Identification and probable role of a single neurone containing the neuropeptide Helix FMRFamide.

Recently, there has been intense interest in the possibility that peptides might function as neurotransmitters. Despite much progress, there remains no clear-cut example in which the production of a chemically characterized peptide may be ascribed to individual identifiable neurones of proven physiological role. Invertebrate systems have proved to be particularly valuable for the study of identified neurones, including those producing peptides.

Distribution of enkephalin-related peptides in rat brain: immunohistochemical studies using antisera to met-enkephalin and met-enkephalin Arg6Phe7.

The enkephalin-related heptapeptide, Tyr-Gly-Gly-Phe-Met-Arg-Phe, forms the C-terminus of a biosynthetic precursor that contains both Met-enkephalin and Leu-enkephalin sequences. We have studied the distribution of heptapeptide-like immunoreactivity in rat brain by immunohistochemistry using a C-terminal specific antiserum. The results were compared with those obtained using an antiserum specific for the C-terminus of Met-enkephalin which does not react with C-terminally-extended variants.

Evidence for periaqueductal cholecystokinin-substance P neurons projecting to the spinal cord.

Using indirect immunofluorescence techniques combined with retrogradely transported fluorescent dyes, cholecystokinin (CCK)-like and substance P immunoreactive cell bodies in the periaqueductal central grey of the rat brain were studied. Data from both adjacent sections and elution-restaining techniques indicated that some of these central grey cells contain both a CCK-like peptide and substance P. Injection of the fluorescent dye, Fast Blue, into the cervical spinal cord indicated that this CCK-substance P cell group is a descending system.

FMRFamide-like immunoreactivity in rat brain: development of a radioimmunoassay and its application in studies of distribution and chromatographic properties.

A radioimmunoassay is described for the molluscan neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide). The antibody used is C-terminal-specific and shows slight but significant (1-2%) cross-reactivity with chicken pancreatic polypeptide (APP). The assay has been used to identify in rat brain extracts a pair of molecules that may represent mammalian counterparts of FMRFamide.

Isolation from porcine antral mucosa of a hexapeptide corresponding to the C-terminal sequence of gastrin.

The present studies were undertaken to confirm reports of high concentrations of the C-terminal tetrapeptide of gastrin in hog antral mucosa. A method was developed whereby synthetic tetrapeptide added to boiling water extracts of hog antral mucosa could be purified to homogeneity by adsorption to Amberlite XAD2 resin, ion exchange chromatography on DEAE cellulose, and reverse phase HPLC. The product had the amino acid composition of gastrin tetrapeptide.

Origin of peptide-containing fibers in the inferior mesenteric ganglion of the guinea-pig: immunohistochemical studies with antisera to substance P, enkephalin, vasoactive intestinal polypeptide, cholecystokinin and bombesin.

After different denervation procedures the guinea-pig inferior mesenteric ganglion was analysed by immunohistochemistry using antisera to substance P, enkephalin, vasoactive intestinal polypeptide, cholecystokinin and bombesin. The results demonstrate that each of the nerve trunks connected to the ganglion carries specific peptidergic pathways. Thus, the lumbar nerves contain substance P-immunoreactive primary afferent neurons, which to a large extent traverse the ganglion, and enkephalin-immunoreactive preganglionic neurons; the colonic nerves carry vasoactive intestinal polypeptide-, cholecystokinin- and bombesin-immunoreactive fibers from the distal colon to the ganglion; the hypogastric nerves contain vasoactive intestinal polypeptide-positive fibers from the pelvic plexus; and the intermesenteric nerve contains vasoactive intestinal polypeptide, cholecystokinin, substance P and enkephalin from divergent sources.

Cholecystokinin- and secretin-induced pancreatic secretion in normal and diabetic rats.

The influence of the endocrine pancreas on exocrine pancreatic function was studied in rats made diabetic by administration of streptozotocin. In normal urethan-anesthetized rats the C-terminal octapeptide of cholecystokinin (CCK-8) stimulated the rate of flow and rates of amylase and trypsinogen release from the pancreas; the proportions of the two enzymes did not change with repeated stimulation. In diabetic rats CCK-8 (0.

Directional growth of sympathetic nerve fibres in vitro towards enteric smooth muscle and heart from mice with congenital aganglionic colon and their normal littermates.

Neuronotrophic factors in tissues from normal mice and mice with hereditary aganglionic colon (s1/s1) were assayed by examining neurite extension in vitro from sympathetic ganglia (superior cervical and coeliac) towards explants of stomach, atrium and colon in co-culture. Directional growth of neurites from both ganglia towards all 3 target explants was observed. There were no statistically significant differences between normal and s1/s1 ganglia in the capacity to extend neurites, neither were there differences between the target tissues of these mice in ability to promote neurite extension from ganglia.

Effects of conditioned media on extension of substance P-immunoreactive neurites from cultured mouse sensory ganglia.

Media conditioned by heart or stomach smooth muscle cells stimulated elongation of neurites with substance P-immunoreactivity from explants of mouse dorsal root ganglia in culture, but nerve growth factor (NGF) had no effect. However, NGF is known to be needed for sensory neurone survival; at least two types of factor may therefore be required for the normal development of substance P-containing sensory neurones.

Coexistence of cholecystokinin- and substance P-like peptides in neurons of the dorsal root ganglia of the rat.

Using the indirect immunohistochemical technique with antisera to cholecystokinin and to substance P, the spinal dorsal horn and dorsal root ganglia of normal and colchicine-treated rats were studied. In the spinal cord a similar distribution of substance P- and cholecystokinin-positive networks in the superficial layers of the dorsal horn was observed. In the dorsal root ganglia several cholecystokinin and substance P immunoreactive cell bodies were seen in colchicine-treated rats.

Differential distribution in rat basal ganglia of Met-enkephalin- and Met-enkephalin Arg6 Phe7-like peptides revealed by immunohistochemistry.

Antisera to Met-enkephalin and its heptapeptide variant Met-enkephalin Arg6Phe7 were used in immunohistochemistry to study distribution of immunoreactive material in rat basal ganglia. In the pallidum, the Met-enkephalin antiserum, but not the Met-enkephalin Arg6Phe7 antiserum, revealed abundant nerve terminals, whereas in the caudate-putamen the heptapeptide antiserum demonstrated numerous immunoreactive cell bodies that were not shown by the Met-enkephalin antiserum. The differential distribution of immunoreactivity is compatible with distinct physiological roles for the two peptides in basal ganglia.

Substance P and cholecystokinin-like peptides in Helix neurons and cholecystokinin and serotonin in a giant neuron.

Immunohistochemical procedures revealed that the serotonin-containing cell situated in each metacerebral ganglion of the snail Helix also contains a cholecystokinin-like peptide, but is devoid of substance P. In radioimmunoassays the cholecystokinin-like material showed similarities to the carboxy terminal regions of mammalian cholecystokinin and gastrin. Since much is known about the morphology and synaptic connections of this serotonin neuron, the role of the cholecystokinin-like peptides can now be investigated by neurophysiological methods, thus opening the way to discovering whether a neuron can use more than one transmitter.

Capsaicin depletes CCK-like immunoreactivity detected by immunohistochemistry, but not that measured by radioimmunoassay in rat dorsal spinal cord.

In rats treated neonatally with capsaicin there were significant reductions in substance P measured by radioimmunoassay in dorsal spinal cord, spinal ganglia, and coeliac ganglia, but concentrations of cholecystokinin octapeptide (CCK8) measured by radioimmunoassay were not decreased. However, immunohistochemical studies using antisera to both substance P and CCK8, respectively, demonstrated decreased immunoreactive material in the dorsal horn of the spinal cord. The nature of the material localized by CCK8 antiserum in immunohistochemistry remains to be resolved.

Distribution and development of peptidergic nerves and gut endocrine cells in mice with congenital aganglionic colon, and their normal littermates.

Studies were made of the distribution of gut hormones and neuropeptides in the gastrointestinal tract of mice with hereditary aganglionic colon (s1/s1) and their normal littermates. Antisera to substance P, vasoactive intestinal polypeptide, and enkephalins demonstrated markedly diminished numbers of immunofluorescent nerve fibers in the aganglionic segment of colon; in contrast, in proximal colon and small intestine the distribution of peptidergic nerve fibers was essentially normal. Mucosal endocrine cells were demonstrated in the colon by antisera to substance P, somatostatin, glucagon, and cholecystokinin; in each case there were similar numbers of cells in s1/s1 and normal mice.

Bombesin-like immunoreactivity in specific neurones of the snail Helix aspersa and an example of the coexistence of substance P and serotonin in an invertebrate neurone.

Serial sections of the suboesophageal ganglia from Helix aspersa were processed immunohistochemically for the localisation of bombesin-, substance P-, cholecystokinin-, vasoactive intestinal polypeptide- and serotonin-related substances. All of these substances were localized in specific neurones and processes. Bombesin-like immunoreactivity was found in both perikarya and terminals throughout the suboesophageal ganglia.

A rational approach to the fixation of peptidergic nerve cell bodies in the gut using parabenzoquinone.

Study of the possible chemical mechanisms by which parabenzoquinone acts as a fixative in the localisation of neuronal peptides by immunohistochemistry suggested several possible improvements to standard techniques. In particular, by using favourable conditions for regenerative oxidation reactions, i.e.

Identification of NH2-terminal fragments of big gastrin in plasma.

Molecular forms of gastrin in human plasma were studied by radioimmunoassay using antisera specific for the NH2-terminus of big gastrin (gastrin 34). A plasma enzyme system that destroys immunoreactivity of NH2-terminal fragments of gastrin 34, but not of gastrin 34 itself, has been demonstrated and shown to be inhibited by phenanthroline. Endogenous immunoreactive human plasma gastrin that had been concentrated by immunoaffinity adsorption and separated by gel filtration was shown to consist of gastrin 34, together with peptides corresponding to its NH2-terminal tryptic peptide and a shorter NH2-terminal fragment.

FMRFamide-like immunoreactivity in the nervous system of Hydra.

FMRFamide-like immunoreactivity has been localized in different parts of the hydra nervous system. Immunoreactivity occurs in nerve perikarya and processes in the ectoderm of the lower peduncle region near the basal disk, in the ectoderm of the hypostome and in the ectoderm of the tentacles. The immunoreactive nerve perikarya in the lower peduncle region form ganglion-like structures.

Identification and measurement of molecular variants of cholecystokinin in duodenal mucosa and plasma. Diminished concentrations in patients with celiac disease.

The amount and type of cholecystokinin (CCK) in duodenal extracts and plasma of celiac patients and normal subjects was studied by radioimmunoassay and gel filtration. In both groups there were similar patterns of molecular forms in extracts of duodenal biopsies, but concentrations in celiac disease were significantly depressed. In boiling water extracts of duodenal mucosa from both groups a factor with the properties of the COOH-terminal octapeptide of cholecystokinin predominated, but there were also significant amounts of a larger molecular weight form.

Immunohistochemical demonstration of peptides, serotonin and dopamine-beta-hydroxylase-like material in the nervous system of the leech Hirudo medicinalis.

The central ganglia of the leech, Hirudo medicinalis, were processed for the immunohistochemical localisation of bombesin-, substance P-, cholecystokinin-, vasoactive intestinal polypeptide-, enkephalin-, serotonin- and dopamine-beta-hydroxylase-related substances. To varying extents all of the substances were localised in neuropile processes, and all, with the exception of substance P, were associated with specific perikarya. The most prominent neuropeptides, in terms of the number of immunoreactive neurones, were cholecystokinin and vasoactive intestinal peptide.