alpha-Conotoxins: nicotinic acetylcholine receptor antagonists as pharmacological tools and potential drug leads.

Conotoxins are small disulfide rich peptides from the venoms of marine cone snails. They target specific nicotinic acetylcholine receptor (nAChR) subtypes with high affinity and potency and are therefore valuable as neuropharmacological probes and potential drug leads. This article gives a general overview of the chemical and biological features of alpha-conotoxins, including their pharmacology, binding interactions and structure. A detailed analysis of recently reported three-dimensional structures from members of different subfamilies of the alpha-conotoxins, including those with 3/5, 4/3, 4/6 and 4/7 spacings of their two intracysteine loops is given. The structures are generally well defined and represent useful frameworks for the display of amino acid residues to target molecules.

P2X(1) receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras.

The P2X(1) purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X(1) and a chimera of P2X(1) and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X(1)-GFP chimera was used to study the time course of P2X(1) receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X(1) and P2X(1)-GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4-6 microm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X(1) antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED(50) for the binding of alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) to the P2X(1)-GFP chimera was similar to native P2X(1) receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X(1) or P2X(1)-GFP were similar. Exposure of HEK293 cells to alpha, beta-meATP for 10-20 min in the presence of 5 microM monensin led to the disappearance of P2X(1)-GFP fluorescence from the surface of the cells. These observations using the P2X(1)-GFP chimera demonstrate that P2X(1) receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.

Development of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder.

Postnatal development of the distribution of different isoforms of purinergic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary bladder has been determined with immunofluorescence and confocal microscopy. Antibodies against the extracellular domains of the P2X(1) to P2X(6) receptors were used to detect the receptors in the bladder. Several other antibodies were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X(1) to P2X(6) receptor subtypes (about 0.4 microm diameter) were observed in the muscle which were independent of each other, and sometimes juxtaposed to the rare isolated varicosity strings. At D4 large numbers of strings of varicosities could be discerned throughout the detrusor. Most of these clouds of small P2X(1) to P2X(6) receptor clusters in their immediate vicinity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to tyrosine hydroxylase. Up to D14 there was a gradual coalescence of many of the isolated P2X(1-6) small receptor clusters so that they became colocalized, often at varicosities. Most of the varicosities in isolated strings possessed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P2X small receptor clusters or possessing such clusters in colocalization. However, large numbers of small P2X receptor clusters, many of which consisted of a mixture of isoforms, could be found spatially unrelated to nerve varicosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or these were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different combinations of colocalized P2X receptor isoforms could still be discerned in small clusters unrelated to varicosities. These observations are discussed in relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.

Localisation of P2X receptors in human salivary gland epithelial cells and human embryonic kidney cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting and immunofluorescence.

Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human salivary gland and human embryonic kidney cell line human embryonic kidney (HEK)293 were examined for the purpose of establishing whether they expressed endogenous P2X ionotropic receptors at any stage in their cycles. HSG cells were found to express P2X1-6 subtypes using both Western blotting and immunofluorescence labeling. HEK293 cells had no detectable levels of P2X1-3 and P2X6 under normal circumstances along with very low levels of P2X4 and P2X5 but when the cells were grown past confluence then all subtypes were expressed on the surface membrane with the exception of P2X2. The results are discussed in terms of the likely influence of ATP acting as an intercellular signaling molecule.

P2X (purinergic) receptor distributions in rat blood vessels.

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.

Structural organization and chromosomal localization of three human galanin receptor genes.

Human galanin receptor subtypes GALR1, GALR2, and GALR3 are encoded by separate genes that are located on human chromosomes 18q23, 17q25.3, and 22q13.1, respectively. The exon:intron organization of the gene encoding GALR2 (GALNR2) and GALR3 (GALNR3) is conserved, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 3 and exon 2 encoding the remainder of the receptor, from the second intracellular loop to the COOH-terminus. This conservation of structural organization is indicative of a common evolutionary origin for GALNR2 and GALNR3. The exon:intron organization of the gene encoding GALR1 (GALNR1) is different from that of GALNR2 and GALNR3, with exon 1 encoding the NH2-terminus to the end of transmembrane domain 5, exon 2 encoding the third intracellular loop, and exon 3 encoding the remainder of the receptor, from transmembrane domain 6 to the COOH-terminus. The structural organization of GALNR1 suggests convergent evolution for this gene and represents a structural organization that is unique among genes encoding G-protein-coupled receptors.

Hold that x-ray: aspirate pH and auscultation prove enteral tube placement.

We report here a prospective study evaluating an alternative to the roentgenographic confirmation of "fine-bore" nasoenteral feeding tubes. Of 78 nasoenteral intubations in 46 patients using a Dobbhoff (Biosearch Medical Products) weighted enteral feeding tube, gastric aspirates were evaluated in 28. Auscultation was performed in all 78. Data was collected at initial placement prior to x-ray confirmation. Observers used color-coded pH paper to analyze gastric aspirate (pH < or = 4) and/or auscultation of the epigastrium to determine feeding tube position prior to x-rays. Auscultation alone was ineffective as a confirmatory test with only 6.3% specificity (p = 0.31). Aspiration to ascertain tube position was very accurate when pH < or = 4.0 (p = 0.0005) and when it was performed. A pH value of > 4 was not very helpful in predicting malposition (37%) especially when pH altering medications were used. Aspiration of contents was successful in 85% of patients. We conclude that when the pH of the nasogastric tube aspirate is < 4.0, x-ray films are not needed to prove the accuracy of tube placement. In other situations, a film is indicated since auscultation is inaccurate.