RESUMO
Endothelial dysfunction is a common complication in diabetes in which endothelium-dependent vasorelaxation is impaired. The aim of this study was to examine the involvement of the TRPV4 ion channel in type 1 diabetic endothelial dysfunction and the possible association of endothelial dysfunction with reduced expression of TRPV4, endothelial nitric oxide synthase (eNOS) and caveolin-1. Male Wistar rats (350-450 g) were injected with 65 mg/kg i.p. streptozotocin (STZ) or vehicle. Endothelial function was investigated in aortic rings and mesenteric arteries using organ bath and myograph, respectively. TRPV4 function was studied with fura-2 calcium imaging in endothelial cells cultured from aortas from control and STZ treated rats. TRPV4, caveolin-1 and eNOS expression was investigated in these cells using immunohistochemistry. STZ-treated diabetic rats showed significant endothelial dysfunction characterised by impaired muscarinic-induced vasorelaxation (aortic rings: STZ-diabetics: Emax = 29.6 ± 9.3%; control: Emax = 77.2 ± 2.5% PË0.001), as well as significant impairment in TRPV4-induced vasorelaxation (aortic rings, 4αPDD STZ-diabetics: Emax = 56.0 ± 5.5%; control: Emax = 81.1 ± 2.1% PË0.001). Furthermore, STZ-diabetic primary aortic endothelial cells showed a significant reduction in TRPV4-induced intracellular calcium elevation (PË0.05) compared with the control group. This was associated with significantly lower expression of TRPV4, caveolin-1 and eNOS and this was reversed by insulin treatment of the endothelial cultures from STZ -diabetic rats. Taken together, these data are consistent with the hypothesis that signalling through TRPV4, caveolin-1, and eNOS is downregulated in STZ-diabetic aortic endothelial cells and restored by insulin treatment.
Assuntos
Caveolina 1/metabolismo , Diabetes Mellitus Experimental/metabolismo , Endotélio Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Insulina/farmacologia , Insulina/uso terapêutico , Masculino , Ratos , Ratos Wistar , EstreptozocinaRESUMO
Bowel dysfunction is a common non-motor symptom in Parkinson's disease (PD). The main contractile neurotransmitter in the GI tract is acetylcholine (ACh), while nitric oxide (NO) causes the relaxation of smooth muscle in addition to modulating ACh release. The aim of this study was to characterise functional and neurochemical changes in the isolated ileum of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated marmoset, an established model of PD motor dysfunction. While NO-synthase inhibitor L-NAME concentration dependently augmented the neurogenically-evoked contractions and inhibited the relaxations in normal tissues, it had no effects on the MPTP ileum. Immunohistochemical analyses of the myenteric plexus showed that ChAT-immunoreactivity (-ir) was significantly reduced and the density of the enteric glial cells as shown by SOX-10-ir was increased. However, no change in TH-, 5-HT-, VIP- or nNOS-ir was observed in the MPTP tissues. The enhancement of the neurogenically-evoked contractions and the inhibition of the relaxation phase by L-NAME in the control tissues is in line with NO's direct relaxing effect on smooth muscle and its indirect inhibitory effect on ACh release. The absence of the relaxation and the inefficacy of L-NAME in the MPTP tissues suggests that central dopaminergic loss dopamine may eventually lead to the impairment of NO signal coupling that affects bowel function, and this may be the result of a complex dysregulation at the level of the neuroeffector junction.
RESUMO
Diabetic vascular dysfunction is a major complication of diabetes. Methylglyoxal (MGO) is a dicarbonyl metabolite elevated in diabetic plasma that reacts with interstitial molecules to form advanced glycation end products (AGE). We investigated whether MGO affects the release of nitric oxide (NO) from rat aortic smooth muscle cells (ASMCs), and if L-arginine can prevent these effects of MGO. MGO was significantly elevated in serum from streptozotocin (STZ)-treated rats (121⯱â¯11.2⯵M) compared with vehicle control rats (27.5⯱â¯9.2⯵M). The pathological concentration of MGO (100⯵M) was then applied to investigate its effect on inducible nitric oxide synthase (iNOS) expression and NO release on interferon-gamma (IFN-γ) (100 IU/ml) and lipopolysaccharide (LPS) (100⯵g/ml)-stimulated control ASMCs. MGO (100⯵M) inhibited IFN-γ and LPS-stimulated iNOS expression through inhibiting Akt phosphorylation and inhibition of iNOS expression was prevented by L-arginine (100⯵M) co-treatment. These findings show for the first time that MGO inhibits IFN-γ and LPS-stimulated iNOS expression in ASMCs, in addition to inhibiting IFN-γ and LPS-induced Akt phosphorylation. The actions of MGO might contribute to the vascular dysfunction induced by MGO in diabetes.
Assuntos
Aorta/efeitos dos fármacos , Aorta/metabolismo , Óxido Nítrico/biossíntese , Aldeído Pirúvico/sangue , Estreptozocina/farmacologia , Animais , Aorta/citologia , Aorta/fisiologia , Arginina/farmacologia , Glicemia/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Músculo Liso Vascular/citologia , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Wistar , Vasoconstrição/efeitos dos fármacosRESUMO
BACKGROUND: Diabetes is a complex progressive disease characterized by chronic hyperglycaemia and dyslipidaemia associated with endothelial dysfunction. Oxidized LDL (Ox-LDL) is elevated in diabetes and may contribute to endothelial dysfunction. The aim of this study was to relate the serum levels of Ox-LDL with endothelial dysfunction in streptozotocin (STZ)-diabetic rats and to further explore the changes in endothelial nitric oxide synthase (eNOS) and caveolin-1 (CAV-1) expression in primary aortic endothelial cells. METHODS: Diabetes was induced with a single intraperitoneal injection of STZ in male Wistar rats. During the hyperglycaemic diabetes state serum lipid markers, aortic relaxation and aortic endothelial cell eNOS and CAV-1 protein expressions were measured. RESULTS: Elevated serum Ox-LDL (STZ 1486 ± 78.1 pg/mL vs control 732.6 ± 160.6 pg/mL, P < .05) was associated with hyperglycaemia (STZ 29 ± 0.9 mmol/L vs control: 7.2 ± 0.2 mmol/L, P < .001) and hypertriglyceridaemia (STZ 9.0 ± 1.5 mmol/L vs control: 3.0 ± 0.3 mmol/L, P < .01) in diabetic rats. A significant reduction was observed in STZ-diabetic aortic endothelial cell eNOS and CAV-1 of 40% and 30%, respectively, accompanied by a compromised STZ-diabetic carbachol-induced vasodilation (STZ 29.6 ± 9.3% vs control 77.2 ± 2.5%, P < .001). CONCLUSIONS: The elevated serum Ox-LDL in hyperglycaemic STZ-diabetic rats may contribute to diabetic endothelial dysfunction, possibly through downregulation of endothelial CAV-1 and eNOS.
Assuntos
Caveolina 1/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Dislipidemias/fisiopatologia , Endotélio Vascular/patologia , Hiperglicemia/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Doenças Vasculares/patologia , Animais , Endotélio Vascular/metabolismo , Hiperglicemia/metabolismo , Masculino , Óxido Nítrico/metabolismo , Estresse Oxidativo , Ratos , Ratos Wistar , Doenças Vasculares/metabolismoRESUMO
Bladder hyperreflexia is a common non-motor feature of Parkinson's disease. We now report on the contractility of the isolated primate detrusor strips devoid of nerve input and show that following MPTP, the amplitude and frequency of spontaneous contraction was increased. These responses were unaffected by dopamine D1 and D2 receptor agonists A77636 and ropinirole respectively. Contractions by exogenous carbachol, histamine or ATP were similar and no differences in the magnitude of noradrenaline-induced relaxation were seen in detrusor strip obtained from normal and MPTP-treated common marmosets (Callithrix jacchus). However, the neurogenic contractions following electrical field stimulation of the intrinsic nerves (EFS) were markedly greater in strips obtained from MPTP treated animals. EFS evoked non-cholinergic contractions following atropine were also greater but the contribution of the cholinergic innervation as a proportion of the overall contraction was smaller in the detrusor strips of MPTP treated animals, suggesting a preferential enhancement of the non-cholinergic transmission. Although dopaminergic mechanism has been proposed to underlie bladder hyperreflexia in MPTP-treated animals with intact bladder, the present data indicates that the increased neurogenically mediated contractions where no extrinsic innervation exists might be due to long-term adaptive changes locally as a result of the loss of the nigrostriatal output.
Assuntos
Intoxicação por MPTP/fisiopatologia , Contração Muscular , Reflexo Anormal , Bexiga Urinária/fisiopatologia , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Atropina/farmacologia , Benzopiranos/farmacologia , Callithrix , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/fisiologia , Agonistas de Dopamina/farmacologia , Feminino , Indóis/farmacologia , Masculino , Antagonistas Muscarínicos/farmacologia , Músculo Liso/inervação , Músculo Liso/fisiologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/inervaçãoRESUMO
A simple synthetic route affording 27%-85% yields of benzo[6,7][1,5]diazocino[2,1-a]isoindol-12(14H)-one ring systems from readily available 3-(2-oxo-2-phenylethyl) isobenzofuran-1(3H)-ones and 2-(aminomethyl)aniline starting materials in toluene and catalysed by p-toluene-sulfonic acid is developed. The ¹H- and (13)C-NMR spectra of the final products were assigned using a variety of one and two-dimensional NMR experiments. The distinction between the two potential isomers of the final products was made on the basis of heteronuclear multiple bond connectivity (HMBC) NMR spectra.
Assuntos
Isoindóis/síntese química , Isoindóis/química , Isomerismo , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
BACKGROUND: Diabetes mellitus (DM) is a pandemic metabolic disease characterized by a chronically elevated blood glucose concentration (hyperglycemia) due to insulin dysfunction. Approximately 50% of diabetics show diabetes complications by the time they are diagnosed. Vascular dysfunction, nephropathy and neuropathic pain are common diabetes complications. Chronic hyperglycemia contributes to reactive oxygen species (ROS) generation such as methylglyoxal (MGO). METHODS: Peer reviewed research papers were studied through bibliographic databases searching focused on review questions and inclusion/exclusion criteria. The reviewed papers were appraised according to the searching focus. The characteristics of screened papers were described, and a deductive qualitative content analysis methodology was applied to the included studies using a conceptual framework to yield this comprehensive systematic review. RESULTS: Sixty-six papers were included in this review. Eleven papers related methylglyoxal generation to carbohydrates metabolism, ten papers related lipid metabolism to methylglyoxal and 5 papers showed the proteolytic pathways that contribute to methylglyoxal generation. Methylglyoxal metabolism was derived from 7 papers. Descriptive figure 1 was drawn to explain methylglyoxal sources and how diabetes increases methylglyoxal generation. Furthermore, twenty-six papers related methylglyoxal to diabetes complications from which 9 papers showed methylglyoxal ability to induce insulin dysfunction, an effect which was described in schematic figure 2. Additionally, fifteen papers revealed methylglyoxal contribution to vascular dysfunction and 3 papers showed methylglyoxal to cause neuropathic pain. Methylglyoxal-induced vascular dysfunction was drawn in a comprehensive figure 3. This review correlated methylglyoxal with diabetes and diabetes complications which were summarised in table 1. CONCLUSION: The findings of this review suggesting methylglyoxal as an essential therapeutic target for managing diabetes in the future.
Assuntos
Diabetes Mellitus/metabolismo , Angiopatias Diabéticas/metabolismo , Neuropatias Diabéticas/metabolismo , Resistência à Insulina , Aldeído Pirúvico/metabolismo , Glicemia/metabolismo , Diabetes Mellitus/sangue , Angiopatias Diabéticas/sangue , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/metabolismo , Neuropatias Diabéticas/sangue , Humanos , Hiperglicemia/sangue , Hiperglicemia/metabolismo , Insulina/metabolismo , Neuralgia/sangue , Neuralgia/metabolismo , Aldeído Pirúvico/sangueRESUMO
Cannabinoid receptor 2 (CB2) agonists provide the potential for treating chronic pain states without CNS effects associated with CB1 receptor activation. Animal models suggest that they act mainly via non-neuronal cells, possibly inhibition of inflammatory cells in the periphery or CNS, or via release of beta-endorphin; however, the clinical relevance and mechanism of analgesic action is uncertain. Here, we demonstrate colocalisation of CB2 with CB1 and the capsaicin receptor TRPV1 in human dorsal root ganglion (DRG) sensory neurons and increased levels of CB2 receptors in human peripheral nerves after injury, particularly painful neuromas. In primary cultures of human DRG neurons, selective CB2 agonists blocked activation of inward cation currents and elevation of cytoplasmic Ca2+ in response to capsaicin. These inhibitory effects were reversed by GW818646X a CB2 antagonist, and 8-bromo cAMP, but not by SR141716 a CB1 antagonist, or naloxone. Thus CB2 receptor agonists functionally inhibited nociceptive signalling in human primary sensory neurons via a mechanism shared with opioids, of adenylyl cyclase inhibition, but not via mu-opioid receptors. We conclude that CB2 agonists deserve imminent clinical trials for nociceptive, inflammatory and neuropathic chronic pain, in which capsaicin or heat-activated responses via TRPV1 may provide a clinical marker.
Assuntos
Capsaicina/antagonistas & inibidores , Capsaicina/farmacologia , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/análise , Células Receptoras Sensoriais/química , Adolescente , Adulto , Idoso , Animais , Células CHO , Canabinoides/farmacologia , Células Cultivadas , Criança , Cricetinae , Cricetulus , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Receptor CB2 de Canabinoide/antagonistas & inibidores , Células Receptoras Sensoriais/efeitos dos fármacosRESUMO
Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.
Assuntos
Células/citologia , Animais , Fenômenos Biomecânicos , Calibragem , Sobrevivência Celular , Células Epiteliais/citologia , Eritrócitos/citologia , Modelos Biológicos , Miócitos Cardíacos/citologia , Neurônios/citologia , Pressão , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Transient receptor potential (TRP) receptors expressed by primary sensory neurons mediate thermosensitivity, and may play a role in sensory pathophysiology. We previously reported that human dorsal root ganglion (DRG) sensory neurons co-expressed TRPV1 and TRPV3, and that these were increased in injured human DRG. Related receptors TRPV4, activated by warmth and eicosanoids, and TRPM8, activated by cool and menthol, have been characterised in pre-clinical models. However, the role of TRPs in common clinical sensory neuropathies needs to be established. METHODS: We have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods. RESULTS: TRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons. CONCLUSION: The accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.
Assuntos
Neuropatias Diabéticas/metabolismo , Traumatismos dos Nervos Periféricos , Nervos Periféricos/metabolismo , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/metabolismo , Axônios/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Valores de ReferênciaRESUMO
The relationship between an initial mechanical event causing brain tissue deformation and delayed neurodegeneration in vivo is complex because of the multiplicity of factors involved. We have used a simplified brain surrogate based on rat hippocampal slices grown on deformable silicone membranes to study stretch-induced traumatic brain injury. Traumatic injury was induced by stretching the culture substrate, and the biological response characterized after 4 days. Morphological abnormalities consistent with traumatic injury in humans were widely observed in injured cultures. Synaptic function was significantly reduced after a severe injury. The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 attenuated neuronal damage, prevented loss of microtubule-associated protein 2 immunoreactivity and attenuated reduction of synaptic function. In contrast, the NMDA receptor antagonists 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) and GYKI53655, were neuroprotective in a moderate but not a severe injury paradigm. Nifedipine, an L-type voltage-dependent calcium channel antagonist was protective only after a moderate injury, whereas omega-conotoxin attenuated damage following severe injury. These results indicate that the mechanism of damage following stretch injury is complex and varies depending on the severity of the insult. In conclusion, the pharmacological, morphological and electrophysiological responses of organotypic hippocampal slice cultures to stretch injury were similar to those observed in vivo. Our model provides an alternative to animal testing for understanding the mechanisms of post-traumatic delayed cell death and could be used as a high-content screen to discover neuroprotective compounds before advancing to in vivo models.
Assuntos
Lesões Encefálicas/metabolismo , Canais de Cálcio/metabolismo , Hipocampo/metabolismo , Degeneração Neural/metabolismo , Fármacos Neuroprotetores/farmacologia , Receptores de Glutamato/metabolismo , Animais , Lesões Encefálicas/complicações , Lesões Encefálicas/fisiopatologia , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Hipocampo/fisiopatologia , Membranas Artificiais , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Neurológicos , Degeneração Neural/etiologia , Degeneração Neural/fisiopatologia , Fármacos Neuroprotetores/uso terapêutico , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Técnicas de Cultura de Órgãos , Estimulação Física/métodos , Ratos , Receptores de Glutamato/efeitos dos fármacos , Silicones , Estresse Mecânico , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Mechanosensitive ion channels convert external mechanical force into electrical and chemical signals in cells, but their physiological function in different tissues is not clearly understood. One reason for this is that there is as yet no satisfactory physiological method to stimulate these channels in living cells. Using the nanopipette-probe of the Scanning Ion Conductance Microscope (SICM), we have developed a new technique to apply local mechanical stimulus to living cells to an area of about 0.385 microm2, determined by the pipette diameter. Our method prevents any physical contact and damage to the cell membrane by use of a pressure jet applied via the nanopipette. The study used whole-cell patch-clamp recordings and measurements of intracellular Ca2+ concentration to validate the application of the mechanical stimulation protocols in human and rat dorsal root ganglia (DRG) sensory neurons. We were able, for the first time, to produce a non-contact, controlled mechanical stimulation on living neurites of human DRG neurons. Our methods will enable the identification and characterisation of compounds being developed for the treatment of clinical mechanical hypersensitivity states.
Assuntos
Gânglios Espinais/fisiologia , Neurônios Aferentes/fisiologia , Estimulação Física/instrumentação , Adulto , Animais , Sinalização do Cálcio/fisiologia , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Células Cultivadas , Dendritos/fisiologia , Eletrofisiologia , Gânglios Espinais/citologia , Humanos , Técnicas In Vitro , Masculino , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Neuritos/fisiologia , Estimulação Física/métodos , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos WistarRESUMO
Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.
Assuntos
Canais de Cátion TRPM/genética , Sequência de Bases , Primers do DNA/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade , Canais de Cátion TRPM/classificação , Distribuição TecidualRESUMO
We report the detailed expression profile of TRPM2 mRNA within the human central nervous system (CNS) and demonstrate increased TRPM2 mRNA expression at 1 and 4 weeks following ischemic injury in the rat transient middle cerebral artery occlusion (tMCAO) stroke model. Microglial cells play a key role in pathology produced following ischemic injury in the CNS and possess TRPM2, which may contribute to stroke-related pathological responses. We show that TRPM2 mRNA is present in the human C13 microglial cell line and is reduced by antisense treatment. Activation of C13 cells by interleukin-1beta leads to a fivefold increase of TRPM2 mRNA demonstrating transcriptional regulation. To confirm mRNA distribution correlated with functional expression, we combined electrophysiology, Ca2+ imaging, and antisense approaches. C13 microglia exhibited, when stimulated with hydrogen peroxide (H2O2), increased [Ca2+]i, which was reduced by antisense treatment. Moreover, patch-clamp recordings from C13 demonstrated that increased intracellular adenosine diphosphoribose (ADPR) or extracellular H2O2 induced an inward current, consistent with activation of TRPM2. In addition we confirm the functional expression of a TRPM2-like conductance in primary microglial cultures derived from rats. Activation of TRPM2 in microglia during ischemic brain injury may mediate key aspects of microglial pathophysiological responses.
Assuntos
Microglia/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Animais , Sequência de Bases , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Interleucina-1/farmacologia , Masculino , Microglia/efeitos dos fármacos , Artéria Cerebral Média , RNA Antissenso/administração & dosagem , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
BACKGROUND: The recent identification of the cold-menthol sensory receptor (TRPM8; CMR1), provides us with an opportunity to advance our understanding of its role in the pathophysiology of bladder dysfunction, and its potential mediation of the bladder cooling reflex. In this study, we report the distribution of the cool and menthol receptor TRPM8 in the urinary bladder in patients with overactive and painful bladder syndromes, and its relationship with clinical symptoms. METHODS: Bladder specimens obtained from patients with painful bladder syndrome (PBS, n = 16), idiopathic detrusor overactivity (IDO, n = 14), and asymptomatic microscopic hematuria (controls, n = 17), were immunostained using specific antibodies to TRPM8; nerve fibre and urothelial immunostaining were analysed using fibre counts and computerized image analysis respectively. The results of immunohistochemistry were compared between the groups and correlated with the Pain, Frequency and Urgency scores. RESULTS: TRPM8-immunoreactive staining was observed in the urothelium and nerve fibres scattered in the suburothelium. The nerve fibre staining was seen in fine-calibre axons and thick (myelinated) fibres. There was marked increase of TRPM8-immunoreactive nerve fibres in IDO (P = 0.0249) and PBS (P < 0.0001) specimens, compared with controls. A significantly higher number of TRPM8-immunoreactive axons were also seen in the IDO (P = 0.0246) and PBS (P < 0.0001) groups. Urothelial TRPM8 and TRPM8-immunoreactive thick myelinated fibres appeared unchanged in IDO and PBS. The relative density of TRPM8-immunoreactive nerve fibres significantly correlated with the Frequency (r = 0.5487, P = 0.0004) and Pain (r = 0.6582, P < 0.0001) scores, but not Urgency score. CONCLUSION: This study demonstrates increased TRPM8 in nerve fibres of overactive and painful bladders, and its relationship with clinical symptoms. TRPM8 may play a role in the symptomatology and pathophysiology of these disorders, and may provide an additional target for future overactive and painful bladder pharmacotherapy.
Assuntos
Canais de Cátion TRPM/metabolismo , Doenças da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Axônios/metabolismo , Hematúria , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Pessoa de Meia-Idade , Medição da Dor , Índice de Gravidade de Doença , Canais de Cátion TRPM/fisiologia , Bexiga Urinária/inervação , Bexiga Urinária/patologia , Doenças da Bexiga Urinária/patologia , Doenças da Bexiga Urinária/fisiopatologia , Urotélio/metabolismoRESUMO
TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.
Assuntos
Lisofosfolipídeos/química , Canais de Cátion TRPC/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Colagenases/metabolismo , DNA Complementar/metabolismo , Detergentes/farmacologia , Eletrofisiologia , Proteínas de Ligação ao GTP/química , Humanos , Íons , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , Técnicas de Patch-Clamp , Fosfolipídeos/química , Ligação Proteica , Isoformas de Proteínas , Espécies Reativas de Oxigênio , Transdução de Sinais , Canais de Cátion TRPC/fisiologia , Fatores de TempoRESUMO
Traumatic brain injury (TBI) is caused by rapid deformation of the brain, resulting in a cascade of pathological events and ultimately neurodegeneration. Understanding how the biomechanics of brain deformation leads to tissue damage remains a considerable challenge. We have developed an in vitro model of TBI utilising organotypic hippocampal slice cultures on deformable silicone membranes, and an injury device, which generates tissue deformation through stretching the silicone substrate. Our injury device controls the biomechanical parameters of the stretch via feedback control, resulting in a reproducible and equi-biaxial deformation stimulus. Organotypic cultures remain well adhered to the membrane during deformation, so that tissue strain is 93 and 86% of the membrane strain in the x- and y-axis, respectively. Cell damage following injury is positively correlated with strain. In conclusion, we have developed a unique in vitro model to study the effects of mechanical stimuli within a complex cellular environment that mimics the in vivo environment. We believe this model could be a powerful tool to study the acute phases of TBI and the induced cell degeneration could provide a good platform for the development of potential therapeutic approaches and may be a useful in vitro alternative to animal models of TBI.
Assuntos
Lesões Encefálicas , Hipocampo/lesões , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Animais , Modelos Animais de Doenças , Hipocampo/patologia , Masculino , Degeneração Neural/patologia , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Silicones , Estresse MecânicoRESUMO
TRPM2, a member of the TRP ion channel family, is expressed both in the brain and immune cells of the monocyte lineage. Functionally, it is unique in its activation by intracellular ADP-ribose and both oxidative and nitrosative stress. To date studies of this channel have concentrated on human recombinant channels and rodent native preparations. This provides the potential for cross-species complications in the interpretation of native tissue observations based on recombinant channel phenotype. Consequently, we have cloned and heterologously expressed rat TRPM2 (rTRPM2) in HEK293 cells. We find that, like hTRPM2, it responds to intracellular ADP-ribose in a manner dependent on extracellular Ca(2+). At the single channel level rTRPM2 is a slow gating, large conductance (84pS) channel that rapidly runs down in isolated membrane patches. Pharmacologically, rTRPM2 is rapidly and irreversibly blocked by clotrimazole (10muM), thus resembling hTRPM2 but not the TRPM2-like current of the rat-derived insulinoma CRI-G1, which exhibits reversible inhibition by this agent. We show that cultured rat striatal neurones exhibit an ADP-ribose-activated conductance at both the whole cell and single channel level. Pharmacologically this neuronal current can be irreversibly inhibited by clotrimazole. It is also sensitive to removal of extracellular Ca(2+), suggesting that it is mediated by TRPM2-containing channels. These data provide a functional characterisation of heterologously expressed rTRPM2 and demonstrate that, in addition to the previous descriptions in immune cells, microglia and insulinomas, a TRPM2-like conductance can be found in neurones derived from the rodent CNS.