Characterization of the vasoactive intestinal peptide receptor in rat submandibular gland: radioligand binding assay in membrane preparations

Journal Article · Tue Sep 01 04:00:00 EDT 1987 · J. Pharmacol. Exp. Ther.; (United States)

OSTI ID:5842166

Turner, J T; Bylund, D B

The vasoactive intestinal peptide (VIP) receptor in membranes from rat submandibular gland was studied using radioligand binding assays with /sup 125/I-VIP and various unlabeled competing ligands. In addition to the necessity of working within the parameters under which all radioligand binding assays should be performed, binding studies with /sup 125/I-VIP, as with other peptide hormones and neurotransmitters, are subject to additional technical difficulties. Specific problems that were addressed included radioligand proteolysis, the identification of an effective protease inhibitor (leupeptin) and the deleterious effects of a commonly used inhibitor (bacitracin); avid radioligand absorption to incubation tubes that was eliminated by precoating of the tubes with a combination of polyethylenimine and an organosilane; and a disproportionate effect of increasing membrane protein concentration on affinity estimates. Under optimized conditions, the affinity (Kd) and density Bmax values for /sup 125/I-VIP obtained from saturation assays (76 pM, 2.0 pmol/mg) were in excellent agreement. Membrane protein (or receptor) levels beyond the linear portion of the receptor concentration curve are often used in radioligand binding assays. Results from /sup 125/I-VIP binding studies at elevated receptor concentrations revealed the predicted marked decrease in receptor affinity. In addition, the rank order potency of unlabeled ligands in inhibition binding assays was changed. The optimization of the assay for measuring VIP receptors in submandibular gland membrane provides a reliable method for studying the role of receptor regulation in stimulus-secretion coupling for this neuropeptide.

• Research Organization: Univ. of Missouri-Columbia
• OSTI ID: 5842166
• Journal Information: J. Pharmacol. Exp. Ther.; (United States), Journal Name: J. Pharmacol. Exp. Ther.; (United States) Vol. 242:3; ISSN JPETA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ANIMALS
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BUFFERS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYME INHIBITORS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGANDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDES
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
TRACER TECHNIQUES
VERTEBRATES