S. St-Pierre

Characterization of CGRP1 and CGRP2 Receptor Subtypesa

Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide generated from the alternate tissue-specific processing of the calcitonin gene mRNA..? Various forms (a and p ) of CGRP have been characterized from human tissue^.^-^ In the periphery, CGRP is co-localized and co-released with calcitonin in the parafollicular C cell of the thyroid gland, with substance P in sensory nerves and with acetylcholine in motoneurons.* In the brain, CGRP-like immunoreactivity is broadly distributed and especially concentrated in hypothalamic areas and some brainstem nuclei.9 A variety of biological effects have been claimed for CGRP, including the modulation of nicotinic receptor activities at the neuromuscularjunctiono*ll and of substance P in inflammation, a reduction of gastric acid s e c r e t i ~ n , ~ . ~ peripheral blood vessel dilation,Is cardiac a~celeration, ~ a regulation of calcium metabolismh and insulin secretion, an increase in body temperature, and a decrease in food intake. Iy-? In spite of the various biological actions induced by CGRP, relatively little is known concerning the characteristics of its receptor~. ~,? -?~ Consequently, a few years ago, our group undertook the development of selective agonists and antagonists which prove to be most useful for the identification of the existence of CGRP receptor s ~ b t y p e s . ~ ~ ~ ~ T h e various criteriaused to classify these receptor classes are reviewed here.

Autoradiographic distribution of [125I]Leu31,Pro34]PYY and [125I]PYY3–36 binding sites in the rat brain evaluated with two newly developed Y1 and Y2 receptor radioligands

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.

PEPTIDE YY DERIVATIVES AS SELECTIVE NEUROPEPTIDE Y/PEPTIDE YY Y1 AND Y2 AGONISTS DEVOIDED OF ACTIVITY FOR THE Y3 RECEPTOR SUB-TYPE

Peptide YY derivatives were evaluated for their respective ability to bind and activate the NPY/PYY receptor sub-types (Y1, Y2 and Y3) present in various preparations. The analogue [Leu31,Pro34]PYY demonstrated high (nM) affinity in rat frontoparietal cortical membrane preparations (Y1-enriched tissue) and the rabbit saphenous vein (Y1 in vitro bioassay) but only low affinity in a Y2-enriched preparation (rat hippocampus). In contrast, PYY C-terminal fragments such as PYY3-36 and PYY13-36 were more potent in Y2 than Y1 assays. Interestingly, and in contrast to [Leu31,Pro34]NPY and NPY13-36, the PYY derivatives [Leu31,Pro34]PYY and PYY3-36 were inactive in a purported Y3 bioassay (rat colon). These results suggest that [Leu31,Pro34]PYY and PYY3-36 respectively represent the first selective and potent Y1 and Y2 agonists, devoided of significant affinity/activity for the Y3 receptor class.

Mapping of hypothalamic sites involved in the effects of NPY on body temperature and food intake

The objective of the present study was to identify hypothalamic sites that might be implicated in the effects of neuropeptide Y (NPY) on both body temperature and food intake. For this purpose, the effects of direct microinjections of NPY in several doses (0.156-20 micrograms) into discrete hypothalamic nuclei on body temperature were examined in rats. To examine specificity of effects, food consumption of animals following injections was also measured. Results indicate that the influence of NPY on body temperature varies with the hypothalamic region where the peptide is administered. NPY had no effect on temperature after administration into the ventromedial (VMH) and the perifornical hypothalamus (PeF). However, a significant hypothermia was seen following administration into the preoptic (POA) and arcuate nucleus (Arc), and hyperthermia was seen after injection into the paraventricular nucleus (PVN). Finally, a biphasic effect was observed after injection into the lateral hypothalamus (LH): hyperthermia with relatively small doses and hypothermia with higher doses. Similar effects were obtained when administered into the third ventricle (3V) but in an inverted dose-related fashion: hypothermia at low and hyperthermia at higher doses. For feeding, NPY consistently increased food intake in all regions examined, with the strongest effect obtained after administration into the PeF. The present results clearly dissociate the effects of NPY on food intake and body temperature, and demonstrate that these effects are related to specific hypothalamic nuclei.

Effects of NPY and NPY2-36 on body temperature and food intake following administration into hypothalamic nuclei.

Our previous in vivo structure-activity studies suggested that the putative receptors mediating the effects of NPY and NPY2-36 on food intake and body temperature are pharmacologically different [17]. In the present study, we examined and compared dose-related effects of NPY and NPY2-36 on ad lib food intake and rectal temperature after administration into discrete hypothalamic nuclei of the rat. Results indicate that NPY and NPY2-36 have opposite effects on body temperature to those of NPY when injected in the preoptic area (POA): hypothermia and hyperthermia, respectively. When administered in the paraventricular nucleus (PVN), both increased body temperature. When injected into the third ventricle (3V), NPY produced a biphasic effect: hypothermia at low doses and hyperthermia at high doses. Similar effects were obtained with NPY2-36, but in an inverted dose-related fashion: hyperthermia at low and hypothermia at higher doses. In the arcuate nucleus (Arc), NPY induced a significant hypothermia whereas NPY2-36 had no effect. Finally, neither peptide affected body temperature when injected into the ventromedial (VMH) and perifornical (PeF) nuclei. Both NPY and NPY2-36 increased food intake after injection in all regions examined. In general, NPY was more potent and efficacious than NPY2-36. The present results clearly dissociate the effects of NPY on food intake and body temperature. Furthermore, the data support the hypothesis that the putative receptors underlying the effects of NPY and NPY2-36 on food intake are similar, whereas those mediating the effects on body temperature are pharmacologically different.

Heterogeneity of motilin receptors in the gastrointestinal tract of the rabbit

Motilin, a 22-amino acid peptide synthesized in endocrine cells of intestinal mucosa, stimulates GI smooth muscle contractility. To elucidate the mode of action of motilin, we attempted to determine whether motilin receptors are localized on nerve cells or on smooth muscle cells of the GI tract. Mucosa-free tissues from rabbit antrum and duodenum were homogenized separately with a Polytron prior to differential centrifugation to obtain synaptosome or plasma membrane-enriched fractions, as determined by the distribution of [3H]saxitoxin (SAX) binding (neural membranes) and 5 nucleotidase (5N) activity (smooth muscle plasma membranes). Motilin binding was evaluated by the displacement of [125I]motilin by motilin (1-22) on the various membrane fractions. In the antrum, motilin binding was highly correlated with SAX binding (r = 0.81, p < 0.0005), and also significantly with 5N activity (r = 0.54, p < 0.05). In the duodenum, motilin binding correlated significantly with 5N activity (r = 0.67, p < 0.005), but not with SAX binding (r = -0.11, NS). Receptor affinity, for the motilin antagonist MOT(1-12)[CH2NH]10-11, for motilin(1-22), and for the motilin agonist erythromycin lactobionate was significantly (p < 0.001, p < 0.001, and p < 0.05, respectively) higher in SAX-enriched fractions from the antrum than in 5N-enriched fractions from the duodenum. Therefore, in the rabbit: 1) motilin receptors appear to be predominantly located on nerve tissues in the antrum and restricted to smooth muscle cells in the duodenum, and 2) antral receptors and duodenal receptors displayed different pharmacological characteristics, probably corresponding to two specific and heterogeneous motilin receptor subtypes.

Bronchoconstrictor action of neuropeptide Y (NPY) in isolated guinea pig airways

Responses of various preparations of guinea pig and rat airways to synthetic porcine neuropeptide Y were analysed in vitro. NPY in doses up to 10(-6) M, induced a dose dependent contraction in trachea, bronchi and lung parenchymal strips of the guinea pig but did not have any effect in similar preparations obtained from the rat. In guinea pig airways the contractile responses to NPY were small in size and characterized by a slow onset and a long duration. This effect of the peptide was not dependent on pre-junctional nerve stimulation but rather mediated through the secondary generation of cyclooxygenase products. It is concluded that NPY may contribute per se to regulating the resting tone of guinea pig airways.

Neuropeptide tyrosine (NPY)-induced potentiation of the pressor activity of catecholamines in conscious rats

IV bolus administration of 2.5-50 micrograms NPY (0.6-12.5 nmol) to conscious rats produced a dose- and time-dependent increase in systolic and diastolic blood pressure. Following priming with 2.5 micrograms NPY, or larger doses, the subsequent administrations of noradrenaline produced pressor responses that were potentiated both in magnitude and duration. The NPY-induced potentiation of the pressor response to noradrenaline was dose-dependent and extended to the pressor action of adrenaline and angiotensin II but not to the hypotensions produced by bradykinin or isoproterenol. The potentiation was not related to the fact that multiple doses of catecholamines were repeated. Reserpine did not substantially modify the NPY-induced potentiation of the pressor activity of the catecholamines. Chemical sympathectomy following 6-hydroxydopamine caused a marked supersensitivity to the catecholamines and NPY but obliterated the NPY-induced potentiation of the pressor effect of adrenaline. Nifedipine reduced the pressor action of the catecholamines and NPY but did not attenuate the NPY-induced potentiation of the pressor action of catecholamines. It is concluded that the acute pressor effect of NPY and of the potentiation of the catecholamine pressor effects involve different mechanisms.

The rabbit saphenous vein: a tissue preparation specifically enriched in NPY-Y1 receptor subtype

Neuropeptide Y (NPY), a co-transmitter in noradrenergic sympathetic nerves of the cardiovascular system, was tested on isolated segments of rabbit saphenous vein. NPY caused strong, long lasting and concentration dependent contraction resistant to adrenergic blockade. PYY, a NPY related peptide, shared this property. As pressor agents, both peptides were about 100-fold more potent than norepinephrine and at their highest concentrations caused a contraction of a similar magnitude as NE. Gradual shortening of N-terminal end of the NPY molecule caused major loss of potency and reduction of intrinsic activity; which suggests that the entire molecule is required to produce full biological activity in this vascular preparation. Addition of [Leu31,Pro34]pNPY, a NPY analog with specific agonist properties at Y1 receptors, mimicked the effect of NPY whereas NPY (13-36), a selective agonist at Y2 receptors, caused a 2 log unit shift to the right of the concentration response curve. These results suggest that the vasoconstrictor effect of NPY in rabbit saphenous vein results from a direct effect on smooth muscle cells and that the receptors involved are of the Y1 subtype.

CGRP 8-37 blocks the inhibition of gastric emptying induced by intravenous injection of α-CGRP in rats

The receptor subtype mediating rat alpha-calcitonin gene-related peptide (alpha-CGRP)-induced inhibition of gastric emptying of a non nutrient solution was tested in conscious rats using the CGRP1 receptor antagonist, CGRP 8-37, and the CCK antagonist, MK-329. Intravenous injection of alpha-CGRP (0.5 micrograms) decreased gastric emptying to 26.5 +/- 5.8% from 46.4 +/- 3.9% in vehicle-treated group. Intravenous injection of CGRP 8-37 (15 micrograms) did not influence gastric emptying but completely prevented alpha-CGRP inhibitory effect whereas the 47% delay in gastric emptying induced by intravenous cholecystokinin-8 (CCK, 0.25 microgram) was not modified. The CCK antagonist, MK-329 (1 mg) reversed CCK- but not alpha-CGRP-induced delay in gastric emptying. These results demonstrate that CGRP 8-37 is a specific tool to block alpha-CGRP inhibitory action on gastric motor function and suggest that gastric stasis elicited by peripheral injection of alpha-CGRP may involve an interaction with a CGRP1 receptor subtype.