Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons.

C. elegans OSM-9 is a TRPV channel protein involved in sensory transduction and adaptation. Here, we show that distinct sensory functions arise from different combinations of OSM-9 and related OCR TRPV proteins. Both OSM-9 and OCR-2 are essential for several forms of sensory transduction, including olfaction, osmosensation, mechanosensation, and chemosensation. In neurons that express both OSM-9 and OCR-2, tagged OCR-2 and OSM-9 proteins reside in sensory cilia and promote each other's localization to cilia. In neurons that express only OSM-9, tagged OSM-9 protein resides in the cell body and acts in sensory adaptation rather than sensory transduction. Thus, alternative combinations of TRPV proteins may direct different functions in distinct subcellular locations. Animals expressing the mammalian TRPV1 (VR1) channel in ASH nociceptor neurons avoid the TRPV1 ligand capsaicin, allowing selective, drug-inducible activation of a specific behavior.

Molecular aspects of ATP-sensitive K+ channels in the cardiovascular system and K+ channel openers.

ATP-sensitive K+ (K(ATP)) channels are inhibited by intracellular ATP (ATPi) and activated by intracellular nucleoside diphosphates and thus, provide a link between cellular metabolism and excitability. K(ATP) channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K(ATP) channel appears to be activated during ischemic or hypoxic conditions, and may be responsible for the increase of K+ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective, as well as proarrhythmic, effects. These channels are clearly heterogeneous. The cardiac K(ATP) channel is the prototype of K(ATP) channels possessing approximately 80 pS of single-channel conductance in the presence of approximately 150 mM extracellular K+ and opens spontaneously in the absence of ATPi. A vascular K(ATP) channel called a nucleoside diphosphate-dependent K+ (K(NDP)) channel exhibits properties significantly different from those of the cardiac K(ATP) channel. The K(NDP) channel has the single-channel conductance of approximately 30-40 pS in the presence of approximately 150 mM extracellular K+, is closed in the absence of ATPi, and requires intracellular nucleoside di- or triphosphates, including ATPi to open. Nevertheless, K(ATP) and K(NDP) channels are both activated by K+ channel openers, including pinacidil and nicorandil, and inhibited by sulfonylurea derivatives such as glibenclamide. It recently was found that the cardiac K(ATP) channel is composed of a sulfonylurea receptor (SUR)2A and a two-transmembrane-type K+ channel subunit Kir6.2, while the vascular K(NDP) channel may be the complex of SUR2B and Kir6.1. By precisely comparing the functional properties of the SUR2A/Kir6.2 and the SUR2B/Kir6.1 channels, we shall show that the single-channel characteristics and pharmacological properties of SUR/Kir6.0 channels are determined by Kir and SUR subunits, respectively, while responses to intracellular nucleotides are determined by both SUR and Kir subunits.

Crystal structure of human rhinovirus serotype 1A (HRV1A).

The structure of human rhinovirus 1A (HRV1A) has been determined to 3.2 A resolution using phase refinement and extension by symmetry averaging starting with phases at 5 A resolution calculated from the known human rhinovirus 14 (HRV14) structure. The polypeptide backbone structures of HRV1A and HRV14 are similar, but the exposed surfaces are rather different. Differential charge distribution of amino acid residues in the "canyon", the putative receptor binding site, provides a possible explanation for the difference in minor versus major receptor group specificities, represented by HRV1A and HRV14, respectively. The hydrophobic pocket in VP1, into which antiviral compounds bind, is in an "open" conformation similar to that observed in drug-bound HRV14. Drug binding in HRV1A does not induce extensive conformational changes, in contrast to the case of HRV14.

Distribution of the vanilloid receptor (VR1) in the gastrointestinal tract.

The gastrointestinal (GI) tract responds to a variety of stimuli through local and centrally mediated pathways. Changes in the intestinal microenvironment are sensed by vagal, spinal, and intrinsic primary afferent fibers. Sensory nerve endings located close to the lumen of the GI tract respond to pH, chemical composition of lumenal contents, or distortion of the mucosa. Afferents within the muscle layers are thought to be tension sensitive, whereas those located within the myenteric plexus are also thought to respond to changes in chemical composition and humoral substances. Subpopulations of these afferent fibers are activated by capsaicin. However, the exact location of these nerves is currently not known. The vanilloid receptor (VR1) is a nonselective cation channel that is activated by capsaicin, acid, and temperature. Antibodies to VR1 make it possible to determine the location of these afferents, their morphology, and their relationships with enteric nerves and other cell types in the GI tract. VR1-like immunoreactivity was observed on nerves within myenteric ganglia and interganglionic fiber tracts throughout the GI tract. VR1 nerves were also observed within the muscle layers and had an irregular profile, with varicose-like swellings along their lengths. Blood vessels within the GI wall had VR1-immunoreactive nerve fibers associated with them. VR1-like nerves and other immunopositive cells were also observed within the mucosa. In summary, VR1-like immunoreactivity was found in several locations within the GI tract and may provide sensory integration of chemical, physical, or inflammatory stimuli. VR1-like fibers appear to be predominantly spinal in origin, but a few vagal VR1-like fibers exist in the stomach.

Pre- and postsynaptic localizations of the CB1 cannabinoid receptor in the dorsal horn of the rat spinal cord.

Several lines of evidence show that endogenous and exogenous cannabinoids modulate pain transmission at the spinal level through specific cannabinoid-1 (CB1) receptors. Since anatomical data concerning spinal CB1 receptors are rather contradictory, we studied the cellular and subcellular localizations of the CB1 receptors by immunocytochemistry. Results show a dual pre- and postsynaptic localization of CB1 receptors. Presynaptic receptors are evidenced by the labeling of (1) heterogeneous dorsal root ganglion neurons and (2) axons of Lissauer's tract. Postsynaptic receptors are shown by the labeling of numerous interneurons in the outer part of lamina II. Double immunolabelings show that lamina II outer CB1 neurons, probably islet cells, may also contain GABA or nitric oxide synthase. Numerous CB1-containing neurons in lamina X are also immunostained with anti-nitric oxide synthase (NOS) antibody. Under the electron microscope, CB1 immunoreactivity is exclusively localized postsynaptically in both somatic and dendritic compartments. The absence of labeling on primary afferent axon terminals is discussed and compared to the absence of labeling on terminals or vesicle-containing dendrites of islet cells, where a presynaptic localization was expected according to data of the literature.

The vanilloid receptor-1 is expressed in rat spinal dorsal horn astrocytes.

The vanilloid receptor-1 (TRPV1), expressed by nociceptive fibers, is a transducer of thermal and chemical nociceptive messages. However, endogenous ligands excite TRPV1 receptors localized on central nociceptive terminals and interneurons. Using immunocytochemistry at the ultrastructural level, we show that TRPV1 is also expressed in spinal glial cells characterized as astrocyte by double labeling with glial fibrillary acid protein. Quantification of the labeling shows that the most numerous labeling is neuronal and that 7% of the total TRPV1 labeling is localized in astrocytes. The total absence of staining in TRPV1 knock out mice strongly suggests that true TRPV1 protein is present in astrocytes. The localization of TRPV1-containing astrocytes apposed to nociceptive C-terminals suggests that they may be involved in the control of pain transmission.

Tetrameric structure of SUR2B revealed by electron microscopy of oriented single particles.

The ATP-sensitive potassium (K(ATP)) channel is a hetero-octameric complex that links cell metabolism to membrane electrical activity in many cells, thereby controlling physiological functions such as insulin release, muscle contraction and neuronal activity. It consists of four pore-forming Kir6.2 and four regulatory sulfonylurea receptor (SUR) subunits. SUR2B serves as the regulatory subunit in smooth muscle and some neurones. An integrative approach, combining electron microscopy and homology modelling, has been used to obtain information on the structure of this large (megadalton) membrane protein complex. Single-particle electron microscopy of purified SUR2B tethered to a lipid monolayer revealed that it assembles as a tetramer of four SUR2B subunits surrounding a central hole. In the absence of an X-ray structure, a homology model for SUR2B based on the X-ray structure of the related ABC transporter Sav1866 was used to fit the experimental images. The model indicates that the central hole can readily accommodate the transmembrane domains of the Kir tetramer, suggests a location for the first transmembrane domains of SUR2B (which are absent in Sav1866) and suggests the relative orientation of the SUR and Kir6.2 subunits.

Limits and possibilities experienced by nurses in the treatment of women with chronic venous ulcers / Límites y posibilidades vividas por enfermeras en el tratamiento de mujeres con úlcera venosa crónica / Limites e possibilidades vivenciados por enfermeiras no tratamento de mulheres com úlcera venosa crônica

Objective To understand the experiences and expectations of nurses in the treatment of women with chronic venous ulcers. Method Phenomenological research was based on Alfred Schütz, whose statements were obtained in January, 2012, through semi-structured interviews with seven nurses. Results The nurse reveals the difficulties presented by the woman in performing self-care, the perceived limitations in the treatment anchored in motivation, and the values and beliefs of women. It showed professional frustration because venous leg ulcer recurrence, lack of inputs, interdisciplinary work and training of nursing staff. There was an expected adherence to the treatment of women, and it emphasized the need for ongoing care, supported self-care and standard practices in treatment. Conclusion That treatment of chronic venous leg ulcers constitutes a challenge that requires collective investment, involving women, professionals, managers and health institutions. .

Objetivo Comprender las experiencias y expectativas de enfermeras en el tratamiento de mujeres con úlcera venosa crónica. Método Investigación fenomenológica fundamentada en Alfred Schutz, que buscó Se realizó entrevista semiestructurada con siete enfermeras, en enero del 2012. Resultados La enfermera revela dificultades presentadas por la mujer para realizar el autocuidado, percibe limitaciones en el tratamiento relacionadas con la desmotivación, los valores y las creencias de las mujeres. Refiere frustración profesional debido a la recidiva de la lesión, a la falta de insumos, al deficiente trabajo interdisciplinar y a la limitada capacitación del equipo de enfermeras. Espera la adhesión de la mujer al tratamiento y resalta la necesidad del cuidado continuo, del autocuidado apoyado y de estandarizar conductas de tratamiento. Conclusión El tratamiento de la úlcera venosa crónica es un desafío que requiere contribución colectiva, involucrando a las mujeres, a los profesionales, a los gestores y a las instituciones de salud. .

Objetivo Compreender as experiências e expectativas de enfermeiras no tratamento de mulheres com úlcera venosa crônica na Atenção Primária à Saúde. Método Pesquisa fundamentada na fenomenologia social de Alfred Schütz, com depoimentos obtidos em janeiro de 2012, por meio de entrevista semiestruturada com sete enfermeiras. Resultados As enfermeiras revelam dificuldades apresentadas pelas mulheres com úlcera venosa crônica para realizar o autocuidado, percebem limitações na terapêutica ancoradas na desmotivação e nos valores e crenças das mulheres. Referem frustração profissional em razão da recidiva da lesão, falta de insumos e tecnologia, de trabalho interdisciplinar e da capacitação da equipe de enfermagem. Esperam a adesão das mulheres ao tratamento e ressaltam a necessidade do cuidado contínuo, do autocuidado apoiado e da padronização de condutas no tratamento. Conclusão O tratamento da úlcera venosa crônica constitui-se em um desafio que requer investimento coletivo, envolvendo a mulher, os profissionais, os gestores e as instituições de saúde. .

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol.

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.

VRl- and VRL-l-like immunoreactivity in normal and injured trigeminal dental primary sensory neurons of the rat.

The vanilloid receptor VR1 and the vanilloid receptor-like protein VRL-1 are associated with polymodal nociceptors, and may be important for pain processing in normal and injured teeth. Using immunohistochemistry, we have studied the distribution of these receptors in rat pulpal or gingival trigeminal ganglion neurons that were identified through retrograde labeling with fluoro-gold. Twenty-one percent to 34% of tooth pulp-innervating neurons were VRl-positive, while 32%-51% were VRL-1-immunoreactive. However, double-labeling experiments revealed that VR1 and VRL-1 rarely co-existed in the same cells, but rather seemed to be confined to separate subpopulations. Among the gingival neurons, about 25% were VR1-positive and about 41% were VRL-1-immunoreactive. A lesion of the inferior alveolar nerve, which supplies mandibular teeth and gingiva, resulted in a marked down-regulation of VR1 in the affected trigeminal ganglion cells. A down-regulation of VRL-l was also indicated. The results suggest that both VR1 and VRL-1 could have significant roles in pulpal and gingival nociceptive transduction.

Post-natal development of type 1 cannabinoid receptor immunoreactivity in the rat hippocampus.

Type 1 cannabinoid receptors, selectively located on axon terminals of GABAergic interneurons in the hippocampus, are known to be involved in endocannabinoid-mediated retrograde synaptic signalling. The question arises whether type 1 cannabinoid receptors appear on these axons during early post-natal life, when GABAergic transmission is still depolarizing, and whether there are any developmental changes in the cellular or subcellular expression pattern. Here we demonstrate, using single and double immunocytochemical methods at the light and electron microscopic levels, that type 1 cannabinoid receptors are expressed only on the membrane of axon terminals and pre-terminal axons but not on the soma-dendritic membrane at all examined timepoints between post-natal days 0 and 20, similar to the adult distribution. All type 1 cannabinoid receptor-positive boutons formed symmetric synapses. Granular labelling in the somata was already strong at post-natal day 0 and corresponded to multivesicular bodies, lysosomes, Golgi apparatus and rough endoplasmic reticulum. The type 1 cannabinoid receptor-positive axons were shown to originate largely from cholecystokinin-immunoreactive basket and bistratified neurons throughout the hippocampus (90% of all type 1 cannabinoid receptor-containing cells) and dentate gyrus (70% of all type 1 cannabinoid receptor-containing cells). The remaining cells have not been identified but probably belong to the somatostatin- and/or neuropeptide Y-containing subsets, as cholecystokinin-negative, type 1 cannabinoid receptor-positive axons have been observed in strata moleculare and lacunosum-moleculare of the dentate gyrus and CA1-3, respectively, where these neurons are known to arborize. No cell types were found that expressed type 1 cannabinoid receptors transiently at some developmental stage. We conclude that the cellular and subcellular pattern of type 1 cannabinoid receptor expression during early post-natal life is similar to the adult pattern and type 1 cannabinoid receptors are expressed on the cholecystokinin-containing axons as soon as synapse formation begins. This suggests that retrograde synaptic signalling by endocannabinoids is required for the normal operation of GABAergic neurotransmission even before it becomes hyperpolarizing.

Topography of the vanilloid receptor in the human bladder: more than just the nerve fibers.

PURPOSE: We determined the presence and distribution of vanilloid receptor-1 in the human bladder and confirmed or rejected previous findings of other groups that used indirect methods or vanilloid receptor-1 antibodies made by immunizing experimental animals. Also, we tested the reproducibility of results using commercially available antibodies against the N-terminus and C-terminus of the vanilloid receptor. MATERIALS AND METHODS: A total of 11 normal bladder tissue samples were obtained from cystectomy specimens and fresh frozen processed. Specimens were studied by immunohistochemistry and confocal laser microscopy using 3 vanilloid receptor-1 antibodies. Immunohistochemical co-localization studies for neurofilament, neuronal nitric oxide synthase and nerve growth factor were performed. RESULTS: Our results confirm the presence of vanilloid receptor-1 on nonmyelinated and myelinated nerve fibers. There was vanilloid receptor-1 immunoreactivity on smooth muscle cells but different sensitivities for the antibodies. There was immunoreactivity on interstitial cells located in the suburothelium and intermuscular septa of the muscularis. There was co-localization of neuronal nitric oxide synthase with interstitial cells but not with neurofilament. No co-localization was found for nerve growth factor and vanilloid receptor-1. CONCLUSIONS: Vanilloid receptor-1 is located on small unmyelinated and myelinated nerve fibers. In addition, vanilloid receptor-1 is also present on interstitial cells in the suburothelium. There is smooth muscle cell immunoreactivity but a difference in antibodies raised against the C-terminus and N-terminus. These data suggest that the current hypothesis about the mechanism of action of vanilloids is through blocking the afferent reflex arc must be revised and the function of interstitial cells deserves further attention.