neurokinin-a and Astrocytoma

Ligand-receptor binding kinetics (i.e. association and dissociation rates) are emerging as important parameters for drug efficacy in vivo. Awareness of the kinetic behavior of endogenous ligands is pivotal, as drugs often have to compete with those. The binding kinetics of neurokinin 1 (NK1) receptor antagonists have been widely investigated while binding kinetics of endogenous tachykinins have hardly been reported, if at all. Therefore, the aim of this research was to investigate the binding kinetics of endogenous tachykinins and derivatives thereof and their role in the activation of the NK1 receptor. We determined the binding kinetics of seven tachykinins targeting the NK1 receptor. Dissociation rate constants (k

Topics: Algorithms; Animals; Astrocytoma; Binding, Competitive; Cell Line, Tumor; CHO Cells; Cricetulus; Electric Impedance; Humans; Kinetics; Ligands; Nerve Tissue Proteins; Neuroglia; Neurokinin A; Peptide Fragments; Pyrrolidonecarboxylic Acid; Radioligand Assay; Receptors, Neurokinin-1; Recombinant Proteins; Substance P; Tachykinins

Substance P (SP) can produce cytokine-like responses by astrocytes and mononuclear cells. In an effort to identify neurokinin-1-receptors (NK1-R), an antibody to NK1-R was generated by using a linear peptide sequence from the deduced third extracellular region (ECR) corresponding to the seven transmembrane rat brain NK1-R. The ECR-3 peptide was coupled to keyhole-limpet hemocyanin and the antisera produced in rabbits was purified by binding to a peptide-affinity matrix. The specificity for the anti-peptide antibody was shown by its reactivity to the ECR-3 peptide by ELISA. The anti-ECR-3 peptide antibody could detect, by Western blot analysis of SDS-PAGE-separated rat brain membranes, a single band with an apparent molecular weight (MW) of 53-54 kDa. An affinity matrix made from the anti-ECR-3 antibody was used to isolate NK1-R from rat brain membranes which exhibited two products on SDS-PAGE with apparent MW of 54 and 44 kDa. The C6 astrocytes were shown to express NK1-R as determined by [125I]Bolten-Hunter SP binding to intact cells with a Kd = 0.32 nM. These C6 cells did not co-express either NK2-R or NK3-R when analyzed at the mRNA level. The anti-ECR-3 peptide antibody could inhibit [125I]Bolten-Hunter SP binding to intact C6 astrocytes and CHO cells expressing NK1-R by greater than 95% when compared to normal rabbit IgG which failed to inhibit radiolabeled SP binding. Thus, an antibody which recognizes surface determinants to the NK1-R could be generated upon immunization with an NK1-R peptide.

Topics: Amino Acid Sequence; Animals; Antibodies; Antibody Affinity; Astrocytoma; Base Sequence; Binding Sites, Antibody; Binding, Competitive; Blotting, Western; Brain Chemistry; Enzyme-Linked Immunosorbent Assay; Female; Iodine Radioisotopes; Molecular Sequence Data; Neurokinin A; Peptide Fragments; Protein Structure, Tertiary; Rabbits; Rats; Receptors, Neurokinin-1; RNA, Messenger; Substance P; Tachykinins; Tumor Cells, Cultured

Substance P (SP) stimulated [3H]taurine release from human astrocytoma cells (U-373 MG). This effect was concentration dependent and the EC50 was 0.3 nM. This stimulatory effect of SP can be inhibited by spantide, a SP receptor antagonist. The rank order of potencies of related tachykinins and their analogues in stimulating the release of [3H]taurine was SP much greater than neurokinin A much greater than neurokinin B and [Glp6,L-Pro9]SP (6-11) much greater than [Glp6, D-Pro9]SP (6-11) which conformed to that reported for the tachykinin NK-1 receptor. In addition to SP, isoproterenol, a beta-adrenergic agonist, can also increase the release of [3H]taurine from these cells and the effects of SP and isoproterenol were additive.

Topics: Astrocytoma; Cell Line; Cyclic AMP; Humans; Isoproterenol; Kinetics; Neurokinin A; Substance P; Tachykinins; Taurine; Tritium