Histochemistry (1988) 90: 299-307 Histochemis 9 Springer-Verlag 1;98~8 Localisation of mRNA and co-expression and molecular forms of GRP gene products in endocrine cells of fetal human lung M. Bhatnagar ~, D.R. Springail 1, M.A. Ghatei 2, P.W.J. Burnet 2, Q. Hamid 1, A. Giaid ~, N.B.N. lbrahim 3, F. Cuttitta 4, E.R. Spindel 5, R. Penketh 6, C. Rodek 6, S.R. Bloom 2, and J.M. Polak ~* Departments of Histochemistry and Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W/20NN, England Department of Histopathology, Erenchay Hospital, Bristol BSI6 I LE, England NCI Naval Oncology Branch, Bethesda, Maryland, USA Department of Molecular Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA 6 Department of Obstetrics and Gynaecology, Queen Charlotte's Hospital, Goldhawk Road, London we, England Accepted May 12, 1988 Summary. The presence of bombesin (gastrin-releasing pep- tide, GRP)-like immunoreactivity in mucosal endocrine cells of human fetal lung is well established. In this study we have investigated the localisation of pro-GRP mRNA and GRP gene products and compared the distribution and levels of extractable GRP- and C-terminal flanking peptide of human pro-GRP-like immunoreactivity in order to verify synthesis and to investigate their coexistence and molecular forms. Human fetal lungs (14 to 23 weeks gestation) were immunostained, and extracts were assayed using region- specific antisera to pro-GRP. Additional antisera to chro- mogranin and protein gene product 9.5 (PGP 9.5) were used for immunostaining by the peroxidase anti-peroxidase technique and for double immunofluorescence staining us- ing antisera raised in two species. Immunoreactivity for both bombesin (GRP) and flanking peptide was seen mainly in the same endocrine cells, but more cells were stained with antisera to flanking peptide than with antiserum to bombesin (GRP). In situ hybridisation showed that pro- GRP mRNA was present and thus synthesis of the peptides was taking place. Endocrine cells and nerve fibres were PGP 9.5-immunoreactive, and a subset of cells was immunoreac- five for bombesin gene products. Radioimmunoassay and chromatography show that pro-GRP is present in both the uncleaved and cleaved forms, and, in agreement with im- munocytochemistry results, that an excess of C-terminal peptide of pro-GRP is detectable. It is therefore concluded that GRP-like peptides and flanking peptide are co-local- ised in human pulmonary endocrine cells, but the latter is found in larger concentrations than free GRP. Thus GRP-like peptides may be secreted separately from the flanking peptide(s) of pro-GRP. Furthermore PGP 9.5 ap- pears to be a useful marker for endocrine cells in the respira- tory epithelium of human fetal lung. Introduction Bombesin is a 14 amino acid amphibian peptide (Anastasi et al. 1971). Its mammalian homologue is gastrin-releasing peptide (GRP), a 27 amino acid peptide with sequence ho- * To whom offprint requests should be sent mology to the C-terminal region of amphibian bombesin (McDonald et al. 1979). It is now known that three mRNAs encoding pro-GRP are produced from the bombesin gene. The first sequence was deduced by using RNA extracted from a pulmonary endocrine tumour (Spindel et al. 1984). The amino acid se- quence predicted from this showed pro-GRP to consist of a signal sequence at the N-terminus, GRP itself and a C- terminal flanking peptide (Fig. 1). The second and third messages differ from the first in having a 21 and 19 base deletion, respectively, in the region encoding the C-terminal flanking peptide (GRP gene associated peptide - GGAP) (Spindel et al. 1986). All three mRNAs produce peptide sequences that are identical except at the C-terminus of the C-flanking peptide. In this paper the term "flanking peptide" will be used to refer collectively to the three forms of C-terminal flanking peptide of human pro-GRP. 23 1 27 31 125 J GRP : ank ng pept de.,~.~-~:: :: I | | [] 31 98 125 : --.. \ Fo.., I i i i i ;;~ ~Si~\\\\\~.~'~\\\\~..I (~ 104 ~ 118 98 115 0# ~ cleavage and amidation point Fig. 1. Schematic diagram of human prepro-GRP showing the three forms of the flanking peptide. Numbers indicate amino acid residues, beginning with 1 for the first amino acid of GRP rather than the signal peptide. Antisera used in this study were raised to the peptide fragments indicated (see also Table 1): (a) bombesin, (b) N-terminus of flanking peptide, (c) C-terminus of flanking, (at) N-terminal GRP (used for assay only)