Localization of opioid receptor antagonist [3H]-LY255582 binding sites in mouse brain: comparison with the distribution of mu, delta and kappa binding sites.

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.

Introduction to the reviews on neuropeptide Y.

Neuropeptide Y (NPY) was first reported as an abundant peptide in brain tissue in 1982. Shortly thereafter, NPY was found to be a member of a peptide family consisting of the endocrine peptides pancreatic polypeptide (PP) and peptide YY (PYY). These peptides exert most of their biological effects through five G-protein coupled receptors termed Y1, Y2, Y4, Y5 and y6 that mediate either inhibition adenylate cyclase or increases in intracellular calcium. Since the discovery of NPY, a robust a body of literature has developed around the potential functions of this peptide. While initial findings identified NPY is an important contributor to the regulation of feeding, body weight and blood pressure, more recent work as revealed more subtle functions of this peptide and its potential role in affective disorders, bone formation and cravings. The accompanying twelve reviews detail important developments in our understanding of the functional role of NPY.

Neuropeptide Y receptor subtypes in the basolateral nucleus of the amygdala modulate anxiogenic responses in rats.

The behavioral effects induced by intra-amygdala stimulation of the neuropeptide Y (NPY) Y(2) and the NPY Y(5) receptor subtypes were assessed in the social interaction (SI) test. Microinjections of NPY(3-36), an NPY Y(2) preferring agonist, into the basolateral nucleus of the amygdala (BLA) produced bi-directional dose-response curve. At low doses NPY(3-36) has an anxiogenic effect while at higher doses it produced an anxiolytic effect. Pretreatment with the NPY Y(5) receptor antagonist Novartis 1(1 nmol), an analog of CGP71683A synthesized by Eli Lilly and Company, IN, blocked the anxiolytic effects of NPY(3-36) (80 pmol), while pretreatment with BIBO 3304 (200 pmol), a Y(1) antagonist, had no effect, suggesting that the Y(5), but not the Y(1) receptor was involved in the anxiolytic behavior produced following intra-amygdalar NPY(3-36) administration. In addition, the Y(5) antagonist had no behavioral effect when given alone at 1.0 nmol. These findings support the hypothesis that amygdalar Y(2) receptors may play a role in mediating anxiogenic effects, while Y(5) receptors may be involved in the anxiolytic behaviors of NPY.

Characterization of neuropeptide Y Y1-like and Y2-like receptor subtypes in the mouse brain.

To characterize receptor subtypes in the mouse, we performed autoradiographic localization and pharmacological characterization studies using the selective radiolabeled agonists, [(125)I]-Leu(31), Pro(34)-PYY and [(125)I]-PYY 3-36. The pharmacology of [(125)I]-Leu(31), Pro(34)-PYY and [(125)I]-PYY 3-36 binding to mouse brain homogenates were consistent with Y1-like and Y2-like receptors, respectively. Using receptor autoradiography, high Y1-like binding was observed in the islands of Calleja and dentate gyrus. [(125)I]-PYY 3-36 binding was highest in the hippocampus, lateral septum, stria terminalis of the thalamus, and compacta and lateralis of the substantia nigra. In addition, there are differences in receptor distribution in mouse brain compared to other species that may translate into different functional roles for the NPY receptors within each species.

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes.

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.

Astressin, a corticotropin releasing factor antagonist, reverses the anxiogenic effects of urocortin when administered into the basolateral amygdala.

Previous work in our laboratory has shown that Urocortin (Ucn), a peptide related to corticotropin releasing factor (CRF), injected into the basolateral nucleus of the amygdala (BLA) in male Wistar rats would result in an anxiogenic response as measured in the social interaction (SI) test. In addition, it was found that repeated injections of subthreshold doses of Ucn would 'prime' the animal's response. This 'priming' effect induces a sensitivity to sodium lactate infusions that results in a panic-like reaction. Currently, we examined the effects of the CRF(1) and CRF(2) antagonist Astressin (Asn) on the anxiety-like responses produced during 'priming' as well as during a sodium lactate infusion into 'primed' rats. The results showed that Asn (60 pmoles) was able to reverse the anxiogenic effects seen during acute administration of Ucn, but was only able to partially antagonize the same response following 'priming' with Ucn. Furthermore, Asn administered either alone or prior to a sodium lactate infusion had no effect on the primed rat's behavior. Autoradiographic studies, in Ucn primed and sham-primed animals indicated no significant changes in [(125)I]-Sauvagine binding to CRF(1) and CRF(2) receptors in several brain regions. Thus, a 60 pmole dose of Asn blocks the effects of an acute injection of Ucn (100 pmoles), while only partially blocking the behavioral effects after repeated injections of subthreshold doses of Ucn (6 pmoles) are given. Furthermore, Asn has no effect on anxiogenic responses due to sodium lactate infusions in 'primed' rats

Pharmacological characterization of (125)I-1229U91 binding to Y1 and Y4 neuropeptide Y/Peptide YY receptors.

1229U91 (GW1229 or GR231118) [lle,Glu,Pro,Dpr,Tyr, Arg,Leu,Arg, Tyr-NH(2))2 cyclic (2,4'),(2'4)-diamide] has been reported by several research groups to be a potent antagonist at the Y1 neuropeptide Y (NPY) receptor subtype. However, 1229U91 also displaces (125)I-peptide YY (PYY) with high affinity from the Y4 subtype. Previously, we reported that 1229U91 had full agonist properties for the Y4 receptor. To characterize the pharmacological properties of 1229U91 directly, we had it radioiodinated with the chloromine-T method. (125)I-1229U91 bound to cell lines expressing the human Y1 and Y4 receptors with high affinity. The K(d) and B(max) for (125)I-1229U91 binding to Y1 were 14.9 pM and 1458 fmol/mg protein, respectively. The Y4 receptor bound (125)I-1229U91 with a K(d) of 12.5 pM and a B(max) of 1442 fmol/mg protein. When competing (125)I-1229U91 binding from Y1 and Y4 receptors, a similar rank order of potency was observed: 1229U91 > [Leu(31),Pro(34)]-NPY >/= [Leu(31),Pro(34)]-PYY > PYY >/= NPY > NPY(2-36) > PYY(3-36). Pancreatic polypeptide (PP) potently displaced (125)I-1229U91 from the Y4 receptor, but displayed little affinity for Y1. In autoradiographic studies with rat brain sections, (125)I-1229U91 bound with a distribution similar to that reported for the Y1 receptor when localized with (125)I-[Leu(31),Pro(34)]-PYY. Brain regions exhibiting binding sites for (125)I-PP were not detected with this radioligand. Those include the interpeduncular nucleus and the periventricular nucleus of the hypothalamus. Furthermore, (125)I-labeled rat PP was not displaced from these areas with 10 nM 1229U91. Thus, (125)I-1229U91 is a high affinity Y1 and Y4 radioligand and binds with a distribution in the rat brain consistent with the localization of the Y1 receptor.

Differential distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat brain.

Urocortin, a novel 40 amino acid neuropeptide, is a member of the corticotropin-releasing factor family. With 45% homology to corticotropin-releasing factor, urocortin binds with similar affinity to the corticotropin-releasing factor- and corticotropin-releasing factor-2 receptors and may play a role in modulating many of the same systems as corticotropin-releasing factor. To assess whether urocortin and corticotropin-releasing factor are localized in the same regions of the brain, we compared the distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat central nervous system. Polyclonal antibodies to rat corticotropin-releasing factor and rat urocortin were generated and utilized to map the distribution of corticotropin-releasing factor- and urocortin-like immunoreactivities throughout the rat forebrain and brainstem. Characterization of the antibodies by radioimmunoassay showed no cross-reactivity with related peptides. Male Sprague-Dawley rats were treated with colchicine for 18-24 h. Following colchicine treatment, the rats were perfused with paraformaldehyde-lysine-periodate fixative and their brains removed. Serial coronal sections were taken throughout the rat brain and processed for either corticotropin-releasing factor- or urocortin-like immunoreactivity. Urocortin-like immunoreactivity shows a discrete localization within several regions including the supraoptic nucleus, the median eminence, Edinger-Westphal nucleus and the sphenoid nucleus. This is in contrast to the more abundant corticotropin-releasing factor-like immunoreactivity. Regions containing high levels of corticotropin-releasing factor immunoreactivity include the lateral septum, paraventricular nucleus of the hypothalamus, median eminence and locus coeruleus. There are a few regions that contain both urocortin-immunoreactive and corticotropin-releasing factor-immunoreactive cells, such as the supraoptic nucleus and the hippocampus. Therefore, urocortin and corticotropin-releasing factor appear to have different distribution patterns which may be indicative of their respective physiological functions.

Molecular characterization of the human neuropeptide Y Y2-receptor.

Five neuropeptide Y receptors, the Y1-, Y2-, Y4-, Y5- and y6-subtypes, have been cloned, which belong to the rhodopsin-like G-protein-coupled, 7-transmembrane helix-spanning receptors and bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. In this study, the Y2-receptor subtype expressed in a human neuroblastoma cell line (SMS-KAN) and in transfected Chinese hamster ovary cells (CHO-hY2) was characterized on the protein level by using photoaffinity labeling and antireceptor antibodies. Two photoactivatable analogues of NPY were synthesized, in which a Tyr residue was substituted by the photoreactive amino acid 4-(3-trifluoromethyl)-3H-diazirin-3-ylphenylalanine ((Tmd)Phe), [Nalpha-biotinyl-Ahx2,(Tmd)Phe36]NPY (Tmd36), and the Y2-receptor subtype selective [Nalpha-biotinyl-Ahx2,Ahx5-24,(Tmd)Phe27]N PY (Tmd27). Both analogues were labeled with [3H]succinimidyl-propionate at Lys4 and bind to the Y2-receptor with affinity similar to that of the native ligand. A synthetic fragment of the second (E2) extracellular loop was used to generate subtype selective antireceptor antibodies against the Y2-receptor. Photoaffinity labeling of the receptor followed by SDS-PAGE and detection of bound radioactivity and SDS-PAGE of solubilized receptors and subsequent Western blotting revealed the same molecular masses. Two proteins correspondingly have been detected for each cell line with molecular masses of 58 +/- 4 and 50 +/- 4 kDa, respectively.

Role of corticotropin-releasing factor and urocortin within the basolateral amygdala of rats in anxiety and panic responses.

The amygdala is a critical temporal lobe structure involved in the expression of anxiety and stress responses. The basolateral nucleus (BLA) of the amygdala in particular, may play a key role in anxiety. Furthermore, corticotropin-releasing factor (CRF), a 41 amino acid peptide, has been strongly implicated in the regulation of stress and anxiety responses. Centrally administered CRF has been shown to increase the anxiety-like behaviors of rodents in several animal models. A recently cloned related peptide, Urocortin (Ucn), appears to have similar affinity for the CRF1 receptor, but higher affinity at the CRF2 receptor. When microinjected into the BLA, we found Ucn was substantially more potent than CRF in producing anxiogenic-like behavior as assessed in the social interaction test. Furthermore, repetitive administration of subthreshold doses of Ucn and CRF resulted in 'priming'. Once primed, these animals exhibited behavioral and cardiovascular responses to intravenous sodium lactate, a panicogenic agent in susceptible human patients. These results suggest central CRF and Ucn play a role in generating anxiety which may be similar to that seen in pathological conditions such as panic disorder.

Structure-activity relationship of a series of diaminoalkyl substituted benzimidazole as neuropeptide Y Y1 receptor antagonists.

A series of benzimidazoles (4) was synthesized and evaluated in vitro as potent and selective NPY Y1 receptor antagonists. Substitution of the piperidine nitrogen of 4 with appropriate R groups resulted in compounds with more than 80-fold higher affinity at the Y receptor compared to the parent compound 5 (R = H). The most potent benzimidazole in this series was 21 (Ki = 0.052 nM).

Amygdalar neuropeptide Y Y1 receptors mediate the anxiolytic-like actions of neuropeptide Y in the social interaction test.

The effects of intra-amygdalar neuropeptide Y infusions were assessed in rats using the social interaction test. Neuropeptide Y administered into the central nucleus of the amygdala did not alter behavior, while injections into the basolateral nucleus of the amygdala produced an increased social interaction time. Furthermore, the anxiolytic-like effect was antagonized by co-administration of the potent neuropeptide Y Y1 receptor antagonist ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) 3304, but not with the inactive enantiomer ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) 3457. Therefore, neuropeptide Y produces an anxiolytic-like effect in the social interaction test through neuropetide Y Y1 receptors located in the basolateral amygdala.

Structure-activity relationships of a series of benzothiophene-derived NPY Y1 antagonists: optimization of the C-2 side chain.

A series of benzo[b]thiophene-derived NPY-1 receptor antagonists is described. Systematic modification of the C-2 substituent afforded a 1000-fold range in Y1 receptor affinity. Appropriate substitution at the ortho and para positions of the C-2 phenyl ether produced a synergistic effect on Y1 binding affinity, which led to the discovery of the most active ligands, 12t (K(i) = 15 nM), 12u (K(i) = 11 nM), and 12v (K(i) = 13 nM).

Role of hypothalamic neuropeptide Y in feeding and obesity.

The 36-amino-acid peptide, neuropeptide Y (NPY), is the most abundant peptide in the rat brain. When administered into the brain, NPY produces a variety of physiological actions including a pronounced stimulation of feeding in satiated rats. Elevations in hypothalamic NPY have been reported after food deprivation and in genetically obese rodents. NPY is believed to produce its actions through a portfolio of G-protein coupled receptors, Y1, Y2, Y4 and Y5. Studies using peptide analogs, receptor knockout animals and specific receptor antagonists suggest the Y1 and Y5 receptors are important in mediating the effects of NPY on food intake in rats. Development of specific receptor antagonists with improved pharmacokinetic properties will be required to determine the importance of NPY in human obesity and appetite disorders.

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites.

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu31, Pro34]NPY, and NPY(3-36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.

The cloned guinea pig pancreatic polypeptide receptor Y4 resembles more the human Y4 than does the rat Y4.

Pancreatic polypeptide (PP) is involved in gastrointestinal functions and forms, together with neuropeptide Y (NPY) and peptide YY (PYY), the PP-fold family of peptides. The PP-binding receptor subtype Y4 has so far been cloned in human, rat, and mouse, and displays extensive species differences regarding sequence, pharmacology, and distribution. To explore this variability further, we have cloned the Y4 receptor in the guinea pig, which is evolutionarily equally distantly related to both humans and rodents. The guinea pig Y4 receptor is 84% identical to the human Y4 receptor, but only 74-75% identical to the rat and mouse receptors. The two latter are 75-76% identical to human Y4. The guinea pig Y4 receptor bound 125I-hPP with a dissociation constant (Kd) of 29+/-3 pM. The pharmacological profile of guinea pig Y4 has the following rank order of potencies: PP > NPY approximately = PYY approximately = LP-NPY approximately = LP-PYY > NPY2-36 >> [D-Trp32]NPY. Thus, the guinea pig receptor is more similar to the human Y4 than to the rat Y4 both in sequence and pharmacology. This agrees with the greater identity between guinea pig and human PP compared to rat PP. These comparisons suggest that the rodent PPs and Y4 receptors have an accelerated replacement rate.

Characterization of [125I-Tyr0]-corticotropin releasing factor (CRF) binding to the CRF binding protein using a scintillation proximity assay.

We describe the characterization of high affinity [125I-Tyr0]-human CRF binding to purified recombinant human CRF-binding protein (CRF-BP) using a scintillation proximity assay (SPA). For this stable nonseparation technique developed in 96 well microtiter plates, biotinylated CRF-BP is captured by streptavidin-coated SPA beads for the detection of bound [125I-Tyr0]-CRF. Unbound [125I-Tyr0]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of < 100 cpm determined in the presence of excess unlabeled human CRF. A comparison of the SPA method with a charcoal precipitation method confirmed that the biotinylation procedure did not adversely affect affinity of the CRF-BP for [125I-Tyr0]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant (Kd) of 208 +/- 5.0 pM (+/- S.D., n = 3). An inhibition constant (Ki) for unlabeled CRF was calculated to be 0.22 +/- 0.03 nM (+/- S.D., n = 8) and a pharmacological profile for eight CRF-related neuropeptides gave a rank potency similar to previously reported results. Finally, the assay variability was assessed with intra- and inter-plate coefficients of variation which were less than 5% each.

The selective norepinephrine reuptake inhibitor, LY368975, reduces food consumption in animal models of feeding.

The compound, LY368975 ((R)-thionisoxetine) is a potent and selective inhibitor of the norepinephrine (NE) reuptake site. We evaluated the in vivo properties of LY368975 in various animal models. In mice, LY368975 prevented heart NE depletion by 6-hydroxydopamine with an ED50 of 1.22 mg/kg. In rats, orally administered LY368975 inhibited 3H-NE uptake into hypothalamic synaptosomes ex vivo with an ED50 of 2.5 mg/kg and 3H-tomoxetine binding to the NE transporter with an ED50 of 2.7 mg/kg. When rats were deprived of food for 18 hr, 10 mg/kg LY368975 was able to suppress food intake 1, 2 and 4 hr after reintroduction of the feed. In nonfasted rats trained to drink sweetened condensed milk, LY368975 produced a dose-dependent reduction in consumption with a 44% decrease at 3 mg/kg. At doses up to 10 mg/kg p.o., LY368975 produced no significant effects on locomotor activity suggesting the compound does not activate or sedate the animals at pharmacologically relevant doses. Therefore, LY368975 is an orally available and centrally active NE reuptake inhibitor that is capable of reducing food consumption in rodents. Compounds of this class may have use in the treatment of obesity and eating disorders.

Synthesis and evaluation of a series of novel 2-[(4-chlorophenoxy)methyl]benzimidazoles as selective neuropeptide Y Y1 receptor antagonists.

A series of novel benzimidazoles (BI) derived from the indole 2 was synthesized and evaluated as selective neuropeptide Y (NPY) Y1 receptor antagonists with the aim of developing antiobesity drugs. In our SAR approach, the (4-chlorophenoxy)methyl group at C-2 was kept constant and a series of BIs substituted with various piperidinylalkyl groups at N-1 was synthesized to identify the optimal spacing and orientation of the piperidine ring nitrogen relative to the benzimidazole. The 3-(3-piperidinyl)propyl in 33 was found to maximize affinity for the Y1 receptor. Because of the critical importance of Arg33 and Arg35 of NPY binding to the Y1 receptor, the incorporation of an additional aminoalkyl functionality to the structure of 33 was explored. Methyl substitution was used to probe where substitution on the aromatic ring was best tolerated. In this fashion, the C-4 was chosen for the substitution of the second aminoalkyl functionality. Synthesis of such compounds with a phenoxy tether using the 4-hydroxybenzimidazole 11 was pursued because of their relative ease of synthesis. Functionalization of the hydroxy group of 45 with a series of piperidinylalkyl groups provided the dibasic benzimidazoles 55-62. Among them, BI 56 demonstrated a Ki of 0.0017 microM, which was 400-fold more potent than 33. To evaluate if there was a stereoselective effect on affinity for these BIs, the four constituent stereoisomers (69-72) of the BI 60 were prepared using the S- and R-isomers of bromide 17. Antagonist activity of these BIs was confirmed by measuring the ability of selected compounds to reverse NPY-induced forskolin-stimulated cyclic AMP. The high selectivity of several BI antagonists for the Y1 versus Y2, Y4, and Y5 receptors was also shown.

Multiple receptors for the pancreatic polypeptide (PP-fold) family: physiological implications.

The pancreatic polypeptide (PP-fold) family of peptides consists of the endocrine peptides, pancreatic polypeptide (PP) and peptide YY (PYY), and the neuroneally derived peptide, neuropeptide Y (NPY). All three peptides are found in the circulation, with PP found primarily in the pancreas and PYY found principally in the gut. NPY is released into the circulation from neuroneal stores in response to stress. These peptides have broad peripheral actions on a number of organs. Not surprisingly, PYY and PP are believed to play an important role in the function of the gastrointestinal tract while NPY is a potent vasconstrictor and may have effects on the gut through the enteric nervous system. In the brain, NPY has been implicated in anxiety and depression, feeding and obesity, memory retention, neuroneal excitability, endocrine function, and metabolism. Recent advances in the molecular biology of the receptors for these peptides have resulted in the identification of at least six receptor subtypes with varying peptide pharmacology. Compared to other G-protein coupled receptor families, the PP-fold peptide receptors exhibit a relatively low level of sequence identity. Further advances in the development of selective agonists and antagonists for individual receptor subtypes will be needed to understand further their role in physiological function.