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We have identified specific binding sites for pancreatic polypeptide (PP) on the mucosal lining of canine small intestine. The present study was undertaken to further characterize these binding sites (receptors) on purified intestinal membranes and to establish their location on the brush border or basolateral surface of the intestinal enterocyte. Basolateral and brush border membranes were prepared by sorbitol density centrifugation. PP receptors were localized predominately to the vascular surface, and thus binding of PP '25I-labeled on Tyr-27 to the basolateral preparation was used to evaluate receptor characteristics. Binding of PP was calcium, time, temperature, and pH dependent. Maximum specific binding of labeled PP occurred after an 8-hr incubation at 40C with 5 mM calcium at pH 6.8. Data analysis by Scatchard plot showed high-and low-affinit binding sites with relative affinities of 1.5 x 10-9 M and 2.6 x 10-8 M and with corresponding binding capacities of 0.23 pmol/mg and 0.84 pmol/mg of protein, respectively. This receptor was specific for PP since peptide YY and neuropeptide Y, peptides of the PP family, cross-reacted by <3%, as judged from comparisons of half-maximal displacement of label. Structurally dissimilar peptides, insulin and glucagon, did not compete for binding.Specific 12'I-labeled PP binding was localized primarily to basolateral membranes (9.8 ± 0.8%) with little binding by brush border membranes (0.8 ± 0.2%). Thus, we have identified highly specific receptors for PP, located predominately on the vascular surface of the small intestinal mucosa.These data suggest that the mucosal l inig of the small intestine is a target tissue for PP and that PP participates in the hormonal regulation of fuel metabolism and substrate transport in the small intestinal mucosa.Among the known actions of pancreatic polypeptide (PP) are its abilities to inhibit stimulated pancreatic exocrine secretion (for review, see ref. 1) and to augment insulin inhibition of hepatic glucose production (2). Although it is generally agreed that peptide hormones elicit biologic action through interactions with specific cell-surface receptors, there has been no study to date characterizing PP receptors in mammalian tissues. Others have described receptors to avian PP in the chicken (3-6) but were unable to show bovine PP (bPP) binding in rat tissues (4, 7). We have reported (8) specific PP binding to the mucosal lining of canine small intestine by an in vivo animal perfusion technique, but were not able to elucidate the binding characteristics or identify the cellular location of the putative PP receptors.In the present study we describe methods to prepare highly purified basolateral and brush border membranes from the small intestine of the dog. These membranes were used to establish the nature of the PP binding sites as judged by classical receptor criteria obtained from equilibrium binding assays and to determine the location of PP receptors on the intestinal mucosa.MATERIALS AND METHODS Materials. HPLC-purified ...

Little is known about hormonal regulation of substrate transport and metabolism in the mucosal lining of the small intestine. Because insulin regulates these functions in other tissues by binding to its receptor, we have investigated the presence of insulin receptors in canine small intestinal mucosa with basolateral membranes (BLM) and brush border membranes (BBM) prepared by sorbitol density centrifugation. A14-[125I]iodoinsulin was used to study binding and structural characteristics of specific insulin receptors in BLM. Analysis of receptors in BLM identified binding sites with high affinity (Kd 88 pM) and low capacity (0.4 pmol/mg protein) as well as with low affinity (Kd 36 nM) and high capacity (4.7 pmol/mg protein). Binding was time, temperature, and pH dependent, and 125I-labeled insulin dissociation was enhanced in the presence of unlabeled insulin. Cross-reactivity of these receptors to proinsulin, IGF-II, and IGF-I was 4, 1.8, and less than 1%, respectively. Covalent cross-linking of labeled insulin to BLM insulin receptors with disuccinimidyl suberate revealed a single 135,000-Mr band that was completely inhibited by unlabeled insulin. There was a 16-fold greater specific binding of insulin to BLM (39.0 +/- 2.4%) than to BBM (2.5 +/- 0.6%). These results demonstrate the presence of a highly specific receptor for insulin on the vascular, but not the luminal, surface of the small intestinal mucosa in dogs, and suggest that insulin may play an important role in the regulation of gastrointestinal physiology.

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The immunoblot technique permits the visualization of proteins following their separation on acrylamide gels, transfer to cellulose nitrate sheets and subsequent staining with antiserum. We have utilized this technique to demonstrate the presence of four basic proteins in rat PNS myelin with molecular weights of 21K, 18K, 17K, and 14K. Similarly, we demonstrated the presence of two basic proteins in rabbit PNS myelin (molecular weights of 21K and 18K). Exposure of the immunostained cellulose nitrate strips to X-ray film revealed the phosphorylation of four and two basic proteins in rat and rabbit PNS myelin, respectively. These basic proteins were present in the CNS myelin of the two species and were also phosphorylated. Because of the sensitivity of the immunoblot technique, it was also possible for us to visualize the P2 protein in both rat and rabbit PNS myelin.