The candidates long term aims are to make contributions in the understanding of: (1) receptor mediated information transfer, and (2) airway smooth muscle hyperactivity. The immediate objectives are: 1) To solublize functional receptors for VIP from lung; (2) To raise anti-receptor antibody; (3) To purify the receptor and analyze its structure; (4) To rreconstitute signal transduction by the receptor; (5) To determine whether GTP-binding proteins (G- proteins) and phospholipids regulate receptor affinity, selectivity, and coupling with adenylate cyclase; (6) To compare the VIP-neutralizing ability of VIP-autoantibodies identified in asthma patients and healthy subjects, by measuring the effect of these antibodies on VIP binding to receptors, VIP-stimulated cyclic AMP synthesis, and VIP-induced tracheal relaxation; (7) To correlate changes in the affinity and titer of VIP antibodies with the severity of asthma in individual patients. (8) To assess whether autoantibody to the VIP-receptor may be a pathogenetic factor in asthma. An RCDA will ensure that the bulk of the candidate's time (approximately 90%) is applied to achieving these objectives. The candidate's academic environment supplies support mechanisms necessary for intensive research and offers good opportunities of close interaction with investigators in pharmacology, biochemistry, molecular biology, and pulmonary medicine. The candidate has already established fruitful collaborations with established researchers working on neuropeptides, receptor regulation and information transfer, and airway disease. The experimental design is: (1) Solubilization of VIP receptors; (2) Characterization of their 125I-VIP binding properties; (3) Anti-receptor antibody production by hybridoma techniques; (4) Receptor-purification by HPLC and affinity chromatography; (5) Structural analysis by analytical biochemical methods; (6) Receptor-reconstitution with G-proteins, adenylate cyclase and phospholipids followed by assay of GTP-sensitive VIP-binding, and VIP-sensitive GTP binding, GTPase activity and cyclic AMP synthesis; (7) Comparison of receptor affinity, peptide selectivity and receptor-adenylate cyclase coupling, after: (i) reconstitution with and without G-protein, (ii) reconstitution with different phospholipids, (iii) phospholipase-treatment of reconstituted receptors; (8) RIA of VIP-receptor autoantibody; (9) RIA of VIP binding autoantibodies and measurement of their affinity and titer; (10) Spirometric evaluation of the severity of asthma; (11) Delineation of the functional properties of VIP-autoantibodies by measuring their, (i) effect on VIP receptor binding, cyclic AMP synthesis, and tracheal relaxation.
