RESUMO
Diet-induced obesity and hyperlipidemia are a growing public health concern leading to various metabolic disorders. Capsaicin, a major bioactive compound obtained from natural chili peppers, has demonstrated its numerous beneficial roles in treating obesity and weight loss. Current treatment involves either administration of antiobesity drugs or surgical procedures such as Roux-en-Y-gastric bypass or sleeve gastrectomy, both of which are associated with serious side effects and poor patient acceptance. Capsaicin, a pungent molecule, has low oral bioavailability. Therefore, there is a need for the development of site-specific drug delivery system for capsaicin. The present study is aimed at preparing and characterizing 3D-printed capsaicin-loaded rod-shaped implants by thermoplastic extrusion-based 3D printing technology. The implants were printed with capsaicin-loaded into a biodegradable polymer, polycaprolactone, at different drug loadings and infill densities. The surface morphology revealed a smooth and uniform external surface without any capsaicin crystals. DSC thermograms showed no significant changes/exothermic events among the blends suggesting no drug polymer interactions. The in vitro release studies showed a biphasic release profile for capsaicin, and the release was sustained for more than three months (~ 85% released) irrespective of drug loading and infill densities. The HPLC method was stability-indicating and showed good resolution for its analogs, dihydrocapsaicin and nordihydrocapsaicin. The implants were stable for three months at accelerated conditions (40°C) without any significant decrease in the assay of capsaicin. Therefore, capsaicin-loaded implants can serve as a long-acting injectable formulation for targeting the adipose tissue region in obese patients.
Assuntos
Capsaicina , Obesidade , Humanos , Capsaicina/química , Obesidade/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Impressão Tridimensional , Polímeros/uso terapêutico , Liberação Controlada de FármacosRESUMO
ABSTRACT: Painful diabetic peripheral neuropathy (PDPN) is one of the major complications of diabetes. Currently, centrally acting drugs and topical analgesics are used for treating PDPN. These drugs have adverse effects; some are ineffective, and treatment with opioids is associated with use dependence and addiction. Recent research indicates that transient receptor potential vanilloid 1 (TRPV1) expressed in the peripheral sensory nerve terminals is an emerging target to treat pain associated with PDPN. Block of TRPV1 ion channel with specific antagonists, although effective as an analgesic, induced hyperthermia in clinical trials. However, TRPV1 agonists are useful to treat pain by virtue of their ability to cause Ca 2+ influx and subsequently leading to nerve terminal desensitization. Here, we report the effectiveness of an ultrapotent TRPV1 agonist, resiniferatoxin (RTX) nanoparticle, in a topical formulation (RTX-cream; RESINIZIN) that alleviates pain associated with DPN in animal models of diabetes. Resiniferatoxin causes nerve terminal depolarization block in the short term, which prevents pain during application and leading to nerve terminal desensitization/depletion in the long term resulting in long-lasting pain relief. Application of RTX cream to the hind limbs suppresses thermal hyperalgesia in streptozotocin-induced diabetic rats and mini pigs without any adverse effects as compared with capsaicin at therapeutic doses, which induces intense pain during application. Resiniferatoxin cream also decreases the expression of TRPV1 in the peripheral nerve endings and suppresses TRPV1-mediated calcitonin gene-related peptide release in the skin samples of diabetic rats and mini pigs. Our preclinical data confirm that RTX topical formulation is an effective treatment option for PDPN.
Assuntos
Diabetes Mellitus Experimental , Neuropatias Diabéticas , Diterpenos , Suínos , Ratos , Animais , Neuropatias Diabéticas/tratamento farmacológico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Porco Miniatura/metabolismo , Dor , Diterpenos/uso terapêutico , Analgésicos/uso terapêutico , Capsaicina/farmacologia , Canais de Cátion TRPV/metabolismoRESUMO
Transient receptor potential vanilloid subfamily 1 (TRPV1) is a non-selective cation channel protein expressed in neuronal and non-neuronal cells. Although TRPV1 is implicated in thermogenesis and diet-induced obesity (DIO), its precise role remains controversial. TRPV1-/- mice are protected from DIO, while TRPV1 activation enhances thermogenesis to prevent obesity. To reconcile this, we fed wild-type and TRPV1-/- mice for 32 weeks with normal chow or a high-fat diet and analyzed the weight gain, metabolic activities, and thermogenic protein expression in white and brown fats. TRPV1-/- mice became obese, exhibited reduced locomotor activity, reduced energy expenditure, enhanced hepatic steatosis, and decreased thermogenic protein expression in adipose tissues. Our data reveal that lack of TRPV1 does not prevent obesity, but rather enhances metabolic dysfunction.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Redes Reguladoras de Genes/efeitos dos fármacos , Síndrome Metabólica/genética , Obesidade/genética , Canais de Cátion TRPV/genética , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/metabolismo , Camundongos , Obesidade/induzido quimicamente , Obesidade/metabolismo , TermogêneseRESUMO
Kainic acid (KA) is an excitotoxic glutamate analogue produced by a marine seaweed. It elicits neuronal excitotoxicity leading to epilepsy in rodents. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1), a nonselective cation channel protein, by capsaicin, prevents KA-induced seizures in a mouse model of temporal lobe epilepsy. However, the precise mechanism behind this protective effect of capsaicin remains unclear. In order to analyze the direct effect of KA on TRPV1, we evaluated the ability of KA to activate TRPV1 and analyzed its binding to TRPV1 using a molecular modeling approach. In vitro, KA activates a Ca2+ influx into TRPV1 expressing HEK293 cells but not in contsrol HEK293 cells. Pretreatment with either capsaicin (1 M) or capsazepine (10 M; TRPV1 antagonist) prevents the effect of KA. Pharmacological inhibition of phospholipase C (PLC) by U73122 or overexpression of phosphatidylinositol 5 phosphatase (Synaptojanin 1; Synj-1) counters the effect of KA. Further, KA treatment causes actin reorganization in HEKTRPV1 cells and PLC inhibition by U73122 prevents this. Molecular modeling data revealed that KA binds to TRPV1 and prebinding with capsaicin prevents the binding of KA to TRPV1. Consistently, the lack of effect of KA in activating chicken TRPV1, which is insensitive to capsaicin, suggests that there is a significant overlap between the sites of KA and capsaicin activation of TRPV1. However, PLC inhibition did not suppress TRPV1 activation by capsaicin. Collectively, our data suggest that KA binds to and activates TRPV1 and causes actin reorganization via PLC-dependent mechanism in vitro. We propose that KA mediates Ca2+ induced toxicity possibly by activating TRPV1. Therefore, inhibiting TRPV1 will be a beneficial strategy in abating Ca2+-induced neurotoxicity.
Assuntos
Canais de Cátion TRPV , Fosfolipases Tipo C , Capsaicina/farmacologia , Células HEK293 , Humanos , Ácido Caínico/toxicidade , Fosfatidilinositóis , Fosfolipases Tipo C/metabolismoRESUMO
Testosterone regulates dimorphic sexual behaviors in all vertebrates. However, the molecular mechanism underlying these behaviors remains unclear. Here, we report that a newly identified rapid testosterone signaling receptor, Transient Receptor Potential Melastatin 8 (TRPM8), regulates dimorphic sexual and social behaviors in mice. We found that, along with higher steroid levels in the circulation, TRPM8-/- male mice exhibit increased mounting frequency indiscriminate of sex, delayed sexual satiety, and increased aggression compared to wild-type controls, while TRPM8-/- females display an increased olfaction-exploratory behavior. Furthermore, neuronal responses to acute testosterone application onto the amygdala were attenuated in TRPM8-/- males but remained unchanged in females. Moreover, activation of dopaminergic neurons in the ventral tegmental area following mating was impaired in TRPM8-/- males. Together, these results demonstrate that TRPM8 regulates dimorphic sexual and social behaviors, and potentially constitutes a signalosome for mediation of sex-reward mechanism in males. Thus, deficiency of TRPM8 might lead to a delayed sexual satiety phenomenon.
Assuntos
Comportamento Animal/fisiologia , Receptores Androgênicos/metabolismo , Comportamento Sexual Animal/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPM/metabolismo , Testosterona/metabolismo , Agressão/fisiologia , Animais , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Feminino , Masculino , Camundongos , Caracteres Sexuais , Comportamento Social , Área Tegmentar Ventral/metabolismo , Área Tegmentar Ventral/fisiologiaRESUMO
Capsaicin (CAP) activates transient receptor potential vanilloid subfamily 1 (TRPV1) to counter high-fat diet (HFD)-induced obesity. Several studies suggest that CAP induces the browning of white adipocytes in vitro or inguinal white adipose tissue (iWAT) in vivo. However, there is a lack of data on the dose-response for CAP to inhibit HFD-induced obesity. Therefore, we first performed experiments to correlate the effect of various doses of CAP to prevent HFD-induced weight gain in wild-type (WT) mice. Next, we performed a subchronic safety study in WT mice fed a normal chow diet (NCD ± CAP, 0.01% in NCD) or HFD ± CAP (0.01% in HFD) for eight months. We analyzed the expression of adipogenic and thermogenic genes and proteins in the iWAT from these mice, conducted histological studies of vital organs, measured the inflammatory cytokines in plasma and iWAT, and evaluated liver and kidney functions. The dose-response study showed that CAP, at doses above 0.001% in HFD, countered HFD-induced obesity in mice. However, no difference in the anti-obesity effect of CAP was observed at doses above 0.003% in HFD. Also, CAP, above 0.001%, enhanced the expression of sirtuin-1 and thermogenic uncoupling protein 1 (UCP-1) in the iWAT. Safety analyses suggest that CAP did not cause inflammation. However, HFD elevated plasma alanine aminotransferase and creatinine, caused iWAT hypertrophy and hepatic steatosis, and CAP reversed these. Our data suggest that CAP antagonizes HFD-induced metabolic stress and inflammation, while it does not cause any systemic toxicities and is well tolerated by mice.
Assuntos
Capsaicina/efeitos adversos , Capsaicina/farmacologia , Comportamento Alimentar , Metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Citocinas/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Testes de Função Cardíaca , Mediadores da Inflamação/sangue , Metabolismo/efeitos dos fármacos , Camundongos Knockout , Junção Neuromuscular/efeitos dos fármacos , Obesidade/sangue , Obesidade/genética , Tamanho do Órgão/efeitos dos fármacos , Termogênese/genéticaRESUMO
Published research suggests that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) enhances the expression and deacetylation of peroxisome proliferator-activated receptor gamma (PPARγ) to cause browning of white adipose tissue. Here, we show that TRPV1 activation by capsaicin significantly prevents high fat diet-induced obesity in mice. This is associated with an increase in the expression and deacetylation of PPARγ in the epididymal fat of these mice. Consistent with the TRPV1 activation in vivo, overexpression of TRPV1 enhanced the PPARγ and other thermogenic genes in cultured 3T3-L1 preadipocytes. To determine the interaction between TRPV1 and PPARγ signaling, we analyzed the effect of Troglitazone (Trog; a thiazolidinedione derivative and an agonist of PAARγ) treatment on cultured 3T3-L1 cells. Trog enhanced the expression of TRPV1, PPARγ and thermogenic proteins in undifferentiated 3T3-L1 cells but not in differentiated cells. Acute application of Trog stimulated a robust Ca2+ influx into 3T3-L1 cells and TRPV1 inhibition by capsazepine prevented this. More interestingly, Trog or capsaicin treatment caused the deacetylation of PPARγ in 3T3-L1 cells and inhibition of TRPV1 or Sirtuin 1 - prevented this. Our data suggest a novel effect of Trog to induce PPARγ deacetylation by activating TRPV1. This research has a significant implication on the role of TRPV1 and PPARγ signaling in the browning of white adipose tissue.
Assuntos
PPAR gama/metabolismo , Canais de Cátion TRPV/metabolismo , Troglitazona/farmacologia , Células 3T3-L1 , Acetilação/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Dieta Hiperlipídica , Epididimo/efeitos dos fármacos , Epididimo/metabolismo , Comportamento Alimentar , Ativação do Canal Iônico/efeitos dos fármacos , Metabolismo dos Lipídeos , Masculino , Camundongos , PPAR alfa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Termogênese/genética , Proteína Desacopladora 1/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
(1) Background: Capsaicin, a chief ingredient of natural chili peppers, enhances metabolism and energy expenditure and stimulates the browning of white adipose tissue (WAT) and brown fat activation to counter diet-induced obesity. Although capsaicin and its nonpungent analogs are shown to enhance energy expenditure, their efficiency to bind to and activate their receptor-transient receptor potential vanilloid subfamily 1 (TRPV1)-to mediate thermogenic effects remains unclear. (2) Methods: We analyzed the binding efficiency of capsaicin analogs by molecular docking. We fed wild type mice a normal chow or high fat diet (± 0.01% pungent or nonpungent capsaicin analog) and isolated inguinal WAT to analyze the expression of thermogenic genes and proteins. (3) Results: Capsaicin, but not its nonpungent analogs, efficiently binds to TRPV1, prevents high fat diet-induced weight gain, and upregulates thermogenic protein expression in WAT. Molecular docking studies indicate that capsaicin exhibits the highest binding efficacy to TRPV1 because it has a hydrogen bond that anchors it to TRPV1. Capsiate, which lacks the hydrogen bond, and therefore, does not anchor to TRPV1. (4) Conclusions: Long-term activation of TRPV1 is imminent for the anti-obesity effect of capsaicin. Efforts to decrease the pungency of capsaicin will help in advancing it to mitigate obesity and metabolic dysfunction in humans.
Assuntos
Capsaicina , Metabolismo Energético/efeitos dos fármacos , Termogênese/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Capsaicina/química , Capsaicina/metabolismo , Capsaicina/farmacologia , Capsicum/química , Camundongos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al.1 We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Colforsina/farmacologia , Glicerol/análise , Lipólise/efeitos dos fármacos , Espectrofotometria/métodos , Animais , Dieta Hiperlipídica , Glicerol/metabolismo , Masculino , CamundongosRESUMO
Botulinum neurotoxin A (BoNT/A) cleaves SNAP25 at the motor nerve terminals and inhibits stimulus evoked acetylcholine release. This causes skeletal muscle paralysis. However, younger neonatal mice (Assuntos
Toxinas Botulínicas Tipo A/toxicidade
, Colesterol/metabolismo
, Junção Neuromuscular/efeitos dos fármacos
, Animais
, Animais Recém-Nascidos
, Toxinas Botulínicas Tipo A/metabolismo
, Diafragma/efeitos dos fármacos
, Diafragma/metabolismo
, Endocitose
, Camundongos
, Camundongos Endogâmicos C57BL
, Junção Neuromuscular/metabolismo
RESUMO
PURPOSE: Drugs used in the treatment of diseases can cause several unwanted systemic side effects. A site-specific drug delivery system can eliminate such consequences by delivering drugs to certain target areas of the body where therapeutic effects are required. Here we present the preparation and evaluation of magnetic nanoparticles of capsaicin, the active ingredient in chili peppers, coated with poly-L-lactide co-glycolide (PLGA), a FDA-approved biodegradable bioavailable polymer. METHODS: PCMN were prepared by solvent-evaporation/coprecipitation technique and their physicochemical and pharmacological characteristics evaluated in vitro. Further, effective pain/inflammation therapeutics of PCMN in a mouse model of inflammation was also studied. We also prepared and evaluated the subcellular localization of PLGA coated fluorescence magnetic nanoparticle (PFMN) in vitro in HEK293 cells. RESULTS: Transmission electron microscopic images of PCMN showed that the size of the nanoparticles were of the order of 10-20 nm. PCMN showed approximately 9.29% drug loading and 89.15% encapsulation efficiencies. In vitro dissolution studies showed an increased solubility of capsaicin due to the nano-size of the PCMN, while PLGA coating allowed sustained release of capsaicin in vitro. The PCMN also reduced paw edema after injection in mice, and confocal microscopy revealed the successful intracellular localization of PLGA-coated fluorescein magnetic nanoparticles in HEK293 cells. CONCLUSION: The PCMN provided a sustained release of capsaicin in vitro and inhibited carrageenan-induced inflammatory pain in mouse model in vivo. These data suggest that PLGA coating of capsaicin magnetic nanoparticles have the potential to be amenable for a sustained release of capsaicin to relieve pain.
Assuntos
Capsaicina/farmacologia , Ácido Láctico/química , Nanopartículas de Magnetita/química , Dor/tratamento farmacológico , Ácido Poliglicólico/química , Animais , Capsaicina/química , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Portadores de Fármacos , Liberação Controlada de Fármacos , Fluoresceína/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Solubilidade , Propriedades de SuperfícieRESUMO
BACKGROUND AND PURPOSE: The growing epidemic of obesity and metabolic diseases necessitates the development of novel strategies to prevent and treat such diseases. Current research suggests that browning of white adipose tissue (WAT) promotes energy expenditure to counter obesity. Recent research suggests that activation of the TRPV1 channels counters obesity. However, the mechanism by which activation of TRPV1 channels counters obesity still remains unclear. EXPERIMENTAL APPROACH: We evaluated the effect of dietary capsaicin to induce a browning program in WAT by activating TRPV1 channels to prevent diet-induced obesity using wild-type and TRPV1(-/-) mouse models. We performed experiments using preadipocytes and fat pads from these mice. KEY RESULTS: Capsaicin stimulated the expression of brown fat-specific thermogenic uncoupling protein-1 and bone morphogenetic protein-8b in WAT. Capsaicin triggered browning of WAT by promoting sirtuin-1 expression and activity via TRPV1 channel-dependent elevation of intracellular Ca(2) (+) and phosphorylation of Ca(2) (+) /calmodulin-activated protein kinase II and AMP-activated kinase. Capsaicin increased the expression of PPARγ 1 coactivator α and enhanced metabolic and ambulatory activity. Further, capsaicin stimulated sirtuin-1-dependent deacetylation of PPARγ and the transcription factor PRDM-16 and facilitated PPARγ-PRDM-16 interaction to induce browning of WAT. Dietary capsaicin did not protect TRPV1(-/-) mice from obesity. CONCLUSIONS AND INTERPRETATIONS: Our results show for the first time that activation of TRPV1 channels by dietary capsaicin triggers browning of WAT to counteract obesity. Our results suggest that activation of TRPV1 channels is a promising strategy to counter obesity.
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Capsaicina/farmacologia , Capsaicina/uso terapêutico , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Canais de Cátion TRPV/agonistas , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Capsaicina/administração & dosagem , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/metabolismoRESUMO
The transient receptor potential ion channel of the melastatin subfamily, TRPM8, is a major cold receptor in the peripheral nervous system. Along with the sensory neurons, the TRPM8 protein is highly expressed in the prostate epithelial cells, and this expression is regulated by androgens. Here we investigated the expression and intracellular localization of the TRPM8 channel in relationship to androgens. We performed experiments using human prostate tissues obtained from healthy individuals and patients with prostate cancer at various stages of the disease as well as in cultured cells. Using an immunohistochemistry approach, we detected an intensive colocalization pattern of the TRPM8 protein with endogenous androgens in all tissues tested, suggesting possible interactions. Co-immunoprecipitation experiments performed using cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8 further confirmed direct binding of the steroid hormone, testosterone, to the TRPM8 protein. Applications of picomolar concentrations of testosterone to the primary human prostate cells, endogenously expressing TRPM8, elicited Ca(2+) responses and channel currents, and those were inhibited in the presence of TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride. These results indicate that the TRPM8 channel is physically associated with testosterone and suggest that, in addition to a genomic role, testosterone plays a role in direct regulation of the TRPM8 channel function.
Assuntos
Receptores Androgênicos/metabolismo , Canais de Cátion TRPM/metabolismo , Testosterona/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Ligação ProteicaRESUMO
Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.
Assuntos
Receptores Androgênicos/metabolismo , Canais de Cátion TRPM/metabolismo , Testosterona/metabolismo , Cálcio/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Bicamadas Lipídicas/metabolismo , Ligação Proteica/efeitos dos fármacos , Testosterona/farmacologia , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions.
Assuntos
Acetilcolina/metabolismo , Capsaicina/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Animais , Capsaicina/análogos & derivados , Exocitose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/metabolismo , Canais de Cátion TRPV/metabolismo , Estimulação do Nervo Vago/métodosRESUMO
Cyclophilin A (CyPA) is an important mediator in cardiovascular diseases. It possesses peptidyl-prolyl cis-trans isomerase activity (PPIase) and chaperone functions, which regulate protein folding, intracellular trafficking and reactive oxygen species (ROS) production. Platelet glycoprotein receptor αIIbß3 integrin activation is the common pathway for platelet activation. It was our objective to understand the mechanism by which CyPA-regulates αIIbß3 activation in platelets. Mice deficient for CyPA (CyPA-/-) had prolonged tail bleeding time compared to wild-type (WT) controls despite equivalent platelet numbers. In vitro studies revealed that CyPA-/- platelets exhibited dramatically decreased thrombin-induced platelet aggregation. In vivo, formation of occlusive thrombi following FeCl3 injury was also significantly impaired in CyPA-/- mice compared with WT-controls. Furthermore, CyPA deficiency inhibited flow-induced thrombus formation in vitro. Flow cytometry demonstrated that thrombin-induced ROS production and αIIbß3 activation were reduced in CyPA-/- platelets. Coimmunoprecipitation studies showed ROS-dependent increased association of CyPA and αIIbß3. This association was dependent upon the PPIase activity of CyPA. Significantly, fibrinogen-platelet binding, platelet spreading and cytoskeleton reorganisation were also altered in CyPA-/- platelets. Moreover, CyPA deficiency prevented thrombin-induced αIIbß3 and cytoskeleton association. In conclusion, CyPA is an important mediator in platelet function by regulation of αIIbß3 bidirectionalsignalling through increased ROS production and facilitating interaction between αIIbß3 and the cell cytoskeleton.
Assuntos
Plaquetas/fisiologia , Ciclofilina A/metabolismo , Artérias Mesentéricas/patologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/sangue , Animais , Adesão Celular/genética , Células Cultivadas , Cloretos/administração & dosagem , Ciclofilina A/genética , Citoesqueleto/genética , Compostos Férricos/administração & dosagem , Fibrinogênio/metabolismo , Hemorragia/genética , Artérias Mesentéricas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agregação Plaquetária , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Trombina/metabolismo , Trombose/induzido quimicamenteRESUMO
AIMS: Cyclophilin A (CyPA) is a pro-inflammatory mediator involved in oxidative stress-related cardiovascular diseases. It is secreted from vascular smooth muscle cell (VSMC) in response to reactive oxygen species (ROS) in a highly regulated manner. Extracellular CyPA activates VSMCs and endothelial cells (ECs) promoting inflammation, cell growth, and cell death. Recently, it was shown that acetylated CyPA (AcK-CyPA) affects its function. We investigated the role of acetylation of CyPA for its secretion and signalling in vascular cells. METHODS AND RESULTS: We used angiotensin II (Ang II) to create sustained ROS and found significantly increased AcK-CyPA in VSMC. Site-directed mutagenesis showed that lysines K82 and K125 were the predominant CyPA residues acetylated in response to Ang II. Importantly, acetylation of K82 and K125 were required for Ang II-mediated CyPA secretion. ROS inhibitors, Tiron, and N-acetylcysteine inhibited Ang II-induced intracellular CyPA acetylation and also AcK-CyPA secretion. Using secreted CyPA from wild type and K82/125R mutants expressed in transduced VSMC or in vitro acetylated recombinant CyPA, we showed that extracellular AcK-CyPA significantly increased pERK1/2, matrix metalloproteinase-2 activation, and ROS production in VSMC compared with non-acetylated CyPA. Moreover, extracellular AcK-CyPA increased adhesion molecule expression (VCAM-1 and ICAM-1) in EC, which promoted monocyte adhesion. CONCLUSIONS: ROS-dependent acetylation of CyPA is required for the generation of extracellular CyPA. Acetylated extracellular CyPA regulates VSMC and EC activation, suggesting that inhibition of acetylation of CyPA may prevent the pathogenesis of oxidative stress-related cardiovascular diseases.
Assuntos
Ciclofilina A/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Acetilação , Angiotensina II/farmacologia , Animais , Células Cultivadas , Ciclofilina A/química , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
INTRODUCTION: Botulinum neurotoxin A (BoNT/A) cleaves SNAP-25 and inhibits acetylcholine (ACh) release at the neuromuscular junctions (NMJ) to cause neuroparalysis. Previous reports indicate a dyssynchrony between the inhibitory effect of BoNT/A on ACh release and SNAP-25 cleavage. METHODS: We tested the in vitro (acute; 90 min) and in vivo (chronic; 12 h) effects of BoNT/A on stimulus-evoked ACh release (SEAR), twitch tension, and SNAP-25 cleavage in isolated extensor digitorum longus (EDL) nerve-muscle preparations (NMP). RESULTS: In vitro or in vivo BoNT/A poisoning inhibited SEAR and twitch tension. Conversely, SNAP-25 cleavage and inhibition of spontaneous release frequency were observed only in NMP poisoned with BoNT/A in vivo. Moreover, chronic treatment of BoNT/A inhibited ionomycin stimulated Ca(2+) signals in Neuro 2a cells. CONCLUSIONS: These results demonstrate that the inhibition of SEAR precedes SNAP-25 cleavage and suggest involvement of a more complex mechanism for the inhibitory effect of BoNT/A at the NMJ.
Assuntos
Toxinas Botulínicas Tipo A/toxicidade , Potencial Evocado Motor/efeitos dos fármacos , Fármacos Neuromusculares/toxicidade , Junção Neuromuscular/efeitos dos fármacos , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/farmacologia , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Amifampridina , Animais , Ionóforos de Cálcio/farmacologia , Linhagem Celular Tumoral , Estimulação Elétrica , Eletromiografia , Técnicas In Vitro , Ionomicina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma/patologia , Junção Neuromuscular/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Reflexo/efeitos dos fármacos , Fatores de TempoRESUMO
OBJECTIVE: Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. APPROACH AND RESULTS: Overexpression of CyPA in CyPA-deficient VSMC (CyPA(-/-)VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA(-/-)VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA(-/-)VSMC compared with wild-type-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA(-/-)VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. CONCLUSIONS: These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.
Assuntos
Angiotensina II/farmacologia , Cavéolas/efeitos dos fármacos , Ciclofilina A/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/enzimologia , Animais , Western Blotting , Cavéolas/enzimologia , Ciclofilina A/genética , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Músculo Liso Vascular/enzimologia , Mutação , Miócitos de Músculo Liso/enzimologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Oligopeptídeos , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Transporte Proteico , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875-fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.