Neuropeptide Y. Differential binding to rat intestinal laterobasal membranes

Neuropeptide Y (NPY), a neurotransmitter contained within intrinsic nerves of the small intestine, inhibits secretion when added to the serosal side of intestinal mucosa mounted in Ussing chambers. Using NPY radiolabeled with IODO-GEN, lactoperoxidase, or the Bolton-Hunter reagent, we have localized high affinity NPY receptors to the serosal laterobasal membranes of the rat intestinal epithelial cell, isolated according to a recently described protocol that minimizes contamination with endoplasmic reticulum, Golgi, and brush-border membranes. In addition, certain species of radiolabeled NPY, including those labeled with IODO-GEN at the tyrosine residue 36, also demonstrated an ability to bind to brush-border membranes. These receptors were specific for NPY since the homologous peptides, pancreatic polypeptide and peptide YY, were less efficient than NPY in inhibiting the membrane binding of radiolabeled NPY. By cross-linking NPY to its receptor with either disuccinimidyl suberate or dithiobis(succinimidyl propionate) and analyzing the resulting complexes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by radioautography, we identified two NPY receptor species with molecular sizes of 52-59 kDa and 37-39 kDa. The 37-39-kDa species further possesses a disulfide bond which may attach it to a separate approximately 5-kDa subunit, as evidenced by retarded migration in the absence of the reducing agent dithiothreitol. The intestinal NPY receptor is slightly smaller than the rat brain receptor previously characterized using similar techniques. The localization of NPY receptors on laterobasal membranes is consistent with previous anatomic and physiologic findings, and their identification by cross-linking techniques will constitute the basis for detailed characterization.