6-(4-Benzylpiperazin-1-yl)benzodioxanes as selective ligands at cloned primate dopamine D(4) receptors.

A series of novel 6-(4-benzylpiperazin-1-yl)benzodioxanes were prepared and screened at selected dopamine receptor subtypes. 6-(4-[4-Chlorobenzyl]piperazin-1-yl)benzodioxane (2d) had high affinity and selectivity for the D(4) dopamine receptor subtype and was identified as a D(4) antagonist via its attenuation of dopamine-induced GTPgamma(35)S binding at the D(4) receptor.

Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana.

Substance P is an important neuropeptide neurotransmitter in the central, autonomic and enteric nervous systems. In sympathetic ganglia, substance P is thought to play a role in modulating synaptic transmission. Release of substance P by neuronal stimulation or direct application of substance P to ganglionic neurons increases neuronal excitability. An amphibian substance P receptor complementary DNA has been cloned and characterized from bullfrog, Rana catesbeiana, sympathetic ganglion complementary DNA libraries. The deduced primary structure contains features indicative of a seven transmembrane domain G-protein-coupled receptor. The deduced protein sequence shows 69% identity to previously cloned mammalian substance P receptors. In situ hybridization analysis performed on bullfrog sympathetic ganglia using digoxigenin-labelled complementary RNA probe demonstrated that approximately 75% of the principal neurons displayed reaction product above background levels. Radioligand binding studies were performed on stably transfected cells with [(125)I]Tyr-1-substance P as the ligand. Substance P had an IC50 of 16 nM and the agonist potency profile was substance P>neurokinin A >> neurokinin B. The order of potency for three tachykinins to increase intracellular calcium when applied to a stably transfected clonal cell line was substance P>neurokinin A >> neurokinin B. This order of agonist potency also held for inhibition of the M-type potassium current in intact bullfrog sympathetic neurons. The non-peptide substance P antagonists CP-96345 and RP-67580 at concentrations that block mammalian substance P receptors had little or no effect on the responses to substance P at the bullfrog receptor. Overall, these results demonstrate that the cloned sequence has the features consistent with and characteristic of a substance P receptor. The results are discussed with reference to the established pharmacology of the bullfrog substance P receptor and known structure activity relationships of mammalian tachykinin receptors.

Residue 78 in the second transmembrane domain of the neurokinin-1 receptor is important in coupling high affinity agonist binding to multiple second messenger responses.

The neurokinin-1 tachykinin receptor is a member of the G protein-coupled receptor superfamily. An unusual feature of the neurokinin-1 receptor is the presence of glutamic acid (residue 78) in the second putative transmembrane domain, at the location of a highly conserved aspartate residue in the G protein-coupled receptor superfamily. The rat neurokinin-1 receptor cDNA was mutated to lysine, aspartate, and glutamine at this site and functionally expressed in Chinese hamster ovary cells, and clonal cell lines were isolated and characterized. Radioligand binding demonstrated that the Asp78 and Lys78 receptors have substance P binding affinities indistinguishable from those of the wild-type receptor and are expressed at roughly the same number of receptors per cell. The Gln78 receptor variant, on the other hand, exhibited no detectable agonist binding. Although wild-type and Asp78 receptors have essentially the same ability to stimulate inositol phospholipid turnover, cAMP production, and arachidonic acid release, the Lys78 variant is markedly attenuated in its ability to activate any of these pathways. These data indicate that residue 78 plays a role in the coupling of the rat neurokinin-1 receptor to cellular effectors. In addition, both Asp78 and Lys78 receptors show a greater percentage of high affinity binding that is resistant to guanosine-5'-O-(3-thio)triphosphate than does the wild-type receptor, indicating a potential difference in G protein coupling between wild-type and mutated receptors.

Lectin affinity high-performance liquid chromatography: interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin.

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.