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
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
(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
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
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
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
NO binds to the receptor sGC (soluble guanylyl cyclase), stimulating cGMP production. The NO-sGC-cGMP pathway is a key component in the cardiovascular system. Discrepancies in sGC activation and deactivation in vitro compared with in vivo have led to a search for endogenous factors that regulate sGC or assist in cellular localization. In our previous work, which identified Hsp (heat-shock protein) 70 as a modulator of sGC, we determined that PDI (protein disulfide-isomerase) bound to an sGC-affinity matrix. In the present study, we establish and characterize this interaction. Incubation of purified PDI with semi-purified sGC, both reduced and oxidized, resulted in different migration patterns on non-reducing Western blots indicating a redox component to the interaction. In sGC-infected COS-7 cells, transfected FLAG-tagged PDI and PDI CXXS (redox active site 'trap mutant') pulled down sGC. This PDI-sGC complex was resolved by reductant, confirming a redox interaction. PDI inhibited NO-stimulated sGC activity in COS-7 lysates, however, a PDI redox-inactive mutant PDI SXXS did not. Together, these data unveil a novel mechanism of sGC redox modulation via thiol-disulfide exchange. Finally, in SMCs (smooth muscle cells), endogenous PDI and sGC co-localize by in situ proximity ligation assay, which suggests biological relevance. PDI-dependent redox regulation of sGC NO sensitivity may provide a secondary control over vascular homoeostasis.
Assuntos
Guanilato Ciclase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Mapeamento de Interação de Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática/fisiologia , Guanilato Ciclase/química , Humanos , Camundongos , Oxirredução , Ligação Proteica/fisiologia , Isomerases de Dissulfetos de Proteínas/química , Mapeamento de Interação de Proteínas/métodos , Ratos , Receptores Citoplasmáticos e Nucleares/química , Guanilil Ciclase SolúvelRESUMO
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
Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.
Assuntos
Angiotensina II , Guanilato Ciclase/metabolismo , Hipertensão/induzido quimicamente , Músculo Liso Vascular/enzimologia , Óxido Nítrico/metabolismo , Estresse Oxidativo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Arteríolas/enzimologia , Arteríolas/fisiopatologia , Pressão Sanguínea , Linhagem Celular , GMP Cíclico/metabolismo , Cisteína , Modelos Animais de Doenças , Ativação Enzimática , Guanilato Ciclase/genética , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Mutação , Miócitos de Músculo Liso/enzimologia , Doadores de Óxido Nítrico/farmacologia , Nitrosação , Estresse Oxidativo/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais , Guanilil Ciclase Solúvel , Fatores de Tempo , Transfecção , Resistência Vascular , VasodilataçãoRESUMO
OBJECTIVE: Carbon monoxide (CO) is a weak soluble guanylyl cyclase stimulator, leading to transient increases in cGMP and vasodilation. The aim of the present work was to measure the effect of CO-releasing molecules (CORMs) on the cGMP/nitric oxide (NO) pathway and to evaluate how selected CORMs affect NO-induced vasorelaxation. METHODS AND RESULTS: Incubation of smooth muscle cells with some but not all of the CORMs caused a minor increase in cGMP levels. Concentration-response curves were bell-shaped, with higher CORMs concentrations producing lower increases in cGMP levels. Although exposure of cells to CORM-2 enhanced cGMP formation, we observed that the compound inhibited NO-stimulated cGMP accumulation in cells and NO-stimulated soluble guanylyl cyclase activity that could be reversed by superoxide anion scavengers. Reactive oxygen species generation from CORMs was confirmed using luminol-induced chemiluminescence and electron spin resonance. Furthermore, we observed that NO is scavenged by CORM-2. When used alone CORM-2 relaxed vessels through a cGMP-mediated pathway but attenuated NO donor-stimulated vasorelaxation. CONCLUSION: We conclude that the CORMs examined have context-dependent effects on vessel tone, as they can directly dilate blood vessels, but also block NO-induced vasorelaxation.
Assuntos
Aorta/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta/citologia , Aorta/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Guanilato Ciclase/metabolismo , Masculino , Modelos Animais , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vasodilatação/fisiologiaRESUMO
Nitric oxide (NO) is involved in the physiology and pathophysiology of the cardiovascular and neuronal systems via activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. Recent structural studies have allowed a better understanding of the residues that dictate the affinity and binding of NO to the heme and the resulting breakage of the bond between the heme iron and histidine 105 (H105) of the ß subunit of sGC. Still, it is unknown how the breakage of the iron-His bond translates into NO-dependent increased catalysis. Structural studies on homologous H-NOX domains in various states pointed to a role for movement of the H105 containing αF helix. Our modeling of the heme-binding domain highlighted conserved residues in the vicinity of H105 that could potentially regulate the extent to which the αF helix shifts and/or propagate the activation signal once the covalent bond with H105 has been broken. These include a direct interaction of αF helix residue aspartate 102 (D102) with the backbone nitrogen of F120. Mutational analysis of this region points to an essential role of the interactions in the vicinity of H105 for heme stability and identifies D102 as having a key role in NO activation following breakage of the iron-His bond.
Assuntos
Ácido Aspártico/metabolismo , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Heme/química , Heme/metabolismo , Óxido Nítrico/farmacologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Alanina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Guanilato Ciclase/genética , Indazóis/farmacologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Homologia de Sequência de Aminoácidos , Guanilil Ciclase SolúvelRESUMO
Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58-2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58-2667. The 2.3-A resolution structure of BAY 58-2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58-2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58-2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58-2667 binding causes a rotation of the alphaF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His(105)-heme covalent bond. The structure provides insights into how BAY 58-2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.
Assuntos
Benzoatos/química , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Heme/química , Mimetismo Molecular , Óxido Nítrico/química , Nostoc/enzimologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Ativadores de Enzimas/química , Modelos Moleculares , Mutagênese , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Guanilil Ciclase Solúvel , Relação Estrutura-AtividadeRESUMO
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
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
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
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