phyllolitorin--leu-8- and phyllolitorin

Pulmonary neuroendocrine cells are localized predominantly at airway branchpoints. Previous work showed that gastrin-releasing peptide (GRP), a major pulmonary bombesin-like peptide, occurred in neuroendocrine cells exclusively in branching human fetal airways. We now demonstrate that GRP and GRP receptor genes are expressed in fetal mouse lung as early as embryonic day 12 (E12), when lung buds are beginning to branch. By in situ hybridization, GRP receptor transcripts were at highest levels in mesenchymal cells at cleft regions of branching airways and blood vessels. To explore the possibility that bombesin-like peptides might play a role in branching morphogenesis, E12 lung buds were cultured for 48 hr in serum-free medium. In the presence of 0.10-10 microM bombesin, branching was significantly augmented as compared with control cultures, with a peak of 94% above control values at 1 microM (P < 0.005). The bombesin receptor antagonist [Leu13- psi(CH2NH)Leu14]bombesin alone (100 nM) had no effect on baseline branching but completely abolished bombesin-induced branching. A bombesin-related peptide, [Leu8]phyllolitorin also increased branching (65% above control values at 10 nM, P < 0.005). [Leu8]Phyllolitorin also significantly augmented thymidine incorporation in cultured lung buds. Fibronectin, which is abundant at branchpoints, induces GRP gene expression in undifferentiated cell lines. These observations suggest that BLPs secreted by pulmonary neuroendocrine cells may contribute to lung branching morphogenesis. Furthermore, components of branchpoints may induce pulmonary neuroendocrine cell differentiation as part of a positive feedback loop, which could account in part for the high prevalence of these cells at branchpoints.

Topics: Animals; Base Sequence; Bombesin; DNA Primers; Female; Gene Expression; Gestational Age; In Situ Hybridization; Lung; Mice; Mice, Inbred Strains; Molecular Sequence Data; Morphogenesis; Oligopeptides; Polymerase Chain Reaction; Pregnancy; Pyrrolidonecarboxylic Acid; Receptors, Bombesin; Transcription, Genetic

The bombesin-like peptides were originally characterized in frog skin, then later found to have a wide distribution and range of actions in mammals. The bombesin-like peptides have classically been divided into three subfamilies, the bombesin subfamily, of which gastrin-releasing peptide (GRP) is the mammalian form; the ranatensin subfamily, of which neuromedin-B (NMB) is the mammalian form; and the phyllolitorin subfamily, which to date has only been characterized in amphibians. As a first step in characterizing mammalian phyllolitorin-like peptides, we have cloned complementary DNAs (cDNAs) encoding Leu8 and Phe8 phyllolitorin from Phyllomedusa sauvagei. Sequence analysis revealed that the amphibian phyllolitorin messenger RNA (mRNA) encodes a precursor of 90 amino acids containing a signal peptide sequence, an amino-terminal extension peptide, the phyllolitorin peptide of nine amino acids, and a carboxy-terminal extension peptide. Northern blot, reverse transcriptase-polymerase chain reaction (PCR), and in situ hybridization analysis showed that the mRNA was present at highest levels in skin, at lower levels in brain, and at lowest levels in gut. Phylogenetic analysis of bombesin-like peptide prohormone sequences showed that the phyllolitorin prohormones are much more closely related to the bombesin and ranatensin prohormones than to the GRP and NMB prohormones. This analysis suggests that the bombesin-like peptides should be reclassified into the GRP subfamily, the NMB subfamily, and the skin peptide subfamily. Surprisingly, the cDNAs encoding Phe8 and Leu8 phyllolitorins were identical except for a single T to C difference in the codon coding for the Phe or Leu residue of phyllolitorin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Anura; Base Sequence; Brain; Cloning, Molecular; DNA, Complementary; Gastric Mucosa; Genes; Liver; Molecular Sequence Data; Neuropeptides; Oligopeptides; Organ Specificity; Polymerase Chain Reaction; Pyrrolidonecarboxylic Acid; RNA Processing, Post-Transcriptional; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Skin

Topics: Amino Acid Sequence; Animals; Blood Pressure; Bombesin; Brain; Feeding Behavior; Molecular Sequence Data; Oligopeptides; Pyrrolidonecarboxylic Acid; Structure-Activity Relationship

In this study we have examined the effect and the possible interaction of ICV injection of Leu8phyllolitorin and SC injection of naloxone on thermoregulation in fasted and fed rats at cold (4 degrees C) and ambient (22 degrees C) temperature. Central injection of Leu8phyllolitorin have been shown to produce hypothermia in animals exposed to a cold environment, but not in animals maintained at 22 degrees C. This suggests that the stressful situation of a cold temperature is an important factor in causing peptide hypothermia. Naloxone enhanced the hypothermic effect of Leu8phyllolitorin at 4 degrees C and 22 degrees C both in fasted and in fed rats, reflecting an opiate receptor interaction for this response.

Topics: Animals; Body Temperature Regulation; Bombesin; Dose-Response Relationship, Drug; Fasting; Male; Naloxone; Oligopeptides; Pyrrolidonecarboxylic Acid; Rats