Bombesin-like immunoreactivity in the avian gut and its localisation to a distinct cell type.

The distribution of a bombesin-like immunoreactive peptide in the avian gastro-intestinal tract was analysed by combined radioimmunoassay and immunocytochemistry. Radioimmunoassay of tissue extracts showed that the largest quantities of bombesin-like immunoreactivity were present in the proventriculus (64.5 +/- 6.0 pmol/g) with smaller but still considerable amounts in the gizzard (40.0 +/- 6.0 pmol/g). Immunocytochemically the extractable bombesin-like immunoreactivity was localised in numerous endocrine cells. These, in the proventriculus, were found mainly in the deeper layers of the mucosa. Further study of these cells by the semi-thin/thin technique revealed the presence of characteristic secretory granules. The functional name BN is proposed for this cell type.     

The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut,pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B

Summary GAWK is a recently discovered peptide isolated from extracts of human pituitary gland and subsequently shown to be identical to sequence 420–493 of human chromogranin B. The distribution of this peptide was studied in human gut, pancreas, adrenal and pituitary glands using antisera to two portions of the 74 amino acid peptide (sequences 1–17 and 20–38). In addition, the co-existence of GAWK immunoreactivity with other peptides and chromogranin B was investigated using comparative immunocytochemistry.In the gut, GAWK was localised mainly to serotonin-containing cells of the mucosal epithelium, where electron microscopy showed it to be stored in typical electron-dense (250 nm diameter) granules, and to a moderate population of nerve fibres in the gut wall. Considerable quantities of GAWK-like immunoreactivity were measured in the gut, up to 36.3±18 pmol GAWK 1–17/g wet weight of tissue (mean±SEM) and 12.4±2.9 pmol GAWK 20–38/g. Chromatography of gut extracts revealed several GAWK-like immunoreactive peaks. GAWK-like immunoreactivity was also detected in endocrine cells of pancreas, pituitary gland and adrenal medulla, where the highest concentrations of GAWK-like immunoreactivity were measured (GAWK 1–17 2071.8±873.2 and GAWK 20–38 1292.7±542.7 pmol/g). Endocrine cells containing GAWK-like immunoreactivity were found also to be immunoreactive for chromogranin B.Our results define a discrete distribution of GAWK immunoreactivity in human endocrine cells and nerves and provide morphological support for the postulated precursor-product relationship between chromogranin B and GAWK. Details of the functions of this peptide are awaited.     

Bombesin-like immunoreactivity in the nervous system of hydra

Summary With immunocytochemical methods, nerve cells have been detected in Hydra attenuata containing bombesin-like immunoreactivity. These nerve cells are located in the ectoderm of all body regions of the animal and are especially abundant in basal disk and tentacles. Radioimmunoassay of extracts of hydra demonstrated at least 0.2 pmol/g wet weight of bombesinlike immunoreactivity. The immunoreactive material elutes from Sephadex G-50 in a similar position to synthetic bombesin. The data show that bombesin-like peptides are among the phylogenetically oldest neuropeptides found so far.     

Mapping,quantitative distribution and origin of substance P- and VIP-containing nerves in the Uvea of guinea pig eye

Summary VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3±4.8 pmol/g, mean ± SEM; substance P:11.1±1.8 pmol/g) in the uveal portion of the guinea pig eye.d Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p<0.0005) than those immunoreactive for substance P (95±7 nm and 82±9 nm respectively; mean ± SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1±1.5 pmol/g, mean ± SEM, control; 5.3±1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.To whom offprint requests should be sent     

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes

The objectives were to evaluate

1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 μg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 μg/dL Cu) and small (82 ± 22.1 μg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 μg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 μg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 μg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10⁶ cumulus cells) and 6 μg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10⁶ cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 μg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 μg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 μg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 μg/mL Cu relative to 0 Cu (IVM alone) and 2 μg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.     

Peptide-immunoreactive nerves in the mammalian female genital tract

Summary Vasoactive intestinal polypeptide, substance P, neuropeptide Y and peptide histidine isoleucine immunoreactivities have been demonstrated in the female genitalia of rat, cat, mouse and guinea-pig using immunocytochemistry and radioimmunoassay. They were localized to nerves. Each type of immunoreactive nerve showed a distinct pattern of distribution, though all were associated to some degree with blood vessels and smooth muscle. Vasoactive intestinal polypeptide-immunoreactive and neuropeptide Y-immunoreactive nerves were the most abundant. Higher concentrations of peptides were detected in the female genitalia of the mouse than those of the other species studied. Vasoactive intestinal polypeptide-immunoreactive nerves were particularly concentrated in the cervix (89.1±17.2 pmol/g, mean±S.E.M.) and the uterus (57.4±14.8 pmol/g) of the mouse, while neuropeptide Y immunoreactivity was more abundant in the Fallopian tube of the mouse (31.6±11.8 pmol/g) and the vagina of the rat (38.6±4.8 pmol/g) than in other regions. Separate populations of ganglion cells in the paracervical ganglia were found to contain vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities. Peptide histidine isoleucine-immunoreactive and vasoactive intestinal polypeptide-immunoreactive nerves were similarly distributed, but the former were much less frequent. Substance P-immunoreactive nerves were seen mainly beneath the epithelium of the vagina and were, in general, more numerous in the guinea-pig than in other species. The significance of these peptide-immunoreactive nerves in the female genital organ remains to be determined.Dr. Wang is on leave from The Institute of Acupuncture, The Academy of Chinese Traditional Medicine, Peking, China.     

Intramural distribution of immunoreactive vasoactive intestinal polypeptide (VIP), substance P,somatostatin and mammalian bombesin in the oesophago-gastro-pyloric region of the human gut

Summary The intramural distribution of vasoactive intestinal polypeptide (VIP), substance P, somatostatin and mammalian bombesin was studied in the oesophago-gastro-pyloric region of the human gut. At each of 21 sampling sites encompassing this entire area, the gut wall was separated into mucosa, submucosa and muscularis externa, and extracted for radioimmunoassay. VIP levels in the mucosa were very high in the proximal oesophagus (1231±174 pmol/g, mean±SEM) and showed varied, but generally decreasing concentrations towards the stomach, followed by a clear-cut increase across the pyloric canal (distal antrum: 73±16 pmol/g, proximal duodenum: 366±62 pmol/ g); consistent levels were found in submucosa and muscle (200–400 pmol/g) at most sites, the stomach again showing lower concentrations. By contrast, substance P was present in small amounts as far as the proximal stomach, but sharply increased across the pyloric canal, especially in mucosa and submucosa (distal antrum: 20±6.5 and 5.5±1.3 pmol/g; proximal duodenum: 62±8.5 and 34±11 pmol/g, respectively). Somatostatin concentrations were very low in the mucosa of the oesophagus and stepwise increased in the cardiac, mid-gastric and pyloric mucosa (cardia: 224±72 pmol/g; distal antrum: 513±152 pmol/g; proximal duodenum: 1013±113 pmol/g); concentrations in the submucosa and muscularis were generally low, with the exception of antrum and duodenum. Mammalian bombesin was comparatively well represented throughout the oesophageal muscularis (5–8 pmol/g), but most abundant in the stomach in all layers (oxyntic mucosa: 24±2.7 pmol/g; submucosa: 20±5.7 pmol/g; muscle: 28±5.0 pmol/g). In conclusion, a distinct differential distribution of the four peptides studied was revealed, indicating a diffuse, but highly differentiated peptide-containing innervation of the proximal human gut.     

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Bombesin-like peptides in small cell lung cancer: biochemical characterization and secretion from a cell line

High intracellular levels of BN-like peptides are present in tumors and cell lines of small cell carcinoma of the lung (SCCL) as well as the putative precursor cells of this tumor, the pulmonary endocrine cell. In cell line NCI-H209 the density of bombesin-like peptides was 8.9 +/- 1.1 pmol/mg total protein. Gel filtration chromatography of an extract of these cells revealed one major peak of immunoreactivity which coeluted with synthetic bombesin (1620 daltons). Also, high pressure liquid chromatography revealed one major peak of immunoreactivity was present which eluted before synthetic peptide. Therefore, SCCL bombesin-like peptides may be of similar size but are more hydrophilic than synthetic peptide. Cells maintained in culture continuously release bombesin-like peptides into the growth medium. Also, high concentrations of K+ stimulated the secretion of immunoreactive bombesin from cell lines in a Ca++-dependent manner. These SCCL bombesin-like peptides may function as important regulatory agents in the malignant lung.     

Enzymatic cleavage prior to antibody incubation as a method for neuropeptide immunocytochemistry

Summary Five anti-gastrin releasing peptide (GRP) sera have been characterized against GRP, bombesin and related polypeptides spotted on cellulose acetate discs. Antibodies reacting with the C-terminal G-14 sequence of bombesin and the 19–27 sequence of GRP, were detected in all sera. Antibodies directed exclsively against the bombesin unrelated 1–17 sequence of GRP were found only in one serum (R-6902). With parallel immunohistochemical tests only the C-terminal immunoreactivity was detected in endocrine-paracrine cells of the chicken proventriculus, while both immunoreactivities were present in nerve fibres and a few nerve cell bodies of the mammalian gut. The distribution of GRP- and bombesin-like immunoreactive nerves in the gastric mucosa of both pyloric and oxyntic type the submucosal and myenteric plexus along the whole gastrointestinal wall and at sphincter regions is detailed.     

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 μg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ±13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 μg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 μg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10⁶ cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 μg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10⁶ cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 μg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 μg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 μg/mL Zn relative to 0 Zn (IVM alone) and 0.7 μg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.     

Distribution of vasoactive intestinal peptide, bombesin, and gastrin-cholecystokinin like peptides in the avian intestinal tract and brain

The distribution of vasoactive intestinal peptide (VIP), bombesin and gastrin-cholecystokinin in the chicken was studied by radioimmunoassay of tissue extracts. VIP was present in high concentrations in colon (186 +/- 29 pmol/g), cloaca (116 +/- 27 pmol/g), jejunum (97 +/- 14 pmol/g) and pancreas (15 +/- 3 pmol/g) but not detected in lung, liver or thymus. The highest concentration of bombesin was in the proventriculus (92 +/- 13 pmol/g), negligible in remaining gut but found in brain. Gel chromatography indicated two forms of bombesin: one form eluting with bombesin-14 and the other with gastrin releasing peptide. Gastrin-like immunoreactivity was found in low levels in the gut and brain. The concentrations were higher with an antiserum which cross reacted with the carboxy terminus common to gastrin-17 and CCK compared to a gastrin specific antisera (P less than 0.01). This suggests that the carboxy terminal region has been conserved during evolution. Each distribution pattern of bombesin, VIP and gastrin CCK is different, and distinct from that found in mammals, suggesting specific roles for these peptides in birds.     

Significant depletion of NPY in the innervation of the rat mesenteric,renal arteries and kidneys in experimentally (aorta coarctation) induced hypertension

Summary The distribution and concentrations of neuropeptide Y (NPY) in kidneys, renal arteries, heart, aorta, mesenteric artery and adrenal glands from aorta-ligated hypertensive rats were studied by immunocytochemistry and radioimmunoassay. Immunocytochemistry showed that in the hypertensive animals NPY-immunoreactive fibres were decreased in both kidney and renal artery, above and below the ligation, and in mesenteric arteries. The depletion of NPY-containing nerves in the kidney was more pronounced around the juxtaglomerular apparatus than in other areas of the organ. By radioimmunoassay, the concentrations of NPY immunoreactivity were significantly lower in the hypertensive animals when compared with the controls, (kidney: hypertensive 1.0±0.1; controls 2.0±0.2 pmol/g, mean±SEM; p<0.05 renal artery: hypertensive 5.0±0.8; controls 12.1±2.0; p<0.05 and mesenteric artery: hypertensive 8.6±1.9; 17.6±3.0; p<0.01). While there were no statistically significant changes in the levels of NPY immunoreactivity in the other areas studied, there was a general trend for the level to fall in the renal artery below the ligation (hypertensive 10.6±1.5; control 15.3±2.4; p>0.05). It is of interest that changes were observed in the vasoconstrictor peptide NPY in this commonly used model of hypertension.     

An ouabain-like factor is secreted from immortalized hypothalamic cells in an aldosterone-dependent manner

Ouabain-like factor (OLF) modulates blood pressure via sodium pump inhibition in the central nervous system and in the peripheral circulation. Ouabain-like factor (OLF) is thought to be produced in the adrenal gland and hypothalamus, and it may relate locally to the renin-angiotensin-aldosterone system. However, the evidence for the latter was obtained from in vivo experiments using animals. In the present study, we investigated ouabain production in the immortalized hypothalamic cell line N1. First, cell culture supernatant was collected from the immortalized hypothalamic cell line N1 at 0.5, 4, 8, and 24 h. A newly developed enzyme-linked immunosorbent assay (ELISA) that used anti-ouabain antibody showed that immunoreactivity in the supernatant was increased significantly at 24 vs. 0.5 h (0.01 ± 0.004 vs. 0.16 ± 0.033 pmol/mg protein, p < 0.01). A combination of HPLC and ELISA was used to characterize N1 cell-derived OLI, showing that the highest peak of OLI had the same retention time as authentic ouabain. Thereafter, N1 cells were cultured with (1-10 μM) aldosterone, and supernatant was collected after 24 h of culture. In addition, N1 cells were cultured with 5 μM eplerenone, a mineralocorticoid receptor blocker, plus aldosterone. OLI was significantly increased in the supernatant of the cells cultured with 10 μM aldosterone (0.40 ± 0.078 pmol/mg protein), and this increase was abolished by the addition of the aldosterone antagonist eplerenone (0.12 ± 0.030 pmol/mg protein). These data suggest that the immortalized hypothalamic N1 cells secrete OLF and that aldosterone stimulates its secretion via mineralocorticoid receptors.     

Immunocytochemical localization of gastrin-releasing peptide/bombesin-like immunoreactive neurons in insects

Summary GRP/bombesin-like immunoreactive material was immunocytochemically detected in neurons of seven insect species belonging to seven orders, while such neurons were not found in three insect species belonging to two other orders. In some insect species certain neurons were found in corresponding places and approximately the same numbers. It seems likely that such neurons have a common evolutionary origin and are homologous. The fact that the GRP-antiserum reveals such homologous neurons in species belonging to different orders, suggests that the part of the GRP/bombesin-like peptide recognized by the antiserum has been relatively stable during evolution. As the GRP-antiserum had to be used in much higher concentrations on insect tissue than for GRP endocrine cells in chicken proventriculus, the chemical resemblance of the insect peptide(s) to GRP and bombesin may be limited.     

Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 µm diameter fraction believed to be composed largely of basal cells, and a 15 µm diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 µm cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 ± 2.7% ciliated cells with 6.5 ± 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 ± 2.1%) and Clara cells (27.0 ± 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 ± 0.09 × 10⁶ cells/ trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 µm cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 ± 66 pmol/ min/ mg protein or 51.2 ± 20.5 pmol/ min/ 10⁶ cells for 7-ethoxycoumarin deethylase and 31.7 ± 15.4 pmol/ min/ mg protein or 10.5 ± 4.8 pmol/ min/ 10⁶ cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.Abbreviations CD cell digest - DNase deoxyribonuclease I - E-1 first elutriator fraction - E-2 second elutriator fraction - E-3 third elutriator fraction - 7-Ec 7-ethoxycoumarin - FCS fetal calf serum - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - HpBS HEPES-buffered salt solution - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PEG Carbowax polyethylene glycol 6000     

Morphological changes during ontogeny of the canine proximal colon

The development of the canine proximal colon from the completion of organogenesis through 43 days after birth was studied using light microscopy, immunofluorescence and electron microscopy. During this period the tunica muscularis increased in thickness from 42±6 m in animals midway through the gestation period to 317±29 m in animals 25–30 days old. This increase in thickness resulted from an increase in the number and size of smooth muscle cells in the circular and longitudinal muscle layers. The cross-sectional thickness of the circular muscle layer increased from 10±2 smooth muscle cells midway through the gestation period to 92±7 cells in animals 25–30 days old. The longitudinal layer increased in thickness from 1.5±1 cells in animals midway through the gestation period to 44±2 cells in animals 25–30 days old. Smooth muscle cells from both layers also increased in diameter and length. Ultrastructural and immunohistochemical studies suggested that many of the smooth muscle cells were undergoing development throughout the fetal period. Midway through the gestation period, the circular layer was positive for desmin-like immunoreactivity (D-LI), while both the circular and longitudinal layers were positive for vimentinlike immunoreactivity (V-LI). By birth, V-LI was suppressed in the circular and longitudinal layers, and both layers expressed D-LI. The enteric nervous system was already established midway through the gestation period, and submucosal and myenteric ganglia could be identified, although the chemical coding and mature morphology of neurons were incomplete. NADPH-diaphorase-positive neurons, indicating the expression of nitric oxide synthase, developed by the time of birth. Interstitial cells of Cajal (IC) could not clearly be identified midway through gestation, however, potential precursors to ICs were observed. Several classes of ICs were identifiable at birth.     

VIP-, Bombesin- and Neurotensin-Like Immunoreactivity in Neurons of the Gut of the Holostean Fish,Lepisosteus platyrhincus

VIP-like immunoreactivity was found in nerve fibres in all layers of the gut wall in both stomach and intestine, and was abundant in the myenteric and submucous plexuses. A few fibres were associated with blood vessels. Nerve cells showing VIP-like immunoreactivity were found in the myenteric plexus. Neurotensin-like immunoreactivity was found in nerve cells and numerous nerve fibres in the myenteric plexus of both stomach and intestine and in nerve fibres of the circular muscle layer, while bombesin-like immunoreactivity was confined to a low number of nerve fibres in the myenteric plexus of the stomach. The results indicate that a VIP-like, a neurotensin-like and a bombesin-like peptide are present in neurons of the gut of Lepisosteus.