RESUMO
Camphor is known to potentiate both heat and cold sensations. Although the sensitization to heat could be explained by the activation of heat-sensitive transient receptor potential (TRP) channels TRPV1 and TRPV3, the camphor-induced sensitization to cooling remains unexplained. In this study, we present evidence for the activation of the cold- and menthol-sensitive channel transient receptor potential melastatin 8 (TRPM8) by camphor. Calcium transients evoked by camphor in HEK293 cells expressing human and rat TRPM8 are inhibited by the TRPM8 antagonists 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide and 2-aminoethyl diphenylborinate. Camphor also sensitized the cold-induced calcium transients and evoked desensitizing outward-rectifying currents in TRPM8-expressing HEK293 cells. In the presence of ruthenium red (a blocker of TRPV1, TRPV3, and TRPA1), the camphor sensitivity of cultured rat dorsal root ganglion neurons was highest in a subpopulation of cold- and icilin-sensitive neurons, strongly suggesting that camphor activates native TRPM8. Camphor has a dual action on TRPM8: it not only activates the channel but also inhibits its response to menthol. The icilin-insensitive chicken TRPM8 was also camphor insensitive. However, camphor was able to activate an icilin-insensitive human TRPM8 mutant channel. The activation and sensitization to cold of mammalian TRPM8 are likely to be responsible for the psychophysical enhancement of innocuous cold and "stinging/burning" cold sensations by camphor.
Assuntos
Cânfora/farmacologia , Pirimidinonas/farmacologia , Canais de Cátion TRPM/metabolismo , Animais , Sequência de Bases , Compostos de Boro/farmacologia , Cálcio/metabolismo , Cânfora/agonistas , Células Cultivadas , Galinhas/genética , Temperatura Baixa , Relação Dose-Resposta a Droga , Eletrofisiologia/métodos , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Mentol/farmacologia , Dados de Sequência Molecular , Ratos , Ratos Wistar , Rutênio Vermelho/farmacologia , Canais de Cátion TRPM/genéticaRESUMO
The global aquatic pet trade encompasses a wide diversity of freshwater and marine organisms. While relying on a continual supply of healthy, vibrant aquatic animals, few sustainability initiatives exist within this sector. Public aquariums overlap this industry by acquiring many of the same species through the same sources. End users are also similar, as many aquarium visitors are home aquarists. Here we posit that this overlap with the pet trade gives aquariums significant opportunity to increase the sustainability of the trade in aquarium fishes and invertebrates. Improving the sustainability ethos and practices of the aquatic pet trade can carry a conservation benefit in terms of less waste, and protection of intact functioning ecosystems, at the same time as maintaining its economic and educational benefits and impacts. The relationship would also move forward the goal of public aquariums to advance aquatic conservation in a broad sense. For example, many public aquariums in North America have been instrumental in working with the seafood industry to enact positive change toward increased sustainability. The actions include being good consumers themselves, providing technical knowledge, and providing educational and outreach opportunities. These same opportunities exist for public aquariums to partner with the ornamental fish trade, which will serve to improve business, create new, more ethical and more dependable sources of aquatic animals for public aquariums, and perhaps most important, possibly transform the home aquarium industry from a threat, into a positive force for aquatic conservation.
Assuntos
Aquicultura/métodos , Organismos Aquáticos , Comércio/economia , Conservação dos Recursos Naturais/métodos , Peixes , Animais de Estimação/economia , Animais , Conservação dos Recursos Naturais/economia , Árvores de Decisões , Especificidade da EspécieRESUMO
Objective: Emergency interim guidance from the British Society for Gastroenterology (BSG) states that a no-biopsy strategy is possible to diagnose coeliac disease (CD) in adults with elevated transglutaminase IgA antibody (TGA-IgA) levels. We aimed to determine if the suggested TGA-IgA ≥10× ULN is safe and robust in making the diagnosis in adult patients in Scotland. We also aimed to establish if any important co-diagnoses would be missed if no biopsy was performed. Method: All positive coeliac serology results for patients aged >15 years in Scotland in 2016 (Grampian 2019) were accessed. Data were collected on demographics, TGA-IgA titres, D1 sampling, histology and macroscopic findings at upper and lower gastrointestinal (GI) endoscopy. Results: 1037/1429 patients with positive serology proceeded to biopsy, of which 796/1037 (76.8%) were diagnosed as CD. A total of 320/322 (99.37%) patients with TGA-IgA ≥10× ULN were diagnosed as CD giving the cut-off a positive predictive value of 99.38%. No significant co-pathology was found at endoscopy in these patients. Conclusion: Our results show that a no-biopsy strategy using a cut-off of TGA-IgA ≥10× ULN is safe to diagnose CD and that no important pathology would be missed. The European Society for Paediatric Gastroenterology, Hepatology and Nutrition 2020 and BSG COVID-19 interim guidelines are applicable to adult patients in Scotland.
RESUMO
Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs - these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science - and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.
Assuntos
Materiais Biocompatíveis , Conservação dos Recursos Naturais/métodos , Criopreservação/métodos , Anfíbios , Animais , BiodiversidadeRESUMO
Nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functional silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.
Assuntos
Fenômenos Químicos , DNA/metabolismo , Nanodiamantes/química , Silanos/metabolismo , Difusão Dinâmica da Luz , Microscopia de Força Atômica , Solubilidade , SonicaçãoRESUMO
A cold- and menthol-activated ionic current has been described in sensory neurones, which probably has a role in temperature sensing. Here we describe the ion channels underlying this current. Cooling activated non-selective cation channels (conductance, about 22 pS; reversal potential, -4.2 mV) in outside-out patches from cold-sensitive rat dorsal root ganglion neurones, and their activity was strongly increased by menthol. The activation threshold was 17.9 degrees C, shifting to 24.3 degrees C in 100 microM (-)-menthol, about 10 degrees C colder than observed in intact neurones. Channels in excised patches did not adapt to sustained cooling, unlike the current in intact neurones. We conclude that the ion channels underlying the cold- and menthol-induced current are directly activated by these stimuli, although other modulatory factors appear to be important in determining threshold and adaptation.
Assuntos
Temperatura Baixa , Gânglios Espinais/fisiologia , Canais Iônicos/fisiologia , Mentol/farmacologia , Neurônios Aferentes/fisiologia , Pele/inervação , Sensação Térmica/fisiologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Animais , Células Cultivadas , Gânglios Espinais/citologia , Hipotermia Induzida , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/fisiologia , Neurônios Aferentes/citologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Ratos , Limiar Sensorial/efeitos dos fármacos , Limiar Sensorial/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sensação Térmica/efeitos dos fármacosRESUMO
The mechanism of sympathetic - sensory coupling after nerve injury is still not well understood. We have studied the changes in resting potential and excitability of sensory neurones induced by adrenergic stimulation, using whole-cell and perforated-patch recordings in cultured dorsal root ganglion neurones from normal rats. Adrenaline (1-100 microM) depolarized 18 of 39 neurones (46%) and hyperpolarized seven neurones (18%); excitability was increased and decreased, respectively. Stimulating the neurones with 10 microM phenylephrine (alpha(1)-agonist) induced depolarization and increased excitability, while 10 microM isoprenaline (beta-agonist) induced hyperpolarization and reduced excitability. We conclude that alpha(1)- and beta-receptors have opposing effects on membrane potential and excitability in cultured dorsal root ganglion neurones, and the differing effects of adrenaline can be explained by different degrees of expression of each receptor type.
Assuntos
Gânglios Espinais/citologia , Neurônios Aferentes/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos beta/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Axotomia , Células Cultivadas , Epinefrina/farmacologia , Isoproterenol/farmacologia , Masculino , Neuralgia/fisiopatologia , Neurônios Aferentes/citologia , Técnicas de Patch-Clamp , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Simpatomiméticos/farmacologiaRESUMO
Whole-cell and single-channel recordings from rat dorsal root ganglion neurones were used to investigate the temperature dependence of currents through the capsaicin receptor (vanilloid receptor 1, VR1). Reducing the temperature from 31 to 14 degrees C inhibited the current induced by 0.5 microM capsaicin by 80%. The Q(10) (temperature coefficient over a 10 degrees C range) of the whole-cell capsaicin-induced current was 2.3 between 10 and 30 degrees C. Single-channel recordings showed that this inhibition by cooling was due to a marked reduction in the open probability (Q(10)=8.2 between 10 and 30 degrees C). This effect can explain the pain relief and reduction in inflammation caused by strong cooling of the skin.
Assuntos
Capsaicina/farmacologia , Gânglios Espinais/fisiologia , Inflamação/metabolismo , Neurônios Aferentes/fisiologia , Nociceptores/fisiologia , Dor/metabolismo , Receptores de Droga/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Células Cultivadas , Temperatura Baixa , Crioterapia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Hipotermia Induzida , Inflamação/fisiopatologia , Neurônios Aferentes/citologia , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/citologia , Nociceptores/efeitos dos fármacos , Dor/fisiopatologia , Manejo da Dor , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Receptores de Droga/efeitos dos fármacos , Canais de Cátion TRPVRESUMO
Two patients with rare sites of metastatic pleural mesothelioma are presented and the literature on similar cases is examined. One patient developed colonic metastases from a sarcomatoid mesothelioma. Another patient with epithelioid mesothelioma developed perineal metastases.
Assuntos
Neoplasias do Colo/secundário , Mesotelioma/secundário , Neoplasias Pélvicas/secundário , Períneo/patologia , Neoplasias Pleurais/patologia , Idoso , Neoplasias do Colo/diagnóstico por imagem , Humanos , Masculino , Mesotelioma/diagnóstico por imagem , Pessoa de Meia-Idade , Neoplasias Pélvicas/diagnóstico por imagem , Períneo/diagnóstico por imagem , Neoplasias Pleurais/diagnóstico por imagem , RadiografiaRESUMO
The primary afferent nociceptive neuron has recently attracted major research interest because of the cloning of very selectively expressed and well-conserved ion channel genes. All parts of the neuron, sensory terminals, axon and cell body, are accessible to validated research techniques in vitro using various isolated tissues or cells taken from laboratory animals. Single-unit recording and measuring stimulated calcitonin gene-related peptide (CGRP) release as well as patch-clamping and calcium imaging of cultured sensory neurons provide different kinds of information, and no model alone answers all questions. In combination, however, consistent results and complementary evidence form a solid basis for translational research to follow.
Assuntos
Eletrofisiologia , Nociceptores , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Gânglios Espinais/citologia , Humanos , Canais Iônicos/metabolismo , Camundongos , Nociceptores/química , Nociceptores/citologia , Nociceptores/metabolismo , RatosRESUMO
Neurotrophic factors (NTFs), beside regulating neuronal survival in the central and peripheral nervous system, are also involved in the modulation of neuronal function in the adult animal. Both brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) levels are altered in pathological pain states, and exogenous BDNF and GDNF have multiple effects on pain behavior, depending on the animal model (i.e. inflammatory vs. neuropathic). Thermally gated TRP channels TRPM8, TRPA1 and TRPV1 play a significant role in pain signaling and their pattern and level of expression as well as their biophysical properties are altered in chronic pain states. Our aim was to investigate the effect of long-term and acute exposure to BDNF and GDNF on the functional expression of these thermoTRP channels in cultured rat dorsal root ganglion (DRG) neurons. We found that while BDNF treatment primarily increased the fraction of capsaicin-sensitive (TRPV1-expressing) neurons, GDNF exposure led to an increase in the allyl isothiocyanate (AITC)-responding (TRPA1-expressing) population. Moreover, BDNF treatment increased the amplitude of the response to both AITC and capsaicin. Acute treatment with both NTFs leads to a reduction in the magnitude of tachyphylaxis to noxious stimuli (heat and AITC). Overall, our data provides evidence for a role of BDNF and GDNF in regulating the pattern of expression and level of activity of the transducer channels TRPA1 and TRPV1, leading to enhanced neuronal sensitivity to painful stimuli and increased co-expression of thermoTRP channels.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Gânglios Espinais/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Dor/metabolismo , Canais de Cátion TRPC/metabolismo , Compostos Alílicos/farmacologia , Animais , Anquirinas , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Temperatura Baixa , Gânglios Espinais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Temperatura Alta , Humanos , Isocianatos/farmacologia , Dor/induzido quimicamente , Ratos , Ratos Wistar , Fármacos do Sistema Sensorial/farmacologia , Canal de Cátion TRPA1 , Canais de Cátion TRPC/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/metabolismoRESUMO
The interaction between cold sensitivity and inflammation in mammals is not entirely understood. We have used adult rat dorsal root ganglion neurones in primary culture together with calcium microfluorimetry to assess the effects of selected inflammatory mediators on cold responses of cold- and menthol-sensitive (most likely TRPM8-expressing) neurones. We observed a high degree of functional co-expression of TRPM8, the receptors for the inflammatory agents bradykinin, prostaglandin E2 and histamine, and TRPA1 in cultured sensory neurones. Treatment with either bradykinin or prostaglandin E2 led to a reduction in the amplitude of the response to cooling and shifted the threshold temperature to colder values, and we provide evidence for a role of protein kinases C and A, respectively, in mediating these effects. In both cases the effects were mainly restricted to the subgroups of cold- and menthol-sensitive cells which had responded to the application of the inflammatory agents at basal temperature. This desensitization of cold-sensitive neurones may enhance inflammatory pain by removing the analgesic effects of gentle cooling.
Assuntos
Gânglios Espinais/fisiologia , Inflamação/fisiopatologia , Neurônios Aferentes/fisiologia , Nociceptores/fisiologia , Dor/fisiopatologia , Sensação Térmica/fisiologia , Analgesia/métodos , Animais , Anquirinas , Antipruriginosos/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Células Cultivadas , Temperatura Baixa , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inflamação/induzido quimicamente , Mediadores da Inflamação/farmacologia , Mentol/farmacologia , Nociceptores/efeitos dos fármacos , Dor/induzido quimicamente , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Tempo de Reação/fisiologia , Limiar Sensorial/efeitos dos fármacos , Limiar Sensorial/fisiologia , Canal de Cátion TRPA1 , Canais de Cátion TRPC , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Sensação Térmica/efeitos dos fármacosRESUMO
Cold sensing in mammals is heterogeneous and more than one type of receptor molecule is likely to be involved in the transduction process. Most features of innocuous cold receptors have been explained by TRPM8, the cold and menthol receptor, but their fast adaptation to cooling has not yet been reproduced in cellular systems. In this study we have used a newly developed system for applying fast thermal stimuli to dissociated dorsal root ganglia (DRG) neurons from young rats (150-200 g) in primary culture. We describe a novel type of cold-sensitive rat DRG neuron with rapid adaptation to cooling. These cells (4.3% of the total DRG population) do not express either TRPM8 or the other cold-activated TRP channel, TRPA1, and the epithelial sodium channel (ENaC) is not involved in their transduction. Increases in intracellular calcium induced by cooling in rapidly adapting neurons are caused by calcium entry. These neurons express a large and rapidly adapting cold-induced inward current with a time constant of adaptation in the seconds range, and may correspond to the rapidly adapting cold receptors described in vivo.
Assuntos
Temperatura Baixa , Gânglios Espinais/metabolismo , Neurônios Aferentes/metabolismo , Sensação Térmica/fisiologia , Adaptação Fisiológica/fisiologia , Amilorida/farmacologia , Animais , Anquirinas , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Cânfora/farmacologia , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Mentol/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Canal de Cátion TRPA1 , Canais de Cátion TRPC , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/metabolismo , Fatores de TempoRESUMO
Cooling is sensed by peripheral thermoreceptors, the main transduction mechanism of which is probably a cold- and menthol-activated ion channel, transient receptor potential (melastatin)-8 (TRPM8). Stronger cooling also activates another TRP channel, TRP (ankyrin-like)-1, (TRPA1), which has been suggested to underlie cold nociception. This review examines the roles of these two channels and other mechanisms in thermal transduction. TRPM8 is activated directly by gentle cooling and depolarises sensory neurones; its threshold temperature (normally approximately 26-31 degrees C in native neurones) is very flexible and it can adapt to long-term variations in baseline temperature to sensitively detect small temperature changes. This modulation is enabled by TRPM8's low intrinsic thermal sensitivity: it is sensitised to varying degrees by its cellular context. TRPM8 is not the only thermosensitive element in cold receptors and interacts with other ionic currents to shape cold receptor activity. Cold can also cause pain: the transduction mechanism is uncertain, possibly involving TRPM8 in some neurones, but another candidate is TRPA1 which is activated in expression systems by strong cooling. However, native neurones that appear to express TRPA1 respond very slowly to cold, and TRPA1 alone cannot account readily for cold nociceptor activity or cold pain in humans. Other, as yet unknown, mechanisms of cold nociception are likely.
Assuntos
Canais de Cálcio/fisiologia , Temperatura Baixa , Proteínas do Tecido Nervoso/fisiologia , Nociceptores/fisiologia , Canais de Cátion TRPM/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Adaptação Fisiológica , Animais , Clonagem Molecular , Humanos , Mentol/metabolismo , Mentol/farmacologia , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Canal de Cátion TRPA1 , Canais de Cátion TRPM/efeitos dos fármacosRESUMO
Skin temperature is sensed by peripheral thermoreceptors. Using the neuronal soma in primary culture as a model of the receptor terminal, we have investigated the mechanisms of cold transduction in thermoreceptive neurones from rat dorsal root ganglia. Cold-sensitive neurones were pre-selected by screening for an increase in [Ca(2+)](i) on cooling; 49 % of them were also excited by 0.5 microM capsaicin. Action potentials and voltage-gated currents of cold-sensitive neurones were clearly distinct from those of cold-insensitive neurones. All cold-sensitive neurones expressed an inward current activated by cold and sensitised by (-)-menthol, which was absent from cold-insensitive neurones. This current was carried mainly by Na(+) ions and caused a depolarisation on cooling accompanied by action potentials, inducing voltage-gated Ca(2+) entry; a minor fraction of Ca(2+) entry was voltage-independent. Application of (-)-menthol shifted the threshold temperatures of the cold-induced depolarisation and the inward current to the same extent, indicating that the cold- and menthol-activated current normally sets the threshold temperature for depolarisation during cooling. The action of menthol was stereospecific, with the (+)-isomer being a less effective agonist than the (-)-isomer. Extracellular Ca(2+) modulated the cold- and menthol-activated current in a similar way to its action on intact cold receptors: lowered [Ca(2+)](o) sensitised the current, while raised [Ca(2+)](o) antagonised the menthol-induced sensitisation. During long cooling pulses the current showed adaptation, which depended on extracellular Ca(2+) and was mediated by a rise in [Ca(2+)](i). This adaptation consisted of a shift in the temperature sensitivity of the channel. In capsaicin-sensitive neurones, capsaicin application caused a profound depression of the cold-activated current. Inclusion of nerve growth factor in the culture medium shifted the threshold of the cold-activated current towards warmer temperatures. The current was blocked by 50 microM capsazepine and 100 microM SKF 96365. We conclude that the cold- and menthol-activated current is the major mechanism responsible for cold-induced depolarisation in DRG neurones, and largely accounts for the known transduction properties of intact cold receptors.
Assuntos
Sinalização do Cálcio/fisiologia , Temperatura Baixa , Gânglios Espinais/fisiologia , Mentol/farmacologia , Termorreceptores/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/farmacologia , Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Capsaicina/farmacologia , Eletrofisiologia , Corantes Fluorescentes , Gânglios Espinais/efeitos dos fármacos , Imidazóis/farmacologia , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Mentol/química , Fator de Crescimento Neural/farmacologia , Compostos Orgânicos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Sódio/fisiologia , Estereoisomerismo , Termorreceptores/efeitos dos fármacosRESUMO
Cold sensing in mammals is not completely understood, although significant progress has been made recently with the cloning of two cold-activated ion channels, TRPM8 and TRPA1. We have used rat DRG neurons in primary culture and calcium fluorimetry to identify distinct populations of cold-sensitive neurons, which may underlie different functions. Menthol sensitivity clearly separated two classes of cold-responding neurons. One group was menthol-sensitive (MS), was activated at warmer temperatures and responded faster and with a larger increase in intracellular calcium concentration during cooling; the fraction of MS neurons in culture and their cold sensitivity were both increased in the presence of nerve growth factor. Neurons in the menthol-insensitive (MI) group required stronger cooling for activation than MS cells and neither their proportion nor their cold sensitivity were significantly altered by nerve growth factor. The two groups of cold-sensitive neurons also had different pharmacology. A larger fraction of MS cells were capsaicin-sensitive and coexpression of menthol and capsaicin sensitivity was observed in the absence of NGF. MI neurons were not stimulated by the super-cooling agent icilin or by the irritant mustard oil. Taken together these findings support a picture in which TRPM8 is the major player in detecting gentle cooling, while TRPA1 does not seem to be involved in cold sensing by MI neurons, at least in the temperature range between 32 and 12 degrees C.
Assuntos
Canais de Cálcio/metabolismo , Temperatura Baixa , Gânglios Espinais/metabolismo , Canais Iônicos/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Neural/metabolismo , Neurônios Aferentes/metabolismo , Sensação Térmica/genética , Animais , Anquirinas , Cálcio/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Capsaicina/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Mentol/farmacologia , Mostardeira , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , Fator de Crescimento Neural/farmacologia , Neurônios Aferentes/classificação , Neurônios Aferentes/efeitos dos fármacos , Extratos Vegetais/farmacologia , Óleos de Plantas , Pirimidinonas/farmacologia , Ratos , Canal de Cátion TRPA1 , Canais de Cátion TRPC , Canais de Cátion TRPM , Canais de Potencial de Receptor TransitórioRESUMO
We have analyzed repellent signal processing in Escherichia coli by flash photorelease of leucine from photolabile precursors. We found that 1). response amplitudes of free-swimming cell populations increased with leucine jump concentration, with an apparent Hill coefficient of 1.3 and a half-maximal dose of 14.4 microM; 2). at a 0-0.5 mM leucine concentration jump sufficient to obtain a saturation motile response, the swimming cell response time of approximately 0.05 s was several-fold more rapid than the motor response time of 0.39 +/- 0.18 s measured by following the rotation of cells tethered by a single flagellum to quartz coverslips; and 3). the motor response time of individual cells was correlated with rotation bias but not cell size. These results provide information on amplification, rate-limiting step, and flagellar bundle mechanics during repellent signal processing. The difference between the half-maximal dose for the excitation response and the corresponding value reported for adaptation provides an estimate of the increase in the rate of formation of CheYP, the phosphorylated form of the signal protein CheY. The estimated increase gives a lower limit receptor kinase coupling ratio of 6.0. The magnitude and form of the motor response time distribution argue for it being determined by the poststimulus switching probability rather than CheYP turnover, diffusion, or binding. The temporal difference between the tethered and swimming cell response times to repellents can be quantitatively accounted for and suggests that one flagellum is sufficient to cause a measurable change of direction in which a bacterium swims.
Assuntos
Proteínas de Bactérias/fisiologia , Quimiotaxia/fisiologia , Escherichia coli/fisiologia , Flagelos/fisiologia , Proteínas de Membrana/fisiologia , Tempo de Reação/fisiologia , Proteínas de Escherichia coli , Leucina/química , Proteínas Quimiotáticas Aceptoras de Metil , FosforilaçãoRESUMO
We have synthesized a set of ATP and ADP analogues that have a fluorophore linked to the nucleotide via the 3'-position of the ribose moiety. Combinations of three different coumarins are each attached via different length linkers. A linker based on propylenediamine increases the separation between the nucleotide and fluorophore relative to that of the previously reported ethylenediamine-linked coumarin nucleotides [Webb, M. R., and Corrie, J. E. T. (2001) Biophys. J. 81, 1562-1569]. A synthesis of 3'-amino-3'-deoxyATP is described using a combination of chemical and enzymatic procedures, mostly from published methods for synthesis of this compound but with some modifications that improved the convenience of the experimental procedures. This compound is used as a basis of a series of analogues with effectively a zero-length linker. Fluorescence properties of all these analogues are described, together with the kinetics of their interaction with rabbit skeletal myosin subfragment 1 in the presence and absence of actin. One particular analogue, deac-aminoATP [3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine 5'-triphosphate], shows a 17-fold enhancement of fluorescence upon binding to this (skeletal) myosin II. As the diphosphate, it exhibits a large signal change upon dissociation from the actomyosin, with kinetics similar to those of natural ADP. The ability of this set of analogues to produce large signals indicated potential uses when scarce proteins are studied in small amounts.