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To investigate structure-function relationships in gamma-glutamyl carboxylases, the enzyme from Drosophila melanogaster was characterized. Four cysteine residues were shown to be important determinants for enzymatic activity. Native Drosophila substrates have not yet been identified, but propeptides of human prothrombin and factor IX are recognized by the Drosophila enzyme. The presence of the propeptide region increased apparent affinity by approximately 200-fold, and mutation of a hydrophobic residue of factor IX propeptide (F-16A) decreased carboxylation by 90%, as in the human enzyme. Substrate recognition appears to be highly conserved between the human and Drosophila gamma-glutamyl carboxylases. Inactivation of Drosophila gamma-glutamyl carboxylase by non-sense mutations or insertional mutagenesis by P-element insertion have no apparent effects on growth and fertility under laboratory conditions.

Degarelix is a potent very long-acting GnRH antagonist after subcutaneous administration. In this paper, we describe the synthesis of two analogs of degarelix incorporating racemic 3-(2-methoxy-5-pyridyl)-alanine (2-OMe-5Pal, 5) at position 3. The two diastereomers were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and the absolute stereochemistry at position 3 in the peptides was determined by enzymatic digestion with proteinase K. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analog 7 with D2-OMe-5Pal was potent in vitro (IC50 = 5.22 nM); however, analog 8 with L2-OMe-5Pal at position 3 in degarelix lost potency as an antagonist of the human GnRH receptor (IC50 = 36.95 nM). Both the analogs were found to be short-acting in vivo.

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.

In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K(i)0.5 nM) for the rat GnRH receptor (rGnRHR). The most favored cycles bridged the side chains of residues (4-10),(1,2) (5-8),(2) (4-10/5-8),(2) (1-3),(3) and (1-3/4-10).(3) Our goal was to identify a consensus model of bioactive conformations of GnRH antagonists, yet these biocompatible constraints did not sufficiently restrain the spatial location of the N-terminal tripeptide with respect to the C-terminal heptapeptide, due largely to the rotational freedom about the bonds connecting these regions. Examination of models derived from NMR studies of cyclo(4-10) analogues suggested a large number of possible cyclic constraints such as cyclo (0-8), (1-8), or (2-8). All analogues tested with these substitutions were inactive as antiovulatory agents at 1 mg/rat (5-9) and had low affinity for rGnRHR. On the other hand, bridging positions 3 and 8 with a [DAsp(3)] to [Dbu(8)] (12, K(i) = 13 nM) or [Orn(8)] (13, K(i) = 14 nM) in the parent compound cyclo(3-8)[Ac-DNal(1),DCpa(2),DXaa(3), Arg(5),DNal(6),Xbb(8),DAla(10)]GnRH yielded analogues that blocked ovulation at 250 microgram/rat. Analogue 14 (K(i) = 2.3 nM), with a [DAsp(3), Lys(8)] bridge, was fully active at 50 microgram/rat. Loss of potency (20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K(i) = 23 nM). Dicyclic analogues possessing the (4-10) cycle and selected (1-6), (2-6), and (2-8) cycles led to analogues that were inactive at doses of 500 microgram/rat or larger. Two analogues with (1-8/4-10) cycles (16, K(i) = 1.1 nM) or (3-8/4-10) cycles (22, K(i) = 17 nM) showed full antiovulatory potency at 250 microgram/rat. None of these substitutions yielded analogues potent enough (80% inhibition of ovulation at 5 microgram/rat or less and K(i)0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1, 1'-5/4-10) analogues met this criterion: dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (32, K(i) = 0.22 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DNal(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (34, K(i) = 0.38 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(betaAla),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (40, K(i) = 0.15 nM), and dicyclo(1, 1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (41, K(i) = 0.24 nM). Since they differed slightly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral condition for teleosts, 2) has the potential for separate regulation of the distinct GnRHs, and 3) may be an evolutionary advantage for refined control of reproduction in different environments.

The characteristics of a high-affinity antagonist radioligand are compared with those a high-affinity agonist in binding to the cloned corticotropin-releasing factor receptor type 1 (CRF-R1) and type 2 (CRF-R2) and to the native receptors that exist in rat cerebellum and brain stem. The relative potencies of CRF antagonists and agonists to the two types of cloned CRF receptors overexpressed stably in Chinese hamster ovary cells are determined using the antagonist radioligand 125I- [DTyr1]astressin (Ast*), and the agonist radioligand, 125I -[Tyr0]rat urocortin (Ucn*). The inhibitory binding constants (Ki) of astressin and urocortin are 1 to 2 nM for all receptors and are independent of which radioligand is employed. Astressin binds with high affinity to the native cerebellar/brain stem receptor and relative potencies of selected CRF analogs determined with Ast* on the native receptor are similar to those obtained for the cloned CRF-R1. The specific binding of Ast* to endogenous brain receptors is greater than that of Ucn*, resulting in more sites being detected by the antagonist than by the agonist. In contrast to another CRF agonist, the binding of Ucn* to the cloned receptors is relatively insensitive to guanyl nucleotides at both 20 degreesC and 37 degreesC; however, its binding to the native receptor is displaced by guanyl nucleotides at 37 degreesC and, to a lesser degree, at 20 degreesC. As expected, the binding of the antagonist Ast* is not affected by guanyl nucleotides. Because it is a high-affinity, specific CRF antagonist, astressin is eminently suitable as a ligand for detection and characterization of both endogenous and cloned CRF receptors.

Urocortin (Ucn) is a newly identified mammalian member of the CRF family of peptides. Ucn activates CRF receptors (both CRF-R1 and CRF-R2) with greater potency than CRF itself, suggesting that Ucn may play an endogenous role in eliciting (at least some) CRF receptor-mediated events. Because the most characterized physiological function of CRF receptors is the activation of pituitary ACTH secretion, we have compared the effects and potential endogenous roles of CRF and Ucn in regulating plasma ACTH concentrations in intact male rats. Synthetic rat Ucn injected i.v. (0.09-9.0 nmol/kg) elicited ACTH secretion in a dose-dependent manner, causing greater ACTH secretion than CRF at each dose tested. The increases in plasma ACTH concentrations produced by CRF or Ucn were virtually abolished by pretreatment with the CRF receptor antagonist, astressin (3 mg/kg), and were partially attenuated (by 27-37%) by an antiarginine vasopressin serum. These data indicate that both Ucn and CRF elicit ACTH secretion via CRF receptor-dependent mechanisms, and that the ACTH-releasing activities of both CRF and Ucn are potentiated by endogenous arginine vasopressin. Intravenous administration of rabbit anti-Ucn serum, which inhibited ACTH secretion produced by Ucn, but not CRF, had no statistically significant effect on either resting (midday) plasma ACTH concentrations or the rise in ACTH levels elicited by 30 min of intermittent electrofootshocks. By contrast, treatment with a rabbit anti-CRF serum that specifically inhibited the ACTH response to CRF lowered plasma concentrations in control unstressed rats and largely prevented the plasma ACTH response to electrofootshocks. These data indicate that although Ucn is a more potent ACTH secretagogue than CRF in the intact male rat, it is not a major endogenous regulator of pituitary ACTH secretion under basal (midday) conditions or during acute footshock stress.

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = gamma-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

Neuropeptide Y (NPY) has neuromodulatory actions on multiple brain functions including endocrine, behavioral, and circadian processes and has been implicated in the pathophysiology of both anxiety and depression. Behavioral studies suggest that NPY is a potent anxiolytic, whereas CRF is anxiogenic, thus it seems that a balance of these two peptides may exert important influences on behavioral state regulation. However, little is known about how the NPY/CRF balance affects general arousal, attention, and/or sleep states. The present study evaluated the effects of CRF alone, and co-administered with NPY, on spontaneous brain activity as well as on auditory processing using electrophysiological measures. Electroencephalographic (EEG) and event-related potentials (ERPs) were obtained in rats following intracerebroventricular administration of CRF (0.5 microgram) and CRF (0.5 microgram)/NPY (5.0 or 15 micrograms). Auditory processing, as assessed by ERPs, was affected most significantly in the frontal cortex where CRF produced increases in the N1 and P3 components of the ERP, and NPY/CRF co-administration produced significant decreases. These data are consistent with a role for CRF in hyperarousal, and further suggest that NPY may be capable of reversing such states. Administration of CRF also produced a significant increase in the time to sleep onset and a decrease in the amount of time spent in non-rapid eye movement (NREM) sleep as quantified by scoring the EEG paper records. Co-administration of NPY with CRF reversed the effects of CRF on sleep duration and sleep onset in a dose-dependent fashion. Spectral analysis revealed that CRF produced quantitative changes in the EEG that were similar to what has previously been reported. CRF-induced increases in fast frequency activity were found to be reversed by co-administration of NPY. Taken together these data suggest that "dysregulation" of sleep and arousal states in depression and anxiety may be consistent with an upset of the balance between hypothalamic neuropeptide systems.

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians.

To test the hypothesis that over a 4-week treatment period, Nal-Glu GnRH antagonist ([AcD2Nal1, D4ClPhe2, D3Pal3, Arg5, DGlu6 [AA], DAla10] GnRH) at a dose of 200 micrograms/kg per day SC would suppress levels of immunologically active and biologically active LH and FSH more completely than a dose of 100 micrograms/kg per day.Placebo controlled clinical study.A university community.Thirty normal male volunteers.We administered Nal-Glu at doses of 0, 100, and 200 micrograms/kg body weight per day in combination with T enanthate, 50 mg IM weekly, to separate groups of men (9 or 10 men per group) for 4 weeks.Serum levels of immunologically active and biologically active gonadotropins were suppressed similarly in both groups of men who received Nal-Glu; this suppression was significantly greater than in the men who received placebo + T. Local side effects were more severe in the Nal-Glu 200 micrograms/kg per day group.Administration of Nal-Glu in combination with T suppresses gonadotropins more completely than does T alone, but at doses100 micrograms/kg, gonadotropins are not suppressed additionally with larger doses of Nal-Glu. Subjects experienced greater local discomfort and side effects with the higher dosage. These findings suggest that dosages of Nal-Glu of100 micrograms/kg per day may have no advantage over the 100-micrograms/kg dose in a male contraceptive regimen.

In goldfish, GnRH stimulates gonadotropin-II (GTH-II) and growth hormone (GH) release. The two native forms of GnRH, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), bind to two classes of GnRH binding sites: high-affinity/low-capacity sites and low-affinity/high-capacity sites. Our previous in vitro perifusion studies of goldfish pituitary fragments showed that [Ac-delta 3-Pro1, 4FD-Phe2, D-Trp3,6]-mGnRH (analog E), [Ac-delta 3-Pro1, 4FD-Phe2, D-Trp3,6]-sGnRH (analog C), and [Ac-D(2)Nal1, 4Cl-D-Phe2, D-(3)Pal3,6]-cGnRH-II (analog N) inhibited both sGnRH- and cGnRH-II-stimulated GTH-II and GH release. Interestingly, analog C stimulated GH release but not GTH-II release. The objectives of the present study were 1) to test the site of action of GnRH antagonists in goldfish, 2) to test the relationship between receptor binding affinity of antagonists and their in vitro inhibitory potencies and apparent duration of action, and 3) to compare the binding characteristics of analog C with its differential action on GTH-II and GH release. As in previous studies, analog E suppressed sGnRH-stimulated GTH-II and GH release from perifused pituitary fragments. Similarly, analog E suppressed both sGnRH- and cGnRH-II-stimulated GTH-II and GH release from perifused dispersed goldfish pituitary cells, indicating the direct action of GnRH antagonists at the pituitary cell level. In the receptor binding studies, analog E displaced 125I-[D-Arg6, Pro9NHEt]-sGnRH (sGnRH-A) from crude goldfish pituitary membrane preparations in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the isolation, characterization, and total synthesis of a small peptide ligand for nicotinic acetylcholine receptors (nAChRs). It is highly active against the neuromuscular receptor in frog but not in mice. In contrast, it induces seizures when injected centrally in mice and rats, suggesting that it may target neuronal nAChRs in mammals. Although such receptors may be important in both normal cognition and the pathophysiology of several neuropsychiatric disorders, there are few ligands to discriminate between the multiple receptor subtypes. The new peptide is a highly divergent alpha-conotoxin from the snail Conus imperialis, which preys on polychaete worms. In this article, the purification, structural analysis, synthesis, and preliminary physiological characterization of alpha-conotoxin ImI (alpha-CTx-ImI) are reported. The sequence of the peptide is: Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2. The peptide shows striking sequence differences from all alpha-conotoxins of fish-hunting Conus, but its disulfide-bridging is similar: [2-8; 3-12]. We suggest that cone venoms may provide an array of ligands with selectivity for various neuronal nAChR subtypes.

In goldfish the two native forms of gonadotropin-releasing hormone (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), stimulate both gonadotropin-II (GTH-II) and growth hormone (GH) release. Modifications of GnRH structure at positions 1, 2, 3, and 6 often result in an antagonist in goldfish, an observation well documented in mammalian studies. In a preliminary study in goldfish, a mammalian GnRH antagonist, [Ac-D(2)Nal1, 4Cl-D-Phe2, D(3)-Pal3,6, Arg5, D-Ala10]-mGnRH (analog L) weakly stimulated GTH-II release, and strongly inhibited GH release. The objectives of the present study were to study the dose-related actions of analog L on GTH-II and GH release in the goldfish, the specificity of inhibition of native GnRH actions, and to test whether analog L can act directly on goldfish pituitary cells. In a goldfish pituitary fragments perifusion system, analog L at different concentrations, given as 2-min pulses or as 30-min prolonged treatments, stimulated GTH-II and inhibited GH release in a dose-dependent manner. Analog L at 2 microM concentration (45 min) significantly suppressed sGnRH- and cGnRH-II-stimulated GTH-II as well as GH release. Analog L specifically inhibited GnRH-stimulated GH release, without having any significant effects on the GH release induced by either SKF38393, a dopamine D1 receptor agonist, or thyrotropin-releasing hormone. The GTH-II stimulatory and GH-inhibitory actions of analog L were significantly suppressed by a 'true' GnRH antagonist (Ac-delta 3-Pro1, 4FD-Phe2, D-Trp3,6)-mGnRH. Further, analog L stimulated GTH-II release and suppressed GH release from the enzymatically dispersed goldfish pituitary cells, indicating the direct actions of analog L at the pituitary cell level. Analog L also displaced 125I-(D-Arg6, Pro9 NHEt)-sGnRH bound to crude goldfish pituitary membrane preparations in a dose-related manner. In conclusion, contrary to its action as a potent GnRH antagonist in mammals, analog L has GTH-II stimulatory action in goldfish. Analog L by acting via GnRH receptors at the pituitary cell level differentially acts on GTH-II and GH release, suggesting functional differences in the properties of the GnRH receptors on GTH and GH cells. Analog L also specifically inhibits sGnRH and cGnRH-II actions on GTH-II and GH release.

The Nal-Glu gonadotropin-releasing hormone (GnRH) antagonist, when given in daily subcutaneous (SC) doses of 5 mg or higher, maximally suppresses serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels to near undetectable levels and induces azoospermia in normal men; lower doses (1.5 and 3.0 mg) are less effective. Cost and convenience are important considerations in contraceptive development. Studies with GnRH agonists suggest that constant delivery is more effective in suppressing gonadal function than equal doses by single daily injection. In this study, we examined whether the constant infusion (CI) of a submaximal suppressive dose (1.5 mg) of Nal-Glu would be more effective in suppressing the pituitary-gonadal axis than its repeated single daily injections (SDI). This (1.5 mg) dose was selected because the 5 mg dose given once daily SC for 21 days led to maximal suppression of LH, FSH, and testosterone (T) levels, whereas 1.5 mg once daily for 21 days gave only partial suppression. It was felt that if continuous infusion was considerably more effective than intermittent administration of this submaximal dose, then the development of long-acting sustained release delivery systems for contraceptives based on GnRH antagonist analogs would allow both reduced cost and enhanced convenience. One and a half mg of Nal-Glu was administered SC either as a SDI or CI over 24 hours for 21 days to two groups of five normal men. Three measurements of serum LH, FSH, and T were performed before antagonist injection and 1, 2, 4, 8, 12, 16, and 24 hours after Nal-Glu injection on days 0, 1, 7, 21.(ABSTRACT TRUNCATED AT 250 WORDS)

To test the hypothesis that corticotropin-releasing hormone (CRH) is linked to stress-associated reproductive dysfunction in the human by determining if the administration of human corticotropin-releasing hormone (hCRH) results in an inhibition of gonadotropin secretion.Twenty-four-hour prospective study with frequent (every 10 minutes) blood sampling.University Clinical Research Center.Sequential 8-hour infusions of normal saline, hCRH (1 to 5 micrograms/kg per hour), and hCRH plus naloxone (2 mg/h).Four normal cycling women and four postmenopausal women.Plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and adrenal and ovarian steroids.In response to hCRH, a prompt and sustained rise in cortisol (F) was noted in both normal cycling women and postmenopausal women. No inhibition of LH or FSH was noted during either the hCRH or hCRH plus naloxone infusion in either group of women. Unexpectedly, elevations in the mean LH peak amplitude and the transverse mean LH concentration were noted in the postmenopausal women during the infusion of hCRH as compared with saline. The infusion of hCRH had no apparent effect on concentrations of PRL, FSH, and gonadal and adrenal steroids (except for F).Under these conditions, intravenously administered hCRH has no inhibitory effect on gonadotropin secretion in either premenopausal or postmenopausal women. The mechanism by which stress exerts its deleterious effect on reproductive function in the human remains unknown.

Neuropeptide Y (NPY) is a 36-amino-acid, C-terminal amidated peptide that is found in bulbospinal pathways and can inhibit the release of the primary afferent C-fiber neurotransmitter, substance P. Based on these observations, the present studies examined the possible antinociceptive effects of this peptide and several NPY fragments after intrathecal administration in rats prepared with chronic intrathecal catheters. In the 52 degrees C hot plate test, NPY produced a dose-dependent elevation in the nociceptive threshold with a median effective dose of 1.1 nmol. The ordering of fragments' activity was: NPY greater than NPY16-36 greater than or equal to NPY19-36 greater than or equal to NPY14-36 greater than or equal to NPY18-36 much greater than NPY1-36-OH = NPY18-36-OH = 0. In the paw pressure test, NPY was not active, even at the highest doses examined (median effective dose greater than 20 nmol), whereas the C-terminal fragments retained their potency and produced significant increases in the pressure required to evoke escape (NPY18-36: median effective dose = 18.7 nmol). The rank ordering of activity in the paw pressure test was: NPY19-36 greater than or equal to NPY14-36 greater than or equal to NPY18-36 greater than or equal to NPY16-36 much greater than NPY = NPY18-36-OH = 0. Peptide YY, human pancreatic polypeptide and avian pancreatic polypeptide behave similarly to NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the possibility of multiple neuropeptide Y (NPY) receptor subtypes. NPY-stimulated Ca2+ mobilization in human erythroleukemia (HEL) cells was used to screen a number of NPY analogues. The potencies of three of these analogues [peptide YY (PYY), [D-Tyr-36]NPY, and NPY-(18-36)] were compared with that of NPY in the following model systems: Ca2+ mobilization and inhibition of adenosine 3',5'-cyclic monophosphate accumulation in HEL cells, potentiation of vasoconstriction in the isolated rabbit ear artery, reduction of cutaneous microvascular perfusion in the rat digit, and inhibition of [3H]serotonin release in rat brain. In each of the five models, PYY was a full agonist that exhibited a similar or slightly higher potency than NPY, whereas [D-Tyr-36]NPY and NPY-(18-36) were partial agonists with lower potencies: NPY-(18-36) had a lower potency and efficacy than [D-Tyr-36]NPY in HEL cells and the rabbit ear artery, but was more effective than [D-Tyr-36]NPY for constricting cutaneous microvasculature and inhibiting serotonin release. Because of its weak partial agonism, we also tested NPY-(18-36) as an antagonist of NPY-stimulated Ca2+ mobilization in HEL cells. NPY-(18-36) shifted the NPY concentration-response curve to the right with a KB affinity value of 297 nM. In summary, [D-Tyr-36]NPY and NPY-(18-36) are partial agonists, the relative potency of which varies between systems. These data demonstrate the presence of multiple NPY receptor subtypes. We propose a modified classification scheme of NPY receptor subtypes.

We have studied the hemodynamic effects of neuropeptide Y (NPY) and its COOH-terminal fragment NPY-(18–36) in conscious rats. Intra-arterial injection of NPY rapidly elevated systemic vascular resistance (SVR), which remained high for greater than 30 min. Cardiac output (CO) decreased, and it remained low for greater than 30 min. Accordingly, blood pressure rose only transiently and returned to base-line values within 5 min. The reduction of CO could be attributed to a decreased stroke volume with an only marginal reduction of heart rate. Thus a direct cardiodepressive effect of NPY rather than baroreflex activation appears to be the major cause of the reduced CO. In vitro experiments excluded the possibility that NPY has direct negative inotropic effects and suggest that its cardiodepressive action is caused by coronary vasoconstriction or by presynaptic inhibition of norepinephrine release. Intra-arterial injections of NPY-(18-36) caused different hemodynamic effects. NPY-(18–36) decreased CO in a manner similar to that seen with NPY but initially did not elevate SVR, resulting overall in a reduced blood pressure. Only later, when blood pressure was reduced, was an elevation of SVR observed, which could be associated with increased plasma levels of catecholamines, angiotensin II, vasopressin, and NPY. Thus NPY-(18–36) mimics the cardiac effects of NPY but does not elicit its vascular effects. As NPY-(18–36) discriminates between NPY receptor subtypes in vitro, we conclude that the cardiac and vascular effects of NPY are mediated by distinct receptor subtypes.

Somatostatin was purified from anglerfish pancreatic islets using acetic acid extraction, gel filtration (Bio-Gel P-10), ion exchange chromatography (CM Bio-Gel A), and reversed phase high pressure liquid chromatography. The resulting peptide was characterized by RIA, bioassay, and determination of amino acid composition. Anglerfish islet somatostatin was found to possess an amino acid composition and immunological and biological activities equivalent to synthetic somatostatin. Sequence analyses revealed that the primary structure was H-Ala-Gly-cyclo-[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys]-OH. These results demonstrate that anglerfish islet somatostatin has the same primary structure as somatostatin from all other sources characterized to date.

The central nervous system effects of thyrotropin-releasing hormone (TRH) on proximal duodenal bicarbonate secretion were studied in freely moving rats. Cerebroventricular administration of TRH (0.5-5.0 nmol) significantly stimulated basal duodenal bicarbonate secretion, whereas intravenous administration of TRH did not. Ganglionic blockade with chlorisondamine and truncal vagotomy abolished TRH-induced bicarbonate secretion, whereas atropine significantly attenuated the response. The vasoactive intestinal peptide (VIP) receptor antagonist, (4Cl-D-Phe6, Leu17) VIP given intravenously completely prevented the stimulatory effect of central TRH on duodenal bicarbonate secretion. In contrast, hypophysectomy, adrenalectomy, opiate and noradrenergic blockade, or indomethacin did not affect the TRH-induced bicarbonate response. Intravenous administration of VIP and carbachol significantly stimulated bicarbonate outputs, and these responses were blocked by the VIP antagonist and atropine, respectively. These results indicate that TRH may serve as a central nervous system mediator that stimulates duodenal bicarbonate secretion in rats by increasing vagal outflow. Vagal stimulation induced by TRH increases duodenal bicarbonate secretion by the release of VIP and, in part, by activation of a muscarinic pathway but not by pituitary, adrenal, and noradrenergic pathways or endogenous opiates and prostaglandins. The actions of peripheral VIP and carbachol appear to be mediated by specific VIP and muscarinic receptors, respectively.

We have studied the effects of 30 peptides administered intracerebroventricularly on basal and pentagastrin-stimulated (8 micrograms/kg s.c.) gastric acid secretion in conscious dogs. None of the peptides significantly increased basal gastric acid secretion. Twelve peptides (2 nmol/kg) significantly (p less than 0.01) decreased the pentagastrin-stimulated 2-h acid output (percentage inhibition in parentheses): human calcitonin (CT) (36%), neurotensin (NT) (52%), rat corticotropin-releasing factor (CRF) (59%), human calcitonin gene-related peptide (CGRP) (59%), ovine CRF (66%), beta-endorphin (beta-End) (80%), urotensin-I (81%), rat CT (81%), porcine gastrin-releasing peptide (GRP) (83%), sauvagine (Svg) (85%), rat CGRP (87%), and bombesin (Bom) (95%). Blockade of the autonomic nervous system with chlorisondamine abolished the gastric inhibitory action induced by CRF, beta-End, CT, and NT, but not by CGRP and Bom (1 nmol/kg each). Corticotropin-releasing factor, beta-End, CT, NT, CGRP, and Bom significantly inhibited gastric acid secretion stimulated by an intragastric 8% peptone meal for 2 h. None of these six peptides significantly altered plasma gastrin concentrations in response to the peptone meal as compared with control experiments. A rise of plasma concentrations of gastrin, CT, CRF, and CGRP could not be detected by radioimmunoassay in animals after intracerebroventricular administration of these four peptides. The results of this study indicate that CT, CGRP, NT, beta-End, and peptides of the CRF and Bom families act within the brain to inhibit pentagastrin- and meal-stimulated gastric acid secretion in conscious dogs. None of the 30 peptides administered intracerebroventricularly increased basal gastric acid secretion in the dog. Inhibition of gastric acid secretion induced by CRF, beta-End, CT, and NT, but not by CGRP and Bom is mediated by the autonomic nervous system. Gastrin does not appear to play a role in gastric acid inhibition induced by the six brain peptides studied.

A radioligand assay employing tritiated gonadotropin-releasing hormone, [3H-Pro9]GnRH or [3H-pGlu1]GnRH is used to investigate the binding of GnRH, its agonists, and its antagonists to male rat anterior pituitary homogenates. The tritiated GnRH purified by high pressure liquid chromatography and stored in 10 mM HOAc is stable for binding for at least 14 weeks. It is found that there is at least one high affinity site with an observed Kd of congruent to 2 nM and another low affinity site whose Kd is congruent to 1 microM. Only approximately 25% of the total specific binding is to the low affinity site. At room temperature, the binding is reduced to 50% of that at 0 C, and at 37 C, there is no measurable binding. Bacitracin has no effect on the binding at any temperature. Maximum binding occurs between pH 7.5--8.5. Quantitative relative binding potencies of several agonists and antagonists are given. These potencies closely parallel their biological potencies, but all antagonists have higher absolute binding affinities when compared to their potencies to inhibit GnRH-mediated LH secretion in vitro.

While chronic treatment with LHRH agonists interferes with reproductive functions (Rivier et al., 1978, 1979; Rivier and Vale, 1979, 1981b; Vale et al., 1979), it is at first associated with an increase in gonadotropin and steroid secretion. This initial stimulation may be considered undesirable under some clinical circumstances. Additionally, LHRH agonists have a multiplicity of sites of action in rats which, if verified in humans, may also prove them unacceptable for human use.

Goldfish pituitary gonadotropin-releasing hormone (GnRH) receptors were characterized by using a superagonist analog of teleost GnRH (tGnRH-A; [D-Arg6, Trp7, Leu8, Pro9-NHEt]-GnRH). Equilibrium binding of 125I-tGnRH-A to a goldfish pituitary membrane preparation was achieved after a 30-min incubation at 4 degrees C; binding was significantly reduced after increasing incubation temperature to 22 degrees C. Binding of the radioligand was a function of tissue concentration, with a linear correlation over the range of 0.5-2 pituitary per tube. Incubation of the pituitary membrane preparation with increasing concentrations of 125I-tGnRH-A indicated saturable binding at radioligand concentrations of 470 pM and above. The binding of 125I-tGnRH-A was found to be reversible after addition of the cold analog, and the dissociation curve could be resolved into two linear components; slower rates of dissociation of 125I-tGnRH-A were observed after the addition of excess unlabeled tGnRH than after the addition of tGnRH-A, indicating that the analog is more effective in displacing the label than the native peptide. Addition of the cold analog displaced bound 125I-GnRH-A, and Scatchard analysis suggested the presence of at least two classes of binding sites: a high-affinity/low-capacity site and a low-affinity/high-capacity site. Bound 125I-GnRH-A was displaced by tGnRH from both sites in parallel to that observed with tGnRH-A, indicating that both peptides bind to the same classes of binding sites; however, tGnRH-A had a greater affinity for the receptors than the native tGnRH. These results demonstrated the presence and provided characterization of GnRH receptors in goldfish pituitary.

The toxic peptide from Conus magus venom (conotoxin MI) is a 14-amino acid peptide (McIntosh, M., Cruz, L. J., Hunkapiller, M. W., Gray, W. R., and Olivera, B. M. (1982) Arch. Biochem. Biophys. 218, 329-334) which inhibits the acetylcholine receptor. In this work we have confirmed the primary structure and established the disulfide bonding configuration (Cys 3-Cys 8; Cys 4-Cys 14) by direct chemical synthesis of the toxin with specific disulfide bridges. Natural and synthetic toxins were compared by several methods. Fast atom bombardment mass spectroscopy confirmed that the synthetic product had the expected molecular mass and number of exchangeable hydrogens. Ultraviolet CD spectra were closely comparable in shape and magnitude for the two materials, which were also identical in biological activity and chromatographic behavior. We have also established that, although the peptide is highly cross-linked with two disulfide bridges, it can slowly equilibrate between two conformations. A simulation analysis suggests that the conformers have half-lives of approximately 12 and approximately 72 min at 0 degrees C, decreasing approximately 2-fold for every 10 degrees C increase in temperature.

We have used a new potent GnRH antagonist to assess the efficacy of suppression of gonadotropin secretion in healthy postmenopausal women at three different doses. The Nal-Glu antagonist produced significant suppression of immunoactive and bioactive LH at a 300 micrograms/kg dose throughout the 30-hr sampling interval with only minimal local (skin) histamine release. This increased potency and decreased skin reactivity of the Nal-Glu antagonist make it a drug with potential clinical use similar to the GnRH agonist, but with the advantage of immediate gonadotropin suppression.

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.

Systematic substitution of the natural L-amino acids in neurotensin by their D isomers reveals that the COOH-terminal portion of this tridecapeptide is required for binding to mast cell receptors: D-amino acid replacements from Pro10 through Leu13 substantially decrease that binding. Either blockage of the COOH-terminal carboxyl group as with N-methylamidation, or formation of a cyclic structure by the inclusion of a disulfide bond, a Cys2,13 substitution, markedly reduces the specific binding to mast cell receptor sites. Modifications in the NH2-terminal portion of neurotensin do not affect the binding to mast cells. However, D-Arg8 and D-Arg9 substitutions increase binding by factors of 5- to 6-fold. The hydroxyl group at position 3 or 11 is not essential for binding since Phe3 or Phe11 is equivalent to Tyr3 or Tyr11. The COOH-terminal penta- and hexapeptides are able to displace approximately 70% 125I-neurotensin relative to the intact peptide. Of 18 other biologically active peptides tested, only xenopsin, a naturally occurring COOH-terminal analog of neurotensin, and bradykinin effectively compete in the binding assay to an extent of 60 and 100%, respectively. Histamine, diphenhydramine, and noradrenaline are ineffective in this regard.

Bullfrog sympathetic ganglion neurons have a “late, slow” excitatory postsynaptic potential (EPSP) that is mediated by a peptide similar to mammalian luteinizing hormone-releasing hormone (M-LHRH) (Jan, Y. N., L. Y. Jan, and S. W. Kuffler (1979) Proc. Natl. Acad. Sci. U. S. A. 76: 1501–1505). On biochemical evidence (Eiden, L. E., E. Loumaye, N. Sherwood, and R. L. Eskay (1982) Peptides 3:323–327) the endogenous LHRH-like peptide in the ganglion may be identical to teleost LHRH (T- LHRH; [Trp7,Leu8]M-LHRH) (Sherwood, N., L. Eiden, M. Brownstein, J. Speiss, J. Rivier, and W. Vale (1983) Proc. Natl. Acad. Sci. U. S. A. 80: 2794–2798). We have found that T-LHRH acts qualitatively like M- LHRH on bullfrog ganglion cells, but it is quantitatively more potent. As for M-LHRH, the primary action of T-LHRH under single-electrode voltage clamp is to inhibit the M-current, a voltage-dependent potassium current that is active in the region between rest and threshold in these cells. The M-current is also inhibited during the late, slow EPSP. Both T-LHRH and the late, slow EPSP can also induce an additional inward current, associated with an increased conductance, in some cells. An LHRH antagonist is effective against both T-LHRH and the late, slow EPSP. These results are consistent with a role for T-LHRH as the natural transmitter for the late, slow EPSP in bullfrog sympathetic ganglia.

A symposium report on the synthesis and structural characterization both by NMR in CD3CN solution. and by x-ray diffraction of the cyclic tetrapeptides cyclo(b-Ala-L-Pro-b-Ala-Aaa) (Aaa = L-Pro, L-Val) in order to verify the usefulness of the sequence b-Ala-Pro-b-Ala as molecular tool to force the peptide in a gamma-turn conformation.