gastrin-releasing-peptide and litorin

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13-14 and 8-9, respectively, with psi[CH2S] and psi[CH2N(CH3)]. [Phe13 psi[CH2S]Leu14]bombesin and [Phe8 psi[CH2S]-Leu9]litorin bound to the murine pancreatic bombesin/gastrin releasing peptide receptor with similar dissociation constants (Kd = 3.9 and 3.4 nM, respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, Kd = 1.6 nM and isomer II, Kd = 0.89 nM. Further oxidation to the sulfone decreased potency ([Phe8 psi[CH2SO2]Leu9]litorin, Kd = 9.9 nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a psi[CH2N(CH3)] at the 8-9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (Kd = 0.1 nM) and [Leu9]litorin (Kd = 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pKa = 6.3 and 8.5). The lower pKa is found in the agonist analogs but not in the psi [CH2S]-containing antagonist.

Topics: Animals; Bombesin; Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Circular Dichroism; Gastrin-Releasing Peptide; Hydrogen-Ion Concentration; Mice; Oligopeptides; Pancreas; Peptides; Protein Conformation; Receptors, Bombesin; Spectrometry, Fluorescence; Structure-Activity Relationship

In the estrogen-treated rat myometrium, bombesin (Bn) and related agonists triggered contraction and the increased generation of inositol phosphates. The relative order of potencies was identical for both responses: Bn = gastrin releasing peptide (GRP) = litorin = neuromedin C >> neuromedin B. Two specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6-13) methyl ester and [Leu14,psi 13-14]Bn were inhibitory for both Bn-mediated tension and generation of inositol phosphates. [125I-Tyr4]Bn bound to myometrial membranes with high affinity (Kd = 104 pM) to a single class of sites in a saturable and reversible manner. The relative potencies for inhibiting binding were GRP = litorin = [Tyr4]Bn (Ki = 0.4 to 0.6 nM) >> neuromedin B (Ki = 10.3 nM). The high affinity displayed by [D-Phe6]Bn-(6-13) methyl ester (Ki = 2.8 nM) and [Leu14,psi 13-14]Bn (Ki = 35 nM) for competing for [Tyr4]Bn binding supported the involvement of a GRP-preferring Bn receptor. Guanine nucleotides decreased the binding of [125I-Tyr4]Bn and accelerated the rate of ligand dissociation, reflecting the coupling of receptors to guanine nucleotide regulatory proteins (G proteins). The results demonstrate that rat myometrium expresses functional GRP-preferring Bn receptors whose activation stimulates the phospholipase C pathway, pertussis toxin-insensitive event that contributes to Bn-mediated uterine contractions.

Topics: Adenosine Triphosphate; Animals; Binding, Competitive; Bombesin; Cell Membrane; Chromatography, High Pressure Liquid; Female; Gastrin-Releasing Peptide; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; In Vitro Techniques; Inositol Phosphates; Kinetics; Muscle, Smooth; Myometrium; Neurokinin B; Oligopeptides; Peptide Fragments; Peptides; Pertussis Toxin; Rats; Rats, Wistar; Receptors, Bombesin; Signal Transduction; Uterine Contraction; Virulence Factors, Bordetella

The high inhibitory potency of the previously developed bombesin antagonist [Leu13, psi CH2NHLeu14]bombesin (analogue I) (IC50 values of 30 and 18 nM for inhibition of bombesin-stimulated amylase secretion from guinea pig acinar cells and Swiss 3T3 cell growth, respectively) diminished considerably when shorter chain lengths were examined. For instance, [Leu13, psi CH2NHLeu14]bombesin-(5-14),[Leu13, psi CH2NHLeu14] bombesin-(6-14), and [Leu9, psi CH2NHLeu10]neuromedin C had IC50 values of 150, 150, and 280 nM, respectively. Incorporation of a D-Phe residue at position 6 of [Leu13, psi CH2NHLeu14] bombesin did not significantly change the various biological parameters. However, its presence in [Leu13, psi CH2NHLeu14]bombesin-(6-14) and at position 2 of psi-neuromedin C-(2-10) resulted in about 10-fold increases in potency up to and above that of the original antagonist. For instance, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) and des-Gly1-[D-Phe2,Leu9,psi CH2NHLeu10]neuromedin C exhibited IC50 values of 5 and 28 nM, respectively. Analogues based on the litorin sequence which contains an NH2-terminal pyroglutamic acid residue at the bombesin position 6 equivalent were also quite potent. The ability of various analogues to interact with bombesin receptors on pancreatic acini correlated reasonably well with potencies derived from inhibition of bombesin-stimulated growth of Swiss 3T3 cells. Additional studies of NH2- and COOH-terminal structure-activity relationships resulted in the synthesis of [D-Phe6,Leu13,psi CH2NHPhe14]bombesin-(6-14), which was particularly effective in inhibiting 3T3 cell growth at high picomolar concentrations (IC50 = 0.72 nM and Ki = 3.1 nM for 3T3 cells; IC50 = 7.5 nM and Ki = 9.9 nM for acini). Detailed investigations with one of the most potent antagonists, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) (Ki = 14 nM for acini cells and 7.1 for 3T3 cells), demonstrated that this analogue was a competitive inhibitor of bombesin and that this activity was specific for the bombesin receptor. Thus, inhibitory potencies have been improved generally up to 25 times over previously reported structures; and, given that bombesin itself has a Ki of 1.2 nM for 3T3 cell binding, some of these analogues are extraordinarily high affinity receptor antagonists. They can also be synthesized more readily and offer fewer proteolytic degradation sites than the original pseudopeptide and should be excellent candidates for in vivo studies aimed at inhibiti

Topics: Amino Acid Sequence; Amylases; Animals; Antineoplastic Agents; Bombesin; Cell Line; Fibroblasts; Gastrin-Releasing Peptide; Guinea Pigs; Mice; Molecular Sequence Data; Oligopeptides; Pancreas; Peptide Fragments; Peptides; Receptors, Bombesin; Receptors, Neurotransmitter

1. Low levels of bombesin-like immunoreactivity (less than 1.10 pmol/g tissue), eluting at the same position as synthetic bombesin and litorin, have been measured in muscle and mucosa of the gastrointestinal canal of the cod, Gadus morhua. 2. Immunohistochemistry showed bombesin-like material in endocrine cells and nerves throughout the gut. Intensity and absorption controls indicated a difference in character between the peptides present in stomach and intestinal muscle respectively. 3. Stomach motility was stimulated by bombesin (pD2 = 8.27 +/- 0.19), litorin (8.03 +/- 0.04) and (less potently) GRP (less than 7.46 +/- 0.22). Litorin (8.21 +/- 0.31) had an inhibitory effect on intestinal motility. 4. In conclusion, one or several bombesin-like peptides may be involved in the control of gastrointestinal motility in the cod, causing opposite effects on stomach and intestine. The structure of the peptide in gastric nerves may differ from that in intestinal nerves.

Topics: Amino Acid Sequence; Animals; Bombesin; Digestive System; Female; Fishes; Gastrin-Releasing Peptide; Gastrointestinal Motility; Immunohistochemistry; Male; Oligopeptides; Peptides; Radioimmunoassay

To examine the biochemical basis for growth factor-induced responses in human lung cancer cells, we used the quin2 technique to study the effect of the amphibian peptide bombesin and its congeners including mammalian gastrin-releasing peptide (GRP) on the intracellular free calcium level [Ca2+]i in small cell lung cancer cell lines. In five of eleven cell lines tested, Tyr4-bombesin or GRP elicited a rapid and transient increase in [Ca2+]i. The response was seen with as little as 1 nM ligand, was not affected by membrane depolarization, and derived in part from internal calcium stores. Desensitization to a second addition of active bombesin congeners occurs subsequent to initial addition of Tyr4-bombesin. Structure-activity analysis showed the carboxyl-terminal octapeptide was the active portion of the peptide. Analogs in which the carboxyl terminus was oxidized or deamidated were inactive. Ranatensin, litorin, alytesin, and GRP, but not physalaemin, were as active as Tyr4-bombesin. A monoclonal antibody to the carboxyl terminus of bombesin selectively blocked the increased [Ca2+]i elicited by Tyr4-bombesin. These studies suggest that bombesin congeners can act on some small cell lung cancer cell lines by a pathway utilizing increased [Ca2+]i.

Topics: Amino Acid Sequence; Bombesin; Calcium; Carcinoma, Small Cell; Cell Line; Dose-Response Relationship, Drug; Gastrin-Releasing Peptide; Humans; Kinetics; Lung Neoplasms; Oligopeptides; Peptides; Pyrrolidonecarboxylic Acid; Structure-Activity Relationship

A technique for preparing a suspension of dispersed functional acini from human pancreas has been developed. The changes in pancreatic enzyme secretion and accumulation of cellular cyclic AMP caused by various secretagogues have been studied. Ca2+-mobilizing agents stimulated amylase release from human pancreatic acini. The relative potencies with which secretagogues increased amylase release were as follows: gastrin-releasing peptide's potency (Ec50, 0.1 +/- 0.01 nM) was greater than bombesin 14's (Ec50, 0.2 +/- 0.01 nM), which was greater than litorin's (Ec50, 0.6 +/- 0.18 nM), which was greater than bombesin 9's (Ec50, 6 +/- 0.1 nM). For CCK-peptides, the relative potencies were as follows: CCK-39's potency (Ec50, 0.28 +/- 0.15 microM) was equal to cerulein's (Ec50, 0.3 +/- 0.07 microM). Both of these potencies were greater than CCK-8's (Ec50, 1.6 +/- 0.1 microM), which was greater than that of CCK-4. Carbamyl choline was poorly potent (Ec50 greater than 1 mM). The 12-O-tetradecanoylphorbol-13-acetate (TPA) was active from 0.1 nM to 0.1 microM. Neither secretin nor VIP increased amylase release from human pancreatic acini but they did cause an accumulation of cellular cyclic AMP, secretin (Ec50, 0.5 +/- 0.2 nM) being more potent than VIP.

Topics: Amylases; Bombesin; Carbachol; Cholecystokinin; Cyclic AMP; Exocrine Glands; Gastrin-Releasing Peptide; Humans; In Vitro Techniques; Oligopeptides; Pancreas; Peptides; Phosphodiesterase Inhibitors; Secretin; Tetradecanoylphorbol Acetate; Vasoactive Intestinal Peptide

When dispersed acini from mouse pancreas are first incubated with bombesin, washed, and then reincubated with fresh incubation solution containing no bombesin, there is significant residual stimulation of amylase release. Induction of residual stimulation is relatively rapid in that significant stimulation occurs as early as after 15 s of first incubation with bombesin. Induction of residual stimulation of amylase release per se is temperature independent, but induction does occur more rapidly when acini are first incubated at 37 degrees C than when they are first incubated at 4 degrees C. Residual stimulation of amylase release persists for at least 75 min in acini that have been first incubated with bombesin at 37 degrees C. The maximal residual stimulation of amylase release obtained with pancreatic acini that have been first incubated with bombesin and then washed is 45% greater than the maximal stimulation obtained when bombesin is added directly to the incubation medium. In terms of their abilities to cause residual stimulation of amylase release, litorin and ranatensin are equal to bombesin in potency and efficacy. Gastrin-releasing peptide is approximately 70% as efficacious as bombesin in causing residual stimulation of amylase release.

Topics: Amylases; Animals; Bombesin; Dose-Response Relationship, Drug; Gastrin-Releasing Peptide; Male; Mice; Mice, Inbred Strains; Oligopeptides; Pancreas; Peptides; Pyrrolidonecarboxylic Acid; Stimulation, Chemical; Time Factors

The amphibian skin tetrapeptide bombesin shows potent action in reducing gastric acid secretion by intracerebral ventricular (ICV) administration in rats. In order to establish a relationship between this action and the amino acid composition of the bombesin-like peptides, most of the natural bombesin-like peptides and some synthetic analogues were tested on their ability to reduce gastric acid secretion by ICV administration. The amphibian peptides bombesin, its [Tyr4]-bombesin analogue, alytesin, ranatensin and litorin, and the mammalian peptide GRP significantly reduced gastric acid output 2 hr after peptide administration (p less than 0.01). The data support the following prerequisites for the maximal neuromodulatory role of bombesin-like peptides on gastric secretion: Trp is required at position 8; Gln and His are important at positions 7 and 12, respectively; Leu replacement by Phe, which occurs in the litorin subfamily, modifies the response; and unspecified amino acids or sequences are also involved in the N-terminal region of bombesin-like peptides. Synthetic analogues are currently being tested to confirm and extend these conclusions.

Topics: Amino Acid Sequence; Animals; Bombesin; Gastric Juice; Gastrin-Releasing Peptide; Hydrogen-Ion Concentration; Male; Neurokinin B; Oligopeptides; Peptide Fragments; Peptides; Pyrrolidonecarboxylic Acid; Rats; Secretory Rate

Peripherally-administered bombesin and gastrin-releasing peptide produce potent, dose-related, and specific reductions of food intake at test meals in rats. Similar effects on meal size are observed after intraperitoneal injections in mice and after intravenous infusions in baboons and humans. The mechanism for this effect is unknown, but the action of bombesin is not blocked by complete subdiaphragmatic vagotomy, by a variety of peripheral endocrine and neural ablations, or by lesions of the area postrema or hypothalamus. Hypothalamic injections of bombesin produce small but specific reductions of food intake; the relationship of this central effect to the peripheral effect of the peptide is unknown. Bombesin and bombesin-like peptides may play roles in the regulation of meal size.

Topics: Animals; Bombesin; Cholecystokinin; Dose-Response Relationship, Drug; Feeding Behavior; Gastrin-Releasing Peptide; Humans; Hypothalamus; Mice; Oligopeptides; Papio; Peptides; Satiation; Vagus Nerve

Topics: Animals; Bombesin; Cats; Electric Stimulation; Gastrin-Releasing Peptide; Guinea Pigs; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Oligopeptides; Peptide Fragments; Peptides; Rats; Swine

Intraperitoneal injections of tetradecapeptide bombesin (BBS) produced large, dose-related suppressions of liquid and solid food intake in rats, with threshold doses of 1--2 micrograms-kg-1. The feeding-associated behaviors of rats receiving BBS by this route at a test meal were normally sequenced, and several other observations suggested that the effect of BBS was specific and not due to malaise. The structurally related amphibian peptide litorin and the structurally related mammalian gastrin-releasing peptide (GRP) produced similar suppressions of food intake. The satiety effect of BBS administered intraperitoneally did not require the accumulation of food in the gut, the presence of intact adrenals, the abdominal vagus, or the release of cholecystokinin. When BBS and cholecystokinin were administered simultaneous, the suppressive effects on food intake were additive. Lateral cerebroventricular injections of BBS also produced large, dose-related suppressions of food intake, with a threshold dose of 100 ng per rat. The effect by this route, however, was not behaviorally specific: BBS produced equivalent inhibitions of food and water intake at every point on the dose-response curve, and produced a marked increase in grooming which dominated the behavioral display. Thus, (1) peripheral BBS is a putative satiety signal in the rat; (2) the class (endocrine, paracrine, or neural) and mechanism of this satiety action is not established; and (3) the differences in specificity and behavior following intraperitoneal and cerebroventricular routes indicate that peripheral BBS does not act solely via the cerebrospinal fluid to elicity satiety.

Topics: Adrenalectomy; Animals; Bombesin; Cholecystokinin; Dose-Response Relationship, Drug; Eating; Feeding Behavior; Gastrin-Releasing Peptide; Gastrointestinal Hormones; Injections, Intraventricular; Oligopeptides; Peptide Fragments; Peptides; Rats; Satiation; Sincalide; Vagotomy