ABSTRACT
We have previously demonstrated that pregnant ovine endometrium expresses the gastrin-releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP 1-46, the primary product of this gene in the pregnant endometrium. Full thickness 125-140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 muBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP 1-46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP(1-46) would require preferential cleavage at the Glu-Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP(18-27) and GRP(1-27) in other tissues. GRP 1-46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP 1-46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP 1-46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.
Subject(s)
Endometrium/chemistry , Gastrin-Releasing Peptide/isolation & purification , Gastrin-Releasing Peptide/pharmacology , Amino Acid Sequence , Animals , Cell Line , Cell Line, Tumor , Female , Gastrin-Releasing Peptide/analysis , Gastrin-Releasing Peptide/metabolism , Humans , Indoles/pharmacology , Inositol Phosphates/metabolism , Mice , Molecular Sequence Data , Molecular Weight , Peptides/genetics , Pregnancy , Protein Binding/physiology , Protein Precursors/genetics , Pyridines/pharmacology , Rats , Receptors, Bombesin/antagonists & inhibitors , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Sheep , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transfection , Type C Phospholipases/metabolismABSTRACT
Two molecular forms of gastrin-releasing peptide (GRP) were isolated from an extract of the intestine of the tetraploid frog Xenopus laevis. The primary structure of GRP-1 (APTSQQHTEQ(10)LSRSNINTRG(20) SHWAVGHLM.NH(2)) differs from that of GRP-2 by a single amino acid substitution (Asn(15)--> Thr(15)). GRP-(20-29) peptide (neuromedin C) was also isolated from the extract. Synthetic GRP-1 produced concentration-dependent contractions of longitudinal smooth muscle strips from Xenopus cardiac stomach (pD(2) = 8.93 +/- 0.32; n = 6). The responses were unaffected by tetrodotoxin, atropine, and methysergide, indicating a direct action of the peptide on smooth muscle cells. GRP-1 elicited concentration-dependent relaxations of precontracted (5 microM carbachol) circular smooth muscle strips from the same region (pD(2) = 8.96 +/- 0.21; n = 8). The responses were significantly (P < 0.05) attenuated (71 +/- 24% decrease in maximum response; n = 6) by indomethacin, indicating mediation, at least in part, by prostanoids. Despite the fact that Xenopus GRP-1 differs from pig GRP at 15 amino acid sites, both peptides are equipotent and equally effective for both contractile and relaxant responses, demonstrating that selective evolutionary pressure has acted to conserve the functional COOH-terminal domain in the peptide. The data suggest a physiologically important role for GRP in the regulation of gastric motility in X. laevis.
Subject(s)
Gastrins/metabolism , Gastrointestinal Hormones/antagonists & inhibitors , Gastrointestinal Hormones/chemistry , Amino Acid Sequence , Animals , Bombesin/chemistry , Bombesin/isolation & purification , Bombesin/pharmacology , Dose-Response Relationship, Drug , Gastrin-Releasing Peptide/chemistry , Gastrin-Releasing Peptide/isolation & purification , Gastrin-Releasing Peptide/pharmacology , Gastrointestinal Hormones/pharmacology , In Vitro Techniques , Indomethacin/pharmacology , Molecular Sequence Data , Muscle Relaxation/drug effects , Muscle Relaxation/physiology , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , Stomach/drug effects , Stomach/physiology , Swine , Xenopus laevisABSTRACT
This immunohistochemical study shows a wide distribution of neuropeptides in the cat amygdala. Neuropeptide Y is present along the whole amygdaloid complex, and fibers and cell bodies containing neuropeptide Y are observed in all the nuclei studied. Leucine-enkephalin-, gastrin-releasing peptide/bombesin-, and calcitonin gene-related peptide-immunoreactive fibers and perikarya are observed only in discrete nuclei of the amygdaloid complex, whereas only fibers -but no cell bodies- containing methionine-enkephalin-Arg6-Gly7-Leu8 have been observed. No immunoreactivity has been found for gamma-melanocyte-stimulating hormone, dynorphin A (1-17), or galanin. These data are compared with those reported in the amygdala of other mammals.
