Characterization of specific pancreatic polypeptide receptors on basolateral membranes of the canine small intestine.

Abstract

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 ...

Functional characterization of receptors with affinity for PYY, NPY, [Leu31, Pro34]NPY and PP in a human colonic epithelial cell line

“…In gastrointestinal epithelia, Y2-receptors are typically involved in reducing electrolyte secretion via a cyclic AMPdependent inhibitory mechanism (Servin et al, 1989) and this is also common for Yj receptor activated responses in other cell lines (van Valen et al, 1992;Krause et al, 1992). A concomitant mobilisation of intracellular Ca2+ is also a common feature of activation of Y1 receptors in certain cells (Krause et (Penner et al, 1993) Rat jejunum and renal epithelia (Sheikh et'al., 1989) (No published data to date in any epithelial preparations) Col-6 adenocarcinoma cell line (Cox & Tough, this study) Canine small intestine epithelia (Gilbert et al, 1988) Rabbit distal colon (Ballantyne et al, 1993) Michel et al, 1992). Since inhibition of accumulated cyclic AMP production is the primary underlying mechanism involved in NPY/PYY antisecretory responses it is possible that functional cross-desensitization observed between particular agonists may not in fact indicate desensitization at a common receptor, but rather that down-regulation of a common transduction pathway has occurred.…”

“…Y2 receptors are also PYY-preferring but are stimulated by C-terminal fragments, whereas theY3 receptor is NPY-preferring and PYY is much less potent. In addition, specific receptors have been described in plasma membranes of canine small intestine epithelia, with high affinity for PP but relatively low affinity for both NPY and PYY (Gilbert et al, 1988). The NPY receptor subtype most commonly expressed by epithelial cells whether renal or gastrointestinal in origin, is Y2-like (Sheikh et al, 1989;) activation of which results in a reduction in prestimulated adenosine 3': 5' cyclic monophosphate (cyclic AMP) accumulation (Servin et al, 1989;Edwards et al, 1990) and as a consequence a reduction in electrolyte secretion.…”

Strong evolutionary conservation of neuropeptide Y: sequences of chicken, goldfish, and Torpedo marmorata DNA clones.

“…All of these peptides consist of 36 amino acids and have a carboxyl-terminal amide. Porcine NPY and PYY show 70% sequence identity and can act on the same receptors with similar potencies (6), whereas porcine PP is only 50% identical to NPY and PYY and appears to have distinct receptors (7)(8)(9). PP differs considerably between species; PP of pig, chicken, and bullfrog share only 44-58% identity.…”

The cloned rat pancreatic polypeptide receptor exhibits profound differences to the orthologous receptor.

“…17). Seemingly distinct PP-preferring receptors have been described based upon binding studies of isolated tissue preparations, namely dog intestinal mucosa (18,19), rat phaeochromocytoma PC12 cells (20), rat brain area postrema (21), rat adrenal cortex and medulla (22), and rat liver (23). A PP receptor was observed in rat vas deferens in a functional assay (24).…”