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1 The action of analogues and C-terminal fragments of neuropeptide Y (NPY) was examined on excitatory synaptic transmission in area CAl of the rat hippocampal slice in vitro, by use of intracellular and extracellular recordings, to determine by agonist profile the NPY receptor subtype mediating presynaptic inhibition. 2 Neither NPY, analogues nor fragments of NPY affected the passive or active properties of the postsynaptic CAL pyramidal neurones, indicating their action is at a presynaptic site. 3 The full-sequence analogues, peptide YY (PYY) and human NPY (hNPY), were equipotent with NPY at the presynaptic receptor, while desamido hNPY was without activity. 4 NPY2-36 was equipotent with NPY. Fragments as short as NPY13-36 were active, but gradually lost activity with decreasing length. NPY16-36 had no effect on extracellular field potentials, but still significantly inhibited excitatory postsynaptic potential amplitudes. Fragments shorter than NPY16-36 had no measurable effect on synaptic transmission. 5 The presynaptic NPY receptor in hippocampal CAL therefore shares an identical agonist profile with the presynaptic Y2 receptor at the peripheral sympathetic neuroeffector junction.

Some evidence has suggested the existence and differential distribution of neuropeptide Y (NPY) receptor subtypes in the mammalian brain (Dumont et al., 1990; Aicher et al., 1991). We now report on the extensive characterization and visualization of at least two classes of NPY receptor sites using a highly selective Y1 analog, [Leu31,Pro34]-NPY or [Pro34]-NPY, and a relatively specific Y2 competitor, NPY13-36. Autoradiographic studies using 125I-peptide YY (125I-PYY) clearly reveal that the Y1 receptor subtype is most abundant in various cortical areas, the dentate gyrus of the hippocampal formation, the claustrum, and the reuniens nucleus of the thalamus. In most other regions, 125I-PYY binding is potently inhibited by increasing concentrations of either NPY2-36 or NPY13-36, suggesting a Y2-like profile. Furthermore, binding assays using homogenates from discrete brain regions clearly demonstrate that various NPY fragments and analogs compete for 125I-PYY labeling with profiles indicative of heterogeneity of NPY receptor subtypes, even in the presence of a selective Y1 blocker. Thus, it is likely that, in addition to the Y1 receptor, which is particularly concentrated in cortical areas, the rat brain is enriched with a receptor class (Y2) that can exist under high- or low-affinity states or with additional receptor subtypes that are recognized by 125I-PYY. These findings cannot be explained by the existence of the very recently reported Y3 receptor subtype, since PYY does not possess significant affinity to this site (Grundemar et al., 1991). Further experiments are currently in progress to determine the nature and functional significance of each of these NPY/PYY receptor sites.

Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.

The role of calcitonin gene-related peptide (CGRP) on colorectal distension-induced visceral pain was investigated in conscious rats. Intracolonic administration of acetic acid (0.6%) resulted in a significantly increased number of abdominal contractions in response to colorectal balloon distension from 5.8 +/- 1.2 in controls to 16.6 +/- 1.0 in acetic acid-treated animals (P < 0.05), evidencing sensitization of visceral afferent pathways and subsequently visceral hyperalgesia. This sensitization phenomenon was not observed in animals previously treated with systemic capsaicin. Likewise, in animals not treated with capsaicin, use of an intravenous antagonist for CGRP [human CGRP-(8-37)], completely reversed the sensitizing effects of acetic acid. Furthermore, intravenous administration of CGRP dose dependently increased the number of abdominal contractions in response to colorectal distension from 3.0 +/- 1.1 (CGRP 250 ng) to 17.0 +/- 1.2 (CGRP 500 ng, P < 0.05), as previously observed in acetic acid-treated animals. Finally, intrathecal administration of hCGRP-(8-37) (mid-lumbar) also resulted in a total dose-dependent reversal of CGRP (500 ng) or acetic acid-induced visceral hypersensitivity. These results demonstrate that CGRP plays a major role in this model of visceral afferent nerve sensitization from gastrointestinal origin.

The peptide YY derivatives [Leu31,Pro34]PYY and PYY3–36 are highly selective Y1 and Y2 agonists, devoid of activity on the Y3 receptor subtype [Dumont et al. (1994) Molec. Brain Res., 26:3220–3324]. These selective ligands were iodinated and used to evaluate the respective quantitative autoradiographic distribution of the Y1 and Y2 receptor subtypes in the rat brain, excluding a potential contamination from Y3 receptor. Specific [125I][Leu31,Pro34]PYY (Y1), and [125I]PYY3–36 (Y2) binding sites are detected in various brain regions, but each showed a differential distribution profile. Y1/[125I][Leu31,Pro34]PYY sites are especially concentrated in superficial layers of the cortex, the olfactory tubercle, islands of Calleja, tenia tecta, molecular layer of the dentate gyrus, several thalamic nuclei, and the posterior part of the medial mammaliary nucleus. These areas generally contained only low densities of Y2/[125I]PYY3–36 binding sites. In contrast, [125I]PYY3–36 binding is most abundant in multiple other regions including the lateral septum, piriform cortex, triangular septal nucleus, bed nucleus of the stria terminalis, oriens layer and stratum radiatum of the dorsal hippocampus, ventral tegmental area, substantia nigra, dorsal raphe nucleus, and the granular cell layer of the cerebellum. Few areas of the rat brain contained significant amounts of both [125I][Leu31,Pro34]‐PYY and [125I]PYY3–36 binding sites such as the anterior olfactory nuclei, oriens layer and stratum radiatum of the ventral hippocampus, nucleus tractus solitarius, area postrema, and inferior olive. Taken together, these results and the use of two selective radioligands demonstrate further the discrete, differential distribution of the Y1 and Y2 receptor subtypes in the rat brain. © 1996 Wiley‐Liss, Inc.

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A structure-activity study was carried out to determine the importance of the N-terminal amino acids of hCGRP8-37 in binding and antagonistic activity to CGRP receptors. Therefore, fragments of hCGRP8-37 as well as analogs obtained by the replacement of residues 9-12 by L-alanine were synthesized by solid-phase peptide synthesis, using BOP as a coupling reagent. The affinities of the peptides to CGRP receptors were evaluated in the rat brain, guinea pig atrium, and guinea pig vas deferens membrane preparations. Their antagonistic activities were measured in the guinea pig atria and rat vas deferens bioassays. The pharmacological characterization showed that arginine-11 and leucine-12 play a crucial role for the affinity of hCGRP8-37. Interestingly, it was observed that [Ala11]hCGRP8-37 was able to potentiate the twitch response of the electrically stimulated rat vas deferens. On the other hand, the substantial antagonistic potencies of analogs [Ala9]-, [Ala10]-, and [Ala12]hCGRP8-37, as compared to those of the fragments hCGRP10-37, hCGRP11-37, and hCGRP12-37, suggest that the side chains of Thr-9, His-10, and Leu-12 assume mainly a structural role. Accordingly, the conformational characterization of these peptides by circular dichroism spectroscopy revealed that the residues 9-12 are important for the integrity of the amphiphilic alpha-helix of hCGRP8-37.