Biomedical Research 4 (1) 93-104, 1983 GASTRIN-RELEASING PEPTIDE (GRP)/BOMBESIN-LIKE IMMUNOREACTIVITY IN THE NEURONS AND PARANEURONS OF THE GUT AND LUNG TOSHIHIKO IWANAGA Department of Anatomy, Niigata University School of Medicine, Niigata 951, Japan ABSTRACT Gastrin-releasing peptide (GRP)/bombesin-like immunoreactivities in the gut and lung were investigated with an immunocytochemical method using antisera showing different specificity for GRP-related peptides and bombesin. GRP-like, but not bombesin-like, immunoreactivity in the gut of birds and frogs was contained in the endocrine cells, which were restricted mainly to the oxyntic mucosa. On the other hand, GRP immunoreactivity in the rat and swine gut was contained in the nerve fibers distributed in the gastric mucosa and in the submucosal and myenteric plex- uses of the gut wall. In the lungs of human fetuses, the endocrine cells, solitary or gathered in groups, were immunoreactive to the GRP antisera. These endocrine cells and nerve fibers in the gut and lung showed the same immunoreactivity to two GRP antisera, but not to a bombesin-specific antiserum. This suggests that the bombesin- like immunoreactivities reported in the previous studies may be due to the existence of GRP or GRP-related peptides. Electron microscopic immunocytochemistry revealed that the GRP-like immunoreactivity was localized in the cytoplasmic gran- ules of the pulmonary endocrine cells and in the dense-cored vesicles of the gut nerve fibers. Bombesin is a tetradecapeptide isolated from the resides in the C-terminal portion of the molecule skin of Bombina bombina and Bombina variegara (17). Moreover, a heptacosapeptide having (1). In mammals, bombesin exerts a wide marked C-terminal homology with porcine GRP variety of actions including stimulation of gas- has been isolated from chicken stomach (15). trin, cholecystokinin and pancreatic polypeptide ‘Bombesin-like’ immunoreactivities have been release, gastrointestinal motility, pancreatic demonstrated in the neurons of the mammalian juice secretion rich in protein and enzymes, brain and gut (4, 19), in some endocrine cells of hypothermia and hyperglycemia (6). the amphibian and avian gut (12, 22, 23), as well Several years after the isolation of bombesin, as in the sensory- and endocrine-like cells (para- gastrin-releasing peptide (GRP), so named be- neurons) in the bronchiolar epithelium of the cause of its intense effect of elevating the blood human fetus (3, 24). With use of an antiserum gastrin level, was isolated from porcine non- specific for bombesin, however, Yanaihara er al. antral gastric mucosa (16). This peptide, (25) detected only extremely low concentrations comprising 27 amino acid residues, was found to of the immunoreactivity in the mammalian gut possess many biological functions in common and postulated that most of the ‘bombesin-like’ with bombesin (15). This is because the C- immunoreactivities demonstrated in mammals terminal decapeptide of GRP is identical with by immunological methods might not corre- that of bombesin, except for the His“ of GRP spond to bombesin but rather to GRP or GRP- which is replaced with Gln in bombesin. It is related peptides. Most of the antisera specific also known that the active site of bombesin for the C-terminus of bombesin which have been 94 T. IWANAGA used by various investigations crossreact coin- ,um in a cryostat. pletely to GRP, and it is impossible to discrimi- Tissues from the stomach and small intestine nate between bombesin and GRP with these of chickens (female, 500-700 g), bullfrogs Rana antisera. caresbeirma (either sex, 300-500 g), and neonates In the present immunohistochemical study, and infants of swine were collected and im- we utilized three kinds of antisera against syn- mersed in Bouin’s fluid for 4-12 h. All the thetic porcine GRP and synthetic bombesin tissues were dehydrated and embedded in par- showing different specificity for GRP-related affin. Paraffin sections were cut at 4-5 um, peptides and bombesin, with which GRP and sometimes serially cut at 1.8-2.0 pm. bombesin could be decisively differentiated. Lungs were obtained from 16 human fetuses ranging in age from 10 to 28 weeks of gestation MATERIALS AND METHODS within 2-1211 after death following therapeutic Antisera or spontaneous abortions. The tissues of human lungs were taken also from two neonates All the antisera and synthetic peptides (antigens) and three adults within 12 h after death. These used in this study were generously provided by tissues were fixed in Bouin’s fiuid for 4-1211 and Professor N. Yanaihara, Shizuoka College of processed for paraffin sections. For electron Pharmacy, Shizuoka, Japan. microscopic immunocytochemistry, the lung The antiserum against bombesin, GP 3303, taken from a 14 week-old fetus about 1 h after was raised in a. guinea pig using N°"-glycyl-[1- death was cut into small pieces and immersed in Glutamine]-bombesin conjugated with bovine 4% paraformaldehyde solution for 6 h. After serum albumin (BSA) as the immunogen. The fixation, they were treated for cryostat sections. antiserum made recognizable mainly the Asn6- Glni’ sequence in the bombesin molecule, and the Gln in position 7 proved to be of importance lmmtmocyfoc/zenvisrry to the antigen-antibody reaction (26). Cryostat sections and dewaxed paraffin sections The antisera against GRP, R 6902 and R 6903, were subjected to immunostaining by the peroxi- were raised in rabbits using synthetic porcine dase-antiperoxidase (PAP) method of Stern- GRP-BSA conjugate as the immunogen. Both berger (21). GRP antisera R 6902 and R 6903 antisera were found to possess the major immu- were used at a dilution of 1: 3,200 and 1: 2,000, nologic determinants in the C-terminal region of respectively. GRP (26). It is, however, shown that besides For immunostaining with bombesin antiserum the C-terminal specific antibodies, the antiserum GP 3303, the sections were processed according R 6902 contains another type of antibodies to the indirect immunoperoxidase method of whose immunologic determinant resides in the Nakane and Pierce (18). GP 3303 was used at central region of GRP molecule, since it hardly various dilutions from 1 : 20 to 1 : 2,000. reacted to bombesin, porcine GRP (14-27) and For electronmicroscopical observation, para- GRP (1-13) (26). It can be expected that the formaldehyde-fixed cryostat sections from the antiserum R 6902, when absorbed with GRP C- oxyntic area of the rat stomach and the lung of terminal fragments or bombesin, may react a human fetus were stained by PAP method specifically to intact GRP. using the GRP antiserum R 6902. After stain- ing, the sections were post-fixed in 1% osmium Tissue Preparation tetroxide for 30 min and embedded in Araldite according to routine procedures. Ultrathin Adult Wista.r rats (male, body weight 250-300 g) sections, after being stained with uranyl acetate and neonates and infants of swine (either sex, and lead citrate, were observed in an electron body weight about 0.6 and 3 kg, respectively) microscope. were anesthetized with sodium pentobarbital (40 mg/kg) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate Specificity of Immunocytoc/remistry buffer, pH 7.4. Various regions of the digestive The conventional staining controls according to tract from the gastric fundus to ileum were Sternberger (21) were employed. In addition, removed and immersed in the fixative for 6-12 h. an absorption test was carried out using antisera The tissues were rinsed overnight at 4°C in 0.1 pretreated with various related peptides. 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