RESUMO
The transient receptor potential vanilloid channel 4 (TRPV4) is associated with the development of several pathologies, particularly gastric disorders. However, there are no studies associating this receptor with the pathophysiology of gastric erosions. The aim of this study was to investigate the role of TRPV4 in the development of ethanol-induced gastric damage in vivo. Gastric lesions were induced by ethanol in Swiss mice pretreated with TRPV4 antagonists, GSK2193874 (0.1; 0.3 and 0.9 mg/kg) or Ruthenium red (0.03; 0.1 or 0.3 mg/kg) or its agonist, GSK1016790A (0.9 mg/kg). Gastric mucosal samples were taken for histopathology, immunohistochemistry, atomic force microscopy and evaluation of antioxidant parameters. The gastric mucus content and TRPV4 mRNA expression were analyzed. Ethanol exposure induced upregulation of gastric mRNA and protein expression of TRPV4. TRPV4 blockade promoted gastroprotection against ethanol-induced injury on macro- and microscopic levels, leading to reduced hemorrhage, cell loss and edema and enhanced gastric mucosal integrity. Moreover, an increase in superoxide dismutase (SOD) and glutathione (GSH) activity was observed, followed by a decrease in malondialdehyde (MDA) levels. TRPV4 blockade during alcohol challenge reestablished gastric mucus content. The combination of TRPV4 agonist and ethanol revealed macroscopic exacerbation of gastric damage area. Our results confirmed the association of TRPV4 with the development of gastric injury, showing the importance of this receptor for further investigations in the field of gastrointestinal pathophysiology and pharmacology.
Assuntos
Úlcera Gástrica/metabolismo , Úlcera Gástrica/fisiopatologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismo , Animais , Edema/induzido quimicamente , Edema/metabolismo , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/lesões , Mucosa Gástrica/metabolismo , Glutationa/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Leucina/uso terapêutico , Masculino , Malondialdeído/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Rutênio Vermelho/farmacologia , Rutênio Vermelho/uso terapêutico , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/patologia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Superóxido Dismutase/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Ruthenium red (RR) is a non-selective antagonist of the temperature-sensitive Transient Receptor Potential (TRP) channels and it is an important pharmacological tool in thermoregulatory research. However, the effect of RR on thermoeffector activity is not well established. Here we evaluated the effect of RR on cold-defense thermoeffectors induced by menthol, an agonist of the cold-sensitive TRPM8 channel. Adult male Wistar rats were used. Epidermal treatment with menthol raised deep body temperature due to an increase in oxygen consumption (an index of thermogenesis), a reduction in heat loss index (an index of cutaneous vasoconstriction), and an induction in warmth-seeking behavior in a two-temperature choice apparatus. Pretreatment with RR attenuated the menthol-induced increase in deep body temperature and oxygen consumption, but it did not affect heat loss index and warmth-seeking behavior. To stimulate brown adipose tissue thermogenesis, rats were treated with CL 316,243, a potent and selective ß3-adrenoceptor agonist. CL 316,243 increased deep body temperature, which was attenuated by RR pretreatment. We conclude that RR reduces brown adipose tissue thermogenesis induced by menthol and CL 316,243, independent of effects at the thermal sensor level (i.e., TRPM8).
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Rutênio Vermelho/farmacologia , Termogênese , Tecido Adiposo Marrom/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Dioxóis/farmacologia , Masculino , Metanol/farmacologia , Ratos , Ratos Wistar , Canais de Cátion TRPM/metabolismoRESUMO
Drugs used to treat pain are associated with adverse effects, increasing the search for new drugs as an alternative treatment for pain. Therefore, we evaluated the antinociceptive behavior and possible neuromodulation mechanisms of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (CLF-1) isolated from Combretum leprosum leaves in zebrafish. Zebrafish (n = 6/group) were pretreated with CLF-1 (0.1 or 0.3 or 1.0 mg/mL; i.p.) and underwent nociception behavior tests. The antinociceptive effect of CFL-1 was tested for modulation by opioid (naloxone), nitrergic (L-NAME), nitric oxide and guanylate cyclase synthesis inhibitor (methylene blue), NMDA (Ketamine), TRPV1 (ruthenium red), TRPA1 (camphor), or ASIC (amiloride) antagonists. The corneal antinociceptive effect of CFL-1 was tested for modulation by TRPV1 (capsazepine). The effect of CFL-1 on zebrafish locomotor behavior was evaluated with the open field test. The acute toxicity study was conducted. CLF-1 reduced nociceptive behavior and corneal in zebrafish without mortalities and without altering the animals' locomotion. Thus, CFL-1 presenting pharmacological potential for the treatment of acute pain and corneal pain, and this effect is modulated by the opioids, nitrergic system, NMDA receptors and TRP and ASIC channels.
Assuntos
Analgésicos/farmacologia , Combretum/química , Locomoção/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Dor/prevenção & controle , Triterpenos/farmacologia , Canais Iônicos Sensíveis a Ácido/metabolismo , Amilorida/farmacologia , Analgésicos/isolamento & purificação , Animais , Cânfora/farmacologia , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Ketamina/farmacologia , Locomoção/fisiologia , Masculino , Azul de Metileno/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Naloxona/farmacologia , Nociceptividade/fisiologia , Dor/metabolismo , Dor/fisiopatologia , Medição da Dor , Extratos Vegetais/química , Folhas de Planta/química , Receptores de N-Metil-D-Aspartato/metabolismo , Rutênio Vermelho/farmacologia , Canais de Cátion TRPV/metabolismo , Triterpenos/isolamento & purificação , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²âº and Mg²âº. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³âº (10 µM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 µM) and SKF 96365 (250 µM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 µM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 µM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²âº signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.
Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Mecanotransdução Celular/fisiologia , Canais de Cátion TRPV/metabolismo , Amilorida/farmacologia , Compostos de Boro/farmacologia , Expressão Gênica , Humanos , Imidazóis/farmacologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Leucemia de Células T/fisiopatologia , Magnésio/metabolismo , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rutênio Vermelho/farmacologia , Sódio/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genéticaRESUMO
This study reports a pharmacological evaluation of anti-inflammatory and anti-ulcer activities of carvacrol, a phenolic monoterpene constituent of essential oils produced by oregano and other several aromatic plants and spices, in experimental models of edema induced by different phlogistic agents and gastric lesions induced by acetic acid. In models of paw edema induced by dextran or histamine, carvacrol was effective at 50 mg/kg (46% and 35%, respectively); in these models, cyproheptadine reduced edema formation (61% and 43%, respectively). In edema induced by substance P, carvacrol (100 mg/kg) and ruthenium red (3 mg/kg) also decreased the edema formation (46% and 40%, respectively). Carvacrol significantly reduced the ear edema induced by 12-O-tetradecanoylphorbol acetate and arachidonic acid at 0.1 mg per ear (43% and 33%, respectively), similar to indomethacin at 0.5 mg per ear or 2.0 mg per ear (55% and 57%, respectively). Carvacrol (at doses of 25, 50, and 100 mg/kg) showed a healing capacity on gastric lesions induced by acid acetic (60%, 91%, and 81%, respectively) after 14 days of treatment. These results suggest that carvacrol acts on different pharmacological targets, probably interfering in release and/or synthesis of inflammatory mediators, such as the prostanoids, and thus favoring the healing process for gastric ulcers.
Assuntos
Anti-Inflamatórios/farmacologia , Antiulcerosos/farmacologia , Inflamação/tratamento farmacológico , Monoterpenos/farmacologia , Óleos Voláteis/farmacologia , Origanum/química , Úlcera Gástrica/tratamento farmacológico , Animais , Ácido Araquidônico/efeitos adversos , Cimenos , Edema/induzido quimicamente , Edema/tratamento farmacológico , Feminino , Indometacina/efeitos adversos , Masculino , Camundongos , Ratos , Ratos Wistar , Rutênio Vermelho/farmacologia , Úlcera Gástrica/induzido quimicamente , Substância P/efeitos adversos , Acetato de Tetradecanoilforbol/efeitos adversos , Acetato de Tetradecanoilforbol/análogos & derivadosRESUMO
We have previously demonstrated that in renal cortical collecting duct cells (RCCD(1)) the expression of the water channel Aquaporin 2 (AQP2) raises the rate of cell proliferation. In this study, we investigated the mechanisms involved in this process, focusing on the putative link between AQP2 expression, cell volume changes, and regulatory volume decrease activity (RVD). Two renal cell lines were used: WT-RCCD(1) (not expressing aquaporins) and AQP2-RCCD(1) (transfected with AQP2). Our results showed that when most RCCD(1) cells are in the G(1)-phase (unsynchronized), the blockage of barium-sensitive K(+) channels implicated in rapid RVD inhibits cell proliferation only in AQP2-RCCD(1) cells. Though cells in the S-phase (synchronized) had a remarkable increase in size, this enhancement was higher and was accompanied by a significant down-regulation in the rapid RVD response only in AQP2-RCCD(1) cells. This decrease in the RVD activity did not correlate with changes in AQP2 function or expression, demonstrating that AQP2-besides increasing water permeability-would play some other role. These observations together with evidence implying a cell-sizing mechanism that shortens the cell cycle of large cells, let us to propose that during nutrient uptake, in early G(1), volume tends to increase but it may be efficiently regulated by an AQP2-dependent mechanism, inducing the rapid activation of RVD channels. This mechanism would be down-regulated when volume needs to be increased in order to proceed into the S-phase. Therefore, during cell cycle, a coordinated modulation of the RVD activity may contribute to accelerate proliferation of cells expressing AQP2.
Assuntos
Aquaporina 2/metabolismo , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Rim/citologia , Animais , Aquaporina 2/genética , Compostos de Bário/farmacologia , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Cloretos/farmacologia , Citometria de Fluxo , Pontos de Checagem da Fase G1 do Ciclo Celular , Microscopia de Vídeo , Pressão Osmótica , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Transporte Proteico , Ratos , Rutênio Vermelho/farmacologia , Fase S , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , TransfecçãoRESUMO
We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.
Assuntos
Aquaporina 2/metabolismo , Rim/citologia , Canais de Cátion TRPV/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Capsaicina/farmacologia , Tamanho Celular , Células Cultivadas , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Expressão Gênica , Pressão Osmótica , Forbóis/farmacologia , Ligação Proteica , Transporte Proteico , Ratos , Rutênio Vermelho/farmacologia , Estresse Fisiológico , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/genética , Moduladores de Tubulina/farmacologiaRESUMO
Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP3Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP3Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP3Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP3Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP3Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.
Assuntos
Inositol 1,4,5-Trifosfato/metabolismo , Oócitos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Bufo arenarum , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Oócitos/efeitos dos fármacos , Rutênio Vermelho/farmacologia , Rianodina/farmacologia , Interações Espermatozoide-Óvulo , Timerosal/farmacologiaRESUMO
Hyperalgesia and allodynia occur as a consequence of peripheral and central sensitization that follows sustained nociceptive activation. The cellular alterations associated to this state of nociceptive network hyperexcitability represent a form of neuronal plasticity, but they are not well understood because of its complexity in situ. In this study, after treating primary spinal neuron cultures with capsaicin (0.5-1 microM) for 48 h fluorimetric recordings were performed. The activation of TRPV1 receptors with capsaicin (0.5-1.0 microM) increased the frequency of calcium transients (0.03+/-0.002 Hz vs. 0.05+/-0.006 Hz, P<0.05), mediated by AMPAergic transmission, as well as the percent of neurons with activity (37+/-3% vs. 65+/-4%, P<0.05). The effect of capsaicin was long lasting and the neurons were found to be hyperfunctional and with increased levels of phosphorylated CREB (cAMP responsive element binding) even after 72 h of treatment with capsaicin (32+/-5% vs. 52+/-5%). The effect of capsaicin was blocked by capsazepine (1 microM), TTX (100 nM) and KN-62 (1 microM), but not by K252a (200 nM) or PD98059 (50 microM) indicating the involvement of TRPV1. The results suggest the participation of Ca2+, CaMKII and CREB on the prolonged enhancement of excitability following chronic exposure to capsaicin. Thus, it is likely that chronic TRPV1 activation is capable of inducing prolonged increases in neurotransmission mediated by glutamatergic receptors.
Assuntos
Cálcio/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Neurônios/efeitos dos fármacos , Medula Espinal/citologia , Canais de Cátion TRPV/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Análise de Variância , Animais , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Rutênio Vermelho/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Tetrodotoxina/farmacologiaRESUMO
The aim of this study was to measure the nociceptive response (avoidance latency) of the land snail Megalobulimus abbreviatus (N=8 in each group) after topical capsaicin exposure (0.1% and 0.5% in 20% ethanol) and to compare it to a well-studied stressful (50 degrees C) thermal stimulus model. We also tested if ruthenium red, and capsazepine, respectively nonselective and selective TRPV1 receptor antagonists, could modify both capsaicin- and thermal-evoked responses. Finally, animals were pretreated with morphine, naloxone or morphine plus naloxone prior to capsaicin stimuli. Latencies were measured when the animal lifted its head-foot complex 1 cm from the substrate. Data were compared using ANOVA and LSD post hoc, and the Student T Test (p<0.05). Capsaicin elicited dose-dependent withdrawal behavior. The capsaicin vehicle (20% ethanol) also evoked a less intense but significant avoidance reaction. Capsazepine and ruthenium red attenuated both capsaicin and heat withdrawal responses, when compared to vehicles. Morphine increased, and naloxone, either alone or in combination with morphine, reduced capsaicin-evoked latencies when compared to morphine or saline. These results indicate that the TRPV1 receptor plays a role in the nociceptive circuits of M. abbreviatus.