Biomedical subjects

J Rivier

Publications and source records attributed to J Rivier.

At least 37 records · Page 2Linked to original sources

PubMed1999

Structural basis for the binding specificity of a SSTR1-selective analog of somatostatin.

The availability of subtype-specific agonists and antagonists for somatostatin (SS) receptors (SSTRs) will be important for elucidation of the function of each receptor isoform in vivo. A SS analog, des-AA1,2,5-[D-Trp8, IAmp9]SS (CH275), has been shown previously to bind preferentially to SSTR1. In this report, we identify structural determinants in the ligand and receptor responsible for the selective binding of CH275 to SSTR1 by modifying both the ligand and the receptor. We propose that IAmp9 in CH275, like Lys9 in SS, interacts with Asp137 in the middle of the third transmembrane domain of SSTR1 to form an ion pair, while other residues unique to SSTR1 conbribute to binding selectivity of CH275 for SSTR1. Replacement of Asp137 with Asn resulted in loss of binding of radiolabeled SS and decreased potencies of both SS and CH275 to induce a change in the extracellular acidification rate measured by microphysiometry. The structural determinants for specific binding to SSTR1 were mapped in chimeric SSTR1/SSTR2 receptors. One chimera, 2beta, with the N-terminus to second transmembrane domain (TM2) from SSTR2 and the remainder of the receptor from SSTR1, had low affinity for CH275. Furthermore, when a single residue, Leu107, in TM2 of SSTR1 was replaced with Phe, the corresponding residue in SSTR2, a 20-fold decrease in affinity for CH275 with no significant change in affinity for SS was observed. A reciprocal change from Phe to Leu in the chimeric receptor 2beta resulted in a 10-fold increase in affinity for CH275. Thus, Leu107 is an important determinant for CH275 binding to SSTR1. To identify the moiety in CH275 which could interact with Leu107, a new analog des-AA1,2,5-[D-Trp8, Amp9]SS was prepared. This analog bound to both SSTR1 and SSTR2 with similar affinities; thus, subtype selectivity was lost. Collectively, these data support a binding model for CH275 in which the positively charged IAmp interacts with the negatively charged Asp137 in TM3 of SSTR1 and the isopropyl group of IAmp forms a hydrophobic interaction with Leu107 in TM2.

Animals↗

PubMed1999

Corticotropin releasing factor (CRF) agonists with reduced amide bonds and Ser7 substitutions.

Strategies to generate competitive antagonists of bioactive peptides include several possible structural modifications such as the introduction of D-residues and of reduced amide bonds, the substitution of amino acid side chains, dimerization of fragments, and deletion of part of the sequence, among others. Whereas we have identified the two most likely residues responsible for receptor activation in corticotropin releasing factor (CRF) (Ser7 and Leu8)1 and generated potent antagonists by deleting residues 1-8,2,3 the question remained as to whether we could generate CRF antagonists with enhanced affinity after reduction of amide bonds at the N-terminus of CRF or through subtle alteration of those residues' side chains. Reduced amide bond replacements (psi[CH2NH]) between residues 6-9 in oCRF(5-41) (11, 12, 15) analogues consistently yielded potencies of <1% that of oCRF. Except for the 10psi11 and 12psi13 analogues 19 and 20, reduced amide bond replacements were generally well-tolerated in the longer hCRF(4-41) analogues, with the 7psi8-, 8psi9-, and 9psi10-modified peptides (13, 14, 18) yielding potencies that were 2-4 times that of hCRF. Although somewhat promising as agonists, they were, however, 3-7 times less potent than the parent [D-Pro4Nle21,38]-hCRF(4-41) (2). Since O-alkylation of Tyr3 in vasopressin yields an antagonist, and since Ser7 is one of the eight fully conserved residues in the CRF family (inclusive of sauvagine, urocortins, and urotensins) and likely to be critical for receptor binding, we synthesized cyclo(30-33)[Ser(OMe)7,D-Phe12,Nle21,Glu30,Lys33 ,Nle38]Ac-hCRF(7-41) (22), which was found to exhibit full efficacy and 40% of the potency of cyclo(30-33)[D-Phe12,Nle21,Glu30,Lys33, Nle38]Ac-hCRF(7-41) (5). Other substitutions at position 7 included aminoglycine (23, 24) and alkylated and/or acylated [alpha or alpha'-methyl (25-28), alpha'-formyl (29, 30), alpha'-formyl, alpha'-methyl (31), alpha'-acetyl (32), alpha'-acetyl, alpha'-methyl (33)], D- or L-aminoglycines. All analogues were active although less potent than the parent compound 2, and all elicited maximal ACTH response as compared to hCRF. The most potent analogue in this series (33) had the bulkiest side chain, Agl(Me, Ac), and was 60% and 80% as potent as the Ser7 analogue 5 and the Ala7 analogue 6, respectively. In conclusion, we found that neither reduction of the individual amide linkages between residues 6-11 and 12-13 nor introduction of a carbamide moiety in lieu of the side chain of Ser7 led to CRF antagonists.

Adrenocorticotropic Hormone↗

PubMed1999

Corticotropin-releasing factor antagonist attenuates the "anxiogenic-like" effect in the defensive burying paradigm but not in the elevated plus-maze following chronic cocaine in rats.

RATIONALE: Chronic cocaine abuse is associated with the development of anxiogenic states in humans. Corticotropin-releasing factor (CRF) is an endogenous neurotropic factor well known to modulate stress responses. It has been postulated that CRF is involved in the neurobiological mechanisms underlying the anxiety and/or stress responses associated with removal of cocaine after chronic administration. OBJECTIVE: The present study investigated the role of endogenous CRF in mediating the "anxiety-like" effect 48 h after the cessation of saline or chronic cocaine treatment in rats, using the defensive burying paradigm and the elevated plus-maze. METHODS: Rats received daily injections of cocaine (20 mg/kg IP, for 14 consecutive days) or vehicle. Forty-eight hours after the last injection, animals were tested in the plus-maze and then in the defensive burying paradigm. In a second experiment, intracerebroventricular (ICV) cannulae were implanted at the lateral ventricle. Animals were allowed a 1-week period for recovery before starting the chronic drug treatment. The defensive burying testing took place 48 h after cessation of the treatment. The CRF antagonist [DPhe12, Nle21,38, CalphaMe Leu37] r/h CRF(12-41), (also known as D-phe CRF(12-41)) (0.04, 0.2 and 1.0 microg/5 microl) was injected 5 min before the 15-min testing. RESULTS: An "anxiogenic-like" effect following chronic cocaine treatment was demonstrated with the defensive burying paradigm, but not with the elevated plus-maze. This "anxiety-like" response was attenuated by ICV pretreatment with the CRF antagonist D-Phe CRF(12-41), with the highest dose of the CRF antagonist reversing the observed "anxiogenic-like" response. CONCLUSIONS: These data suggest that brain CRF may be substantially involved in the development of "anxiety-like" responses related to cocaine withdrawal and could be important for future drug dependence treatments.

Animals↗

PubMed1999

Peripheral urocortin delays gastric emptying: role of CRF receptor 2.

Urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) family has been proposed to be the endogenous ligand for CRF receptor 2 (CRF-R2). We studied the influence of intravenous urocortin on gastric emptying and the role of CRF-R2 in peptide action and postoperative gastric ileus in conscious rats. The intravenous doses of rat CRF and rat urocortin producing 50% inhibition of gastric emptying were 2.5 and 1.1 microgram/kg, respectively. At these intravenous doses, CRF and urocortin have their actions fully reversed by the CRF-R1/CRF-R2 antagonist astressin at antagonist/agonist ratios of 5:1 and 67:1, respectively. Astressin (12 microgram/kg iv) completely prevented abdominal surgery-induced 54% inhibition of gastric emptying 3 h after surgery while having no effect on basal gastric emptying. The selective nonpeptide CRF-R1 antagonists antalarmin (20 mg/kg ip) and NBI-27914 (400 microgram/kg iv) did not influence intravenous CRF-, urocortin- or surgery-induced gastric stasis. These results as well as earlier ones showing that alpha-helical CRF9-41 (a CRF-R2 more selective antagonist) partly prevented postoperative ileus indicate that peripheral CRF-R2 may be primarily involved in intravenous urocortin-, CRF-, and abdominal surgery-induced gastric stasis.

Animals↗

PubMed1999

Corticotropin-releasing factor and the brain-gut motor response to stress.

The characterization of corticotropin-releasing factor (CRF) and CRF receptors, and the development of specific CRF receptor antagonists selective for the receptor subtypes have paved the way to the understanding of the biochemical coding of stress-related alterations of gut motor function. Reports have consistently established that central administration of CRF acts in the brain to inhibit gastric emptying while stimulating colonic motor function through modulation of the vagal and sacral parasympathetic outflow in rodents. Endogenous CRF in the brain plays a role in mediating various forms of stressor-induced gastric stasis, including postoperative gastric ileus, and activates colonic transit and fecal excretion elicited by psychologically aversive or fearful stimuli. It is known that brain CRF is involved in the cross-talk between the immune and gastrointestinal systems because systemic or central administration of interleukin-1-beta delays gastric emptying while stimulating colonic motor activity through activation of CRF release in the brain. The paraventricular nucleus of the hypothalamus and the dorsal vagal complex are important sites of action for CRF to inhibit gastric motor function, while the paraventricular nucleus of the hypothalamus and the locus coeruleus complex are sites of action for CRF to stimulate colonic motor function. The inhibition of gastric emptying by CRF may be mediated by the interaction with the CRF2 receptors, while the anxiogenic and colonic motor responses may involve CRF1 receptors. Hypersecretion of CRF in the brain may contribute to the pathophysiology of stress-related exacerbation of irritable bowel syndrome.

Animals↗

PubMed1999

Peripheral injection of a new corticotropin-releasing factor (CRF) antagonist, astressin, blocks peripheral CRF- and abdominal surgery-induced delayed gastric emptying in rats.

The effect of the corticotropin-releasing factor (CRF) receptor antagonists astressin and D-Phe CRF(12-41) injected i.v. on CRF-induced delayed gastric emptying (GE) was investigated in conscious rats. Gastric transit was assessed by the recovery of methyl cellulose/phenol red solution 20 min after its intragastric administration. The 55% inhibition of GE induced by CRF (0.6 microgram i.v.) was antagonized by 87 and 100% by i.v. astressin at 3 and 10 microgram, respectively, and by 68 and 64% by i.v. D-Phe CRF(12-41) at 10 and 20 microgram, respectively. CRF (0.6 microgram)-injected intracisternally (i.c.) induced 68% reduction of GE was not modified by i.v. astressin (10 microgram) whereas i.c. astressin (3 or 10 microgram) blocked by 58 and 100%, respectively, i.v. CRF inhibitory action. Abdominal surgery with cecal manipulation reduced GE to 7.1 +/- 3.1 and 27.5 +/- 3.3% at 30 and 180 min postsurgery, respectively, compared with 40.3 +/- 4.3 and 59.5 +/- 2.9% at similar times after anesthesia alone. Astressin (3 microgram i.v.) completely and D-Phe CRF(12-41) (20 microgram i.v.) partially (60%) blocked surgery-induced gastric stasis observed at 30 or 180 min. The CRF antagonists alone (i.v. or i.c.) had no effect on basal GE. These data indicate that CRF acts in the brain and periphery to inhibit GE through receptor-mediated interaction and that peripheral CRF is involved in acute postoperative gastric ileus; astressin is a potent peripheral antagonist of CRF when injected i.v. whereas i.c. doses >/=3 microgram exert dual central and peripheral blockade of CRF action on gastric transit.

Animals↗

PubMed1998

Constrained corticotropin-releasing factor (CRF) agonists and antagonists with i-(i+3) Glu-Xaa-DXbb-Lys bridges.

We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an alpha-helical conformation shown to be important for the recognition and binding of the human corticotropin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20-23)[DPhe12,Glu20,Lys23,Nle21,38]hCRF (12-41) and of astressin ¿cyclo(30-33)[DPhe12,Nle21,38,Glu30,Lys33]hCR F(12-41)¿, two potent CRF antagonists, and of cyclo(30-33)[Ac-Leu8,DPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(8-41), the shortest sequence equipotent to CRF reported to date (Rivier et al. J. Med. Chem. 1998, 41, 2614-2620 and references therein). To test the hypothesis that the Glu20-Lys23 and Glu30-Lys33 lactam rings were favoring an alpha-helical conformation rather than a turn, we introduced a D-amino acid at positions 22, 31, and 32 in the respective rings. Whereas the introduction of a D-residue at position 31 was only marginally deleterious to potency (ca. 2-fold decrease in potency), introduction of a D-residue at position 22 and/or 32 was favorable (up to 2-fold increase in potency) in most of the cyclic hCRF, alpha-helical CRF, urotensin, and urocortin agonists and antagonists that were tested and was also favorable in linear agonists but not in linear antagonists; this suggested a unique and stabilizing role for the lactam ring. Introduction of a [DHis32] (6) or acetylation of the N-terminus (7) of astressin had a minor deleterious or a favorable influence, respectively, on duration of action. In the absence of structural data on these analogues, we conducted molecular modeling on an Ac-Ala13-NH2 scaffold in order to quantify the structural influence of specific L- and DAla6 and L- and DAla7 substitutions in [Glu5,Lys8]Ac-Ala13-NH2 in a standard alpha-helical configuration. Models of the general form [Glu5,LAla6 or DAla6,LAla7 or DAla7,Lys8]Ac-Ala13-NH2 were subjected to high-temperature molecular dynamics followed by annealing dynamics and minimization in a conformational search. A gentle restraint was applied to the 0-4, 1-5, and 8-12 O-H hydrogen bond donor-acceptor pairs to maintain alpha-helical features at the N- and C-termini. From these studies we derived a model in which the helical N- and C-termini of hCRF form a helix-turn-helix motif around a turn centered at residue 31. Such a turn brings Gln26 in close enough proximity to Lys36 to suggest introduction of a bridge between them. We synthesized dicyclo(26-36,30-33)[DPhe12,Nle21,Cys26,Glu30 ,Lys33,Cys36, Nle38]Ac-hCRF(9-41) which showed significant alpha-helical content using circular dichroism (CD) and had low, but measurable potency ¿0. 3% that of 6 or ca. 25% that of [DPhe12,Nle21,38]hCRF(12-41)¿. Since the 26-36 disulfide bridge is incompatible with a continuous alpha-helix, the postulate of a turn starting at residue 31 will need to be further documented.

Adrenalectomy↗

PubMed1998

Astressin analogues (corticotropin-releasing factor antagonists) with extended duration of action in the rat.

In earlier reports we identified specific point substitutions (DPhe12,Nle21,38), cyclization strategies [in particular, introduction of lactam rings such as that of cyclo(Glu30,Lys33)], and deletions (residues 1-7) in the CRF molecule that led to agonists. We also noted that further deletions (residues 8-14) produced antagonists such as astressin ¿cyclo(30-33)[DPhe12,Nle21,38, Glu30, Lys33]hCRF(12-41)¿ (1). We hypothesized that the lactam ring promoted conformational stability to yield analogues with increased potency both in vitro and in vivo as compared to that of their linear counterparts. Additionally, we reported that cyclo(30-33)[DPhe12,Nle21,38, Glu30,DHis32,Lys33]hCRF(12-41) (3) and dicyclo(26-36,30-33)[Ac-Asp9,DPhe12,Nle21,38, Cys26, Glu30,Lys33, Cys36]hCRF(9-41) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30-33 (previous paper: Koerber et al. J. Med. Chem. 1998, 41). To increase the potency of 1 and/or 3 in vivo, we extended their chain length by one (5-8), two (9, 10), and three (11, 12) residues at the N-terminus and acetylated (6, 8, 10, 12). Of the compounds tested for duration of action (1, 3-6, 8), we found 6 and 8 to be slightly longer-acting than astressin or [DHis32]astressin, while their potencies in vitro were not significantly different from that of 3. Additionally, we introduced CalphaMe-leucine residues in lieu of leucine at positions 14, 15, 19, 27, and 37 in [DHis32]astressin. The analogue [CalphaMe-Leu27,DHis32]astressin (16) was more potent (although not statistically in all cases) than the other four analogues in vitro. While acetylation of the N-terminus of 16 (i.e., 18) or of [CalphaMe-Leu27]astressin (i.e., 19) did not have a significant effect on in vitro potency, elongation of the N-terminus by one or three residues in addition to acetylation resulted in cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27,Glu3 0,DHis32,Lys33, Nle38]Ac-hCRF(11-41) (21), cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27, Glu30,Lys33,Nle38]Ac-hCRF(9-41) (22), and cyclo(30-33)[DPhe12, Nle21, CalphaMe-Leu27,Glu30,DHis32,Lys33,Nle38 ]Ac-hCRF(9-41) (23) that were longer-acting than 6 and 8 (ca. 2 h inhibition of ACTH secretion at 25 micrograms/adrenalectomized rat). Analogues 22 and 23 were also more potent than astressin at reversing intracisternal CRF- and abdominal surgery-induced delay of gastric emptying in conscious rats.

Adrenalectomy↗

PubMed1998

Inactivation of a serotonin-gated ion channel by a polypeptide toxin from marine snails.

The venom of predatory marine snails is a rich source of natural products that act on specific receptors and ion channels within the mammalian nervous system. A 41-amino acid peptide, final sigma-conotoxin GVIIIA, was purified on the basis of its ability to inactivate the 5-HT3 receptor, an excitatory serotonin-gated ion channel. final sigma-Conotoxin contains a brominated tryptophan residue, which may be important for peptide activity because the endogenous ligand for the 5-HT3 receptor is a hydroxylated derivative of tryptophan. final sigma-Conotoxin inactivates the 5-HT3 receptor through competitive antagonism and is a highly selective inhibitor of this receptor. Serotonin receptors can now be included among the molecular targets of natural polypeptide neurotoxins.

Amino Acid Sequence↗

PubMed1998

Minimal-size, constrained corticotropin-releasing factor agonists with i-(i+3) Glu-Lys and Lys-Glu bridges.

In three earlier publications (Miranda et al. J. Med. Chem. 1994, 37, 1450-1459; 1997, 40, 3651-3658; Gulyas et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10575-10579) we have hypothesized that covalent constraints such as side-chain-to-side-chain lactam rings would stabilize an alpha-helical conformation shown to be important for the recognition and binding of the CRF C-terminus 30 residues, to CRF receptors. These studies led to the discovery of useful CRF antagonists such as alpha-helical CRF (alpha-hel-CRF) and Astressin both in vitro and in vivo. To test the hypothesis that such lactam rings may also be modulating activation of the receptor when introduced at the N-terminus of CRF, we studied the influence of the successive introduction from residues 4 to 14 of a cyclo(i, i+3)[Lysi-Glu(i+3)] and a cyclo(i,i+3)[Glui-Lys(i+3)] bridge on the in vitro potency of the agonist [Ac-Pro4,dPhe12,Nle21,38]hCRF(4-41) and related compounds. We have also introduced the favored cyclo(Glu30-Lys33) substitution found to be remarkable in several families of antagonists (such as Astressin) and in a number of CRF agonists and investigated the role of residues 4-8 on receptor activation using successive deletions. Earlier studies had shown that in both oCRF and alpha-helical CRF, deletion of residues 1-6, 1-7, and 1-8 led to gradual loss of intrinsic activity (IA) (from 50% IA to <10% IA) resulting in alpha-hel-CRF being a potent competitive antagonist. We show that acetylation of the N-terminus of these fragments generally increases potency by a factor of 2-3 with no influence on IA. While cyclo(30-33)[Ac-Leu8,dPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(8-41) (30) is the shortest reported analogue of CRF to be equipotent to CRF (70% IA), the corresponding linear analogue (31) is 120 times less potent (59% IA). Addition of one amino acid at the N-terminus ¿cyclo(30-33)[Ac-Ser7,dPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(7-41) (28)¿ results in a 5-fold increase in agonist potency and full intrinsic activity (113%). The most favored modifications were also introduced in other members of the CRF family including sauvagine (Sau), urotensin (Utn), urocortin (Ucn), and alpha-hel-CRF. Parallel and consistent results were obtained suggesting that the lactam cyclization at residues 29-32 and 30-33 (for the members of the CRF family with 40 and 41 amino acid residues, respectively) will induce (in the shortened agonists) a structural constraint (alpha-helix) that stabilizes a bioactive conformation similar to that shown in the Astressin family of CRF antagonists and that residue 8 (leucine or isoleucine) bears the sole responsibility for activation of the receptor since deletion of that residue leads to potent antagonists (Gulyas et al. Proc. Natl. Acad.Sci. U.S.A. 1995, 92, 10575-10579).

Amino Acid Sequence↗

PubMed1998

A selective analog for the somatostatin sst1-receptor subtype expressed by human tumors.

Somatostatin mediates its actions through five different somatostatin receptor subtypes, sst1-sst5. Recently, the somatostatin analogs des-AA1,2,5-[D-Trp8, IAmp9]somatostatin and des-AA1,5-[Tyr2, D-Trp8, IAmp9]somatostatin were synthesized and shown to be sst1-selective when tested in COS-7 cells transfected with each of the sst subtypes. In the present study, we tested the binding affinity and specificity of the iodinatable analog in primary human tumors expressing various sst subtypes, selected on the basis of in situ hybridization experiments. Des-AA1,5-[Tyr2, D-Trp8, IAmp9]somatostatin was found to have a high affinity, comparable to that of the natural somatostatin-28, for sst1-expressing tumors such as prostate cancers. However, it had no affinity for tumors expressing the sst2, sst3, or sst5 subtypes. For comparison, the somatostatin analogs octreotide or Tyr3-octreotide have no affinity for sst1-expressing tumors, but high affinity for sst2- and sst5-expressing tumors and intermediate affinity for sst3-expressing tumors. These data represent the first characterization of a sst1-selective analog in human tumors; it may be of potential use in the therapy of sst1-expressing tumors as an antiproliferative agent, as well as providing a lead compound for the development of more potent sst1-selective radioligands for in vivo tumor scintigraphy.

Autoradiography↗

PubMed1998

In vitro thyrotropin-releasing activity of corticotropin-releasing hormone-family peptides in coho salmon, Oncorhynchus kisutch.

Investigations of hypothalamic regulation of fish thyrotropin (TSH) secretion and subsequent thyroid activity have been impeded by the lack of a reliable assay for TSH. Using a recently developed radioimmunoassay (RIA) for coho salmon TSH we employed an in vitro pituitary cell culture technique to examine regulation of TSH secretion by corticotropin-releasing hormone (CRH) family peptides [ovine CRH (oCRH), carp urotensin I (UI), and frog sauvagine (SV)] as well as thyrotropin-releasing hormone (TRH), salmon growth hormone-releasing hormone (sGHRH), and salmon gonadotropin-releasing hormone (sGnRH). At concentrations of 0.01 to 100 nM, TRH, sGHRH, and sGnRH did not stimulate TSH secretion from coho salmon pituitary cells. However, at these same concentrations, both oCRH and SV caused a significant and concentration-dependent increase in TSH secretion; whereas, UI was highly stimulatory at all concentrations tested. In a related experiment we examined the effect of alpha-helical CRF(9-41) on oCRH-stimulated TSH release by pituitary cells. alpha-Helical CRF(9-41) is an analogue of CRH that has been shown by others to antagonize the adrenocorticotropic hormone (ACTH)-releasing activity of CRH in goldfish. Preincubation of cells with 1 microM alpha-helical CRF(9-41) for 4 h caused a significant suppression of the TSH-releasing activity of oCRH at 1.0 and 10 nM concentrations. The results of these experiments demonstrate the potency of a CRH-like peptide in the hypothalamic regulation of TSH in fish and reveal similarities in the inhibition of the response of both the thyroid and interrenal axis of fish to alpha-helical CRF(9-41).

Animals↗

PubMed1998

PYY-preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats.

1. Microinjection of peptide YY (PYY, 7-46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane-anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY. 2. [Pro34]rat(r)/porcine(p)PYY and [Pro34]human(h)PYY (23-117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68+/-11 and 89+/-31 micromol 90 min(-1) respectively. 3. Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27+/-8 and 23+/-8 micromol 90 min(-1) respectively) and a higher dose (116 pmol) tended to reduce the response. 4. Pancreatic polypeptide (PP, 4-46 pmol), [Leu31,Pro34]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY3-36, pNPY5-36 and PNPY13-36 (25-168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion. 5. The rank order of potency of PYY > or = [Pro34]r/pPYY = [Pro34]hPYY> r/hNPY = pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu31,Pro34]NPY and C-terminal NPY/PYY fragments suggest that a PYY-preferring receptor subtype may be involved in mediating the stimulating effect.

Animals↗

PubMed1998

Analysis of synthetic peptides by capillary zone electrophoresis in organic/aqueous buffers.

Whereas synthetic peptides have been routinely analyzed for purity by reverse phase high performance liquid chromatography (RPHPLC) for a number of years, it is only in the last decade that the use of capillary zone electrophoresis (CZE) in aqueous buffers has been taken advantage of as an orthogonal method for the detection of impurities. However, we have found that hydrophobic amino acids and peptides often migrate as very broad, tailing absorbances or even precipitate in the aqueous buffers during CZE analysis. As a result, alternative buffer systems containing organic modifiers were sought. Varying concentrations of acetonitrile, methanol and isopropanol in sodium phosphate and triethylammonium phosphate buffers were used to study their effects on the electrophoretic migration of several synthetic peptides [gonadotropin releasing hormone (GnRH), corticotropin releasing factor (CRF) and analogs] and an enantiomeric synthetic amino acid. The organic/aqueous buffers used to obtain the best conditions for separation of porcine gonadotropin-releasing hormone (GnRH) and chicken II GnRH were then used to optimize a separation of nine native forms of GnRH decapeptides. Interestingly, several of these GnRHs have identical formal charges and yet could be separated. This suggests a mixed mechanism of separation that discriminates not only on the basis of peptide charge and structure but also of adsorptive properties (Van der Waals forces, dipole-dipole interactions and hydrogen bonding) of the capillaries.

Buffers↗

PubMed1998

Central CRF inhibits gastric emptying of a nutrient solid meal in rats: the role of CRF2 receptors.

Corticotropin-releasing factor (CRF)-related peptides exhibit different affinity for the receptor subtypes 1 and 2 cloned in the rat brain. We investigated, in conscious rats, the effects of intracisternal (i.c.) injection of CRF (rat/human) on the 5-h rate of gastric emptying of a solid nutrient meal (Purina chow and water ad libitum for 3 h) and the CRF receptor subtype involved. CRF, urotensin I (suckerfish), and sauvagine (frog) injected i.c. inhibited gastric emptying in a dose-dependent manner, with ED50 values of 0.31, 0.13, and 0.08 microgram/rat, respectively. Rat CRF-(6-33) (0.1-10 micrograms i.c.) had no effect. The nonselective CRF1 and CRF2 receptor antagonist, astressin, injected i.c. completely blocked the inhibitory effect of i.c. CRF, urotensin I, and sauvagine with antagonist-to-agonist ratios of 3:1, 10:1, and 16:1, respectively. The CRF1-selective receptor antagonist NBI-27914 injected i.c. at a ratio of 170:1 had no effect. These data show that central CRF and CRF-related peptides are potent inhibitors of gastric emptying of a solid meal with a rank order of potency characteristic of the CRF2 receptor subtype affinity (sauvagine > urotensin I > CRF). In addition, the reversal by astressin but not by the CRF1-selective receptor antagonist further supports the view that the CRF2 receptor subtype is primarily involved in central CRF-induced delayed gastric emptying.

Amphibian Proteins↗

PubMed1998

Peripheral corticotropin-releasing factor mediates the elevation of plasma IL-6 by immobilization stress in rats.

We previously reported the elevation of plasma interleukin (IL)-6 activity in response to immobilization stress in rats. To investigate the role of peripheral corticotropin-releasing factor (CRF) in this response, we examined the effects of CRF antagonists on immobilization-induced IL-6 response. Intravenous pretreatment with either [D-Phe12,Nle21,38,CalphaMeLeu37]-anti-human rat (h/r) CRF12-41 (1.5 mg/kg) or cyclo(30-33)[D-Phe12, Nle21,38,Glu30, Lys33]-h/rCRF12-41 (Astressin, 0.5 mg/kg) attenuated the IL-6 response to immobilization, which confirmed our previous finding that systemic administration of an antiserum against CRF blocked this response. In addition, an intraperitoneal injection of h/rCRF (100 micrograms/kg) or rat urocortin (10 and 100 micrograms/kg) increased the plasma IL-6 activity, mimicking the response to immobilization. An intravenous injection of h/rCRF (100 micrograms/kg) also elevated plasma IL-6 in adrenalectomized rats. These findings suggest that peripheral CRF mediates the plasma IL-6 elevation in response to immobilization.

Animals↗

PubMed1998

Identification and characterization of a receptor from goldfish specific for a teleost growth hormone-releasing hormone-like peptide.

In mammals, growth hormone-releasing hormone (GHRH), acting via the GHRH receptor, plays an important role in the regulation of growth hormone (GH) synthesis and secretion as well as the proliferation and differentiation of somatotropes in the pituitary. In fishes, information concerning the functional role of the characterized GHRHs is limited. For that reason, a putative goldfish GHRH receptor cDNA was characterized in this study. The receptor cDNA is 2,243 bp in length, encoding a 438-amino-acid-long polypeptide with 7 putative transmembrane-spanning regions, which is a characteristic of G-protein-coupled receptors. The receptor, when expressed in COS-7 cells, showed minimal responses (2-fold cAMP responses) when stimulated with 100 nM of human GHRH, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP). However, this receptor was found to be specific for a carp GHRH-like peptide isolated from the brain of common carp (Cyprinus carpio); there was a significant and dose-dependent increase in intracellular cAMP (a maximum response of 22-fold increase with an EC50 of 0.1 nM) when the transfected cells were stimulated with this peptide. As a preliminary study to investigate the functional role of this receptor, the tissue distribution of the mRNA was analyzed by reverse-transcription-polymerase chain reaction. The receptor mRNA was found to be present in the brain, pituitary, gut, gill, heart, liver, skeletal muscle, spleen, ovary and testis. Together with a goldfish PACAP type 1 receptor and a VIP1 receptor recently isolated in our laboratory, characterization of this putative GHRH receptor provides the molecular basis for the future understanding of the neuroendocrine control of growth and reproduction by these neuropeptides in goldfish as well as other teleosts.

Amino Acid Sequence↗

PubMed1997

Constrained corticotropin-releasing factor antagonists with i-(i + 3) Glu-Lys bridges.

Hypothesis driven and systematic structure-activity relationship (SAR) investigations have resulted in the development of effective central nervous system (CNS) antagonists of corticotropin (ACTH)-releasing factor (CRF) such as alpha-helical CRF(9-41) and analogues of our assay standard [DPhe12,Nle21,38]hCRF(12-41). On the other hand, equally potent CRF antagonists that block the hypothalamic/pituitary/adrenal (HPA) axis had not been described until recently. Predictive methods, physicochemical measurements (nuclear magnetic resonance spectrometry and circular dichroism spectroscopy), and SAR studies suggest that CRF and its family members (urotensins and sauvagine) assume an alpha-helical conformation when interacting with CRF receptors. To further test this hypothesis, we have systematically scanned the hCRF(9-41) or hCRF(12-41) sequences with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown could maintain or enhance alpha-helical structure. From this series we have identified seven analogues that are either equipotent to, or 3 times more potent than, the assay standard; in addition, as presented earlier cyclo(30-33)[DPhe12,-Nle21,38,Glu30, Lys33]hCRF(12-41) (astressin) is 32 times more potent than the assay standard in blocking ACTH secretion in vitro (rat pituitary cell culture assay). In vivo, astressin is also significantly more potent than earlier antagonists at reducing hypophysial ACTH secretion in intact stressed or adrenalectomized rats. Since the corresponding linear analogues that were tested are significantly less potent, our interpretation of the increased potency of the cyclic analogues is that the introduction of the side chain to side chain bridging element (Glu30-Lys33, and to a lesser extent that of Glu14-Lys17, Glu20-Lys23, Glu23-Lys26, Glu26-Lys29, Glu28-Lys31, Glu29-Lys32, and Glu33-Lys36) induces and stabilizes in the receptor environment a putative alpha-helical bioactive conformation of the fragment that is not otherwise heavily represented. The effect of the introduction of two favored substitutions [(cyclo(20-23) and cyclo(30-33)] yielded 37 with a potency 8 times that of the assay standard but actually 12 times less than expected if the effect of the two cycles had been multiplicative. These results suggest that the pituitary CRF receptor can discriminate between slightly different identifiable conformations, dramatically illustrating the role that secondary and tertiary structures play in modulating biological signaling through specific protein-ligand interactions.

Amino Acid Sequence↗