAD was performed in male Sprague Dawley (SD) rats at the age of 10 weeks. At 8 weeks after SAD operation, the hemodynamic parameters were determined non-invasively via a Rodent Blood Pressure Analysis System. The TRPC6 expressions in myocardial and thoracic aortic tissue was determined utilizing Western Blot, immunofluorescence and quantitative RT-PCR. The expression of TRPC3 was detected as well. To investigate whether TRPC6 was a causative factor of BPV increase in SAD rats, TRPC6 activator and inhibitor with three progressively increasing doses were intraperitoneally injected to the SAD rats. We found that SAD rats presented significant augmentation of systolic and diastolic BPV with no change of BP level and heart rate. The mRNA and protein expression levels of TRPC6 in myocardial and thoracic aortic tissue in SAD rats were substantially increased, but there was no obvious change in TRPC3 expression. The systolic and diastolic BPV increase were dose-dependently exacerbated after TRPC6 activation with GSK1702934A but were dose-dependently attenuated after TRPC6 inhibition with SAR7334. In Conclusion, the TRPC6 (but not TRPC3) expressions in myocardial and thoracic aortic tissue were substantially increased in SAD rats, and TRPC6 probably played an important role in the development of BPV elevation.
Assuntos
Aorta Torácica/metabolismo , Pressão Arterial , Barorreflexo , Frequência Cardíaca , Miocárdio/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/inervação , Pressão Arterial/efeitos dos fármacos , Denervação Autônoma , Barorreflexo/efeitos dos fármacos , Seio Carotídeo/inervação , Frequência Cardíaca/efeitos dos fármacos , Indanos/farmacologia , Masculino , Ratos Sprague-Dawley , Transdução de Sinais , Canais de Cátion TRPC/agonistas , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Regulação para CimaRESUMO
The nonselective Ca2+ -permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily and is widely expressed in the brain. Compelling evidence reveals that TRPC5 plays crucial roles in depression and other psychiatric disorders. To develop a TRPC5 radioligand, following up on our previous effort, we synthesized the iodine compound TZ66127 and its iodine-125-labeled counterpart [125 I]TZ66127. The synthesis of TZ66127 was achieved by replacing chloride with iodide in the structure of HC608, and the [125 I]TZ66127 was radiosynthesized using its corresponding tributylstannylated precursor. We established a stable human TRPC5-overexpressed HEK293-hTRPC5â cell line and performed Ca2+ imaging and a cell-binding assay study of TZ66127; these indicated that TZ66127 had good inhibition activity for TRPC5, and the inhibitory efficiency of TZ66127 toward TRPC5 presented in a dose-dependent manner. An inâ vitro autoradiography and immunohistochemistry study of rat brain sections suggested that [125 I]TZ66127 had binding specificity toward TRPC5. Altogether, [125 I]TZ66127 has high potential to serve as a radioligand for screening the binding activity of other new compounds toward TRPC5. The availability of [125 I]TZ66127 might facilitate the development of therapeutic drugs and PET imaging agents that target TRPC5.
Assuntos
Ensaio Radioligante , Compostos Radiofarmacêuticos/farmacologia , Canais de Cátion TRPC/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Cálcio/análise , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Radioisótopos do Iodo , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Ratos , Canais de Cátion TRPC/química , Canais de Cátion TRPC/metabolismoRESUMO
BACKGROUND: Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca dyshomeostasis in a mouse model relevant to malignant hyperthermia. METHODS: This study examined the mechanisms responsible for Ca dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. RESULTS: RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene-dose-dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. CONCLUSIONS: Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia-susceptible muscle, both at rest and during the malignant hyperthermia crisis.
Assuntos
Cálcio/metabolismo , Modelos Animais de Doenças , Hipertermia Maligna/metabolismo , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6/metabolismo , Animais , Feminino , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Indanos/farmacologia , Masculino , Hipertermia Maligna/genética , Hipertermia Maligna/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/biossíntese , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6/antagonistas & inibidores , Canal de Cátion TRPC6/genéticaRESUMO
CGA1-78 (Vasostatin-1, VS-1) a N-terminal Chromogranin A (CGA)-derived peptide, has been shown to have a protective effect against TNF-α-induced impairment of endothelial cell integrity. However, the mechanisms of this effect have not yet been clarified. CGA47-66 (Chromofungin, CHR) is an important bioactive fragment of CGA1-78. The present study aims to explore the protective effects of CHR on the vascular endothelial cell barrier response to TNF-α and its related Ca2+ signaling mechanisms. EA.hy926 cells were used as a vascular endothelial culture model. The synthetic peptides CHR and CGA4-16 were assessed for their ability to suppress TNF-α-induced EA.hy926 cells hyper-permeability through Transwell® and TEER assays. Changes in [Ca2+]i were measured through confocal laser scanning microscopy. SOC channel currents (Isoc) were measured via patch-clamp analysis. RT-PCR and western blot were used to analyze mRNA and protein expression of the transient receptor potential channels TRPC1 and TRPC4, respectively. FITC and rhodamine-phalloidin fluorescence were used to assess cell morphology and the distribution of MyPT-1 and F-actin. Compared to untreated cells, TNF-α increased the permeability of EA.hy926 cells that was inhibited by pre-treatment with CHR (10-1000 nM) in concentration-dependent manner, and the effect was most obvious at 100 nM, but CGA4-16 (100 nM) had no effect. TNF-α treatment increased the phosphorylation of MyPT-1 and stress fiber formation. CHR (10-1000 nM) pretreatment inhibited the cytoskeletal rearrangements and increased [Ca2+]i in response to TNF-α treatment. CHR also reduced TRPC1 expression following TNF-α induction. Similar to SOC inhibitor 2-APB, CHR suppressed IP3 mediated SOC activation. These findings suggest that CHR inhibits TNF-α-induced Ca2+ influx and protects the barrier function of vascular endothelial cells, and that these effects are related to the inhibition of SOC and Ca2+ signaling by CHR.