Neurochemistry of visceral afferent neurones.
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Animals↗
Biomedical subjects
G J Dockray
Publications and source records attributed to G J Dockray.
Identification of progastrin in gastrinomas, antrum, and duodenum by a novel radioimmunoassay.
Recent studies on the gene sequence encoding the human pyloric antral hormone, gastrin, indicate a precursor of 101 residues. We have now raised antibodies to a synthetic analogue corresponding to (Tyr)-human progastrin COOH-terminal pentapeptide. The antibodies could be used in radioimmunoassay to measure this peptide, but they did not react with corresponding fragments of procholecystokinin, porcine progastrin, or other human progastrin-derived peptides, notably heptadecapeptide gastrin (G17), and 34-residue gastrin (G34). Radioimmunoassay of human antral and duodenal extracts revealed a major peak of activity that corresponded to the native COOH-terminal fragment of progastrin, and occurred in approximately equimolar amounts with COOH-terminal G17 immunoreactivity. In addition, there was a minor peak of apparently higher molecular weight material. In some gastrinomas the latter material was the predominant immunoreactive form, and it occurred in higher molar concentrations than any other form of gastrin. Digestion of this material with trypsin liberated peptides that reacted with antibodies specific for the NH2-terminus of G34, and G17. On this basis the high molecular weight component was identified as a form of gastrin that extended from the COOH-terminus of the precursor to a point at least beyond the NH2-terminus of G34, and probably included the entire progastrin sequence. The results suggest differences in posttranslational processing pathways of progastrin in antrum, duodenum, and gastrinomas. They also indicate that the present experimental approach allows the identification of progastrin-like substances, which should open the way to studying the mechanisms of gastrin biosynthesis.
Chromatography, High Pressure Liquid↗
CNS effects of circulating CCK8: involvement of brainstem neurones responding to gastric distension.
Cholecystokinin octapeptide (CCK8) given i.v. or i.p. produces a variety of behavioural and CNS effects; these actions are probably exerted at a peripheral site but the neuronal pathways involved are uncertain. We show here that i.v. CCK8 acts on neurones in the n. tractus solitarius with an input from the stomach; cells are either excited or depressed by CCK8 and gastric distension, and responses to the two stimuli are always in the same direction. The responses to close arterial injection of CCK8 indicate a site of action within the splanchnic bed, and most probably a direct action on the vagal afferents mediating gastric mechanoreceptor discharge.
Animals↗
Substance P concentrations in human cerebrospinal fluid vary inversely with age.
The mean concentrations of immunoreactive substance P in cerebrospinal fluid (CSF) of 10 fetuses (gestational age 11-20 weeks) were 22.7 +/- 8.3 pmol/ml, compared with 250.0 +/- 28.2 fmol/ml in premature babies (gestational age 25-31.5 weeks, n = 8), 141.0 +/- 14.2 fmol/ml in full term newborn babies (n = 5), 50.0 +/- 2.3 fmol/ml in children (age 1-6 years, n = 6), and 9.5 +/- 1.5 fmol/ml in 5 adults. The differences between successive age groups were all statistically significant. The high concentration of substance P in human CSF in the early stages of development and its continuous decline towards maturity encourages the idea that substance P plays a role in neuro-development.
Adult↗
Metabolism of the 1-13 sequence of gastrin 17 in humans and failure to influence acid secretion.
The metabolism and action on basal and gastrin 17 (G-17)-stimulated acid secretion of the N-terminal 1-13 sequence of G-17 was studied in human volunteers. Basal acid secretion was not changed by infusion of 1-13 G-17 in doses of 75-1000 pmol X kg-1 X h-1 which gave plasma concentrations of N-terminal G-17 immunoreactivity of 150-2020 pmol X L-1. In addition the acid response to G-17 in a dose that stimulated about 50% maximal acid output was not influenced by 1-13 G-17 (75 and 1000 pmol X kg-1 X h-1). The mean half-time for disappearance of N-terminal immunoreactivity after stopping infusion of 1-13 G-17 was 9.8 +/- 0.5 min. Gel filtration indicated a single peak of N-terminal immunoreactivity in plasma during infusion of 1-13 G-17. Ion exchange chromatography on diethylaminoethyl cellulose, however, revealed two peaks of immunoreactivity. One corresponded to 1-13 G-17, the other eluted earlier. In samples taken after stopping the infusion, the variant predominated. On high-pressure liquid chromatography the variant was resolved into a major component, which had a retention time less than 1-13 G-17, and two minor components. The variants were not produced by incubation of 1-13 G-17 with plasma in vitro. It is concluded that 1-13 G-17 is converted in the circulation to new forms with longer half-lives. Because plasma enzymes cannot account for the formation of these variants, it is possible that enzymes present on cell walls, for example, on capillaries, may be responsible.
Adult↗
Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin.
A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.
Animals↗
FMRFamide- and gastrin/CCK-like peptides in birds.
Antibodies to the molluscan neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide) react with material in extracts of chicken brain. One of the immunoreactive peptides has the sequence Leu-Pro-Leu-Arg-Phe-NH2 (LPLRFamide). We have now raised antibodies to LPLRFamide and used these to examine the distribution and molecular forms of LPLRFamide-like peptides in the chicken. Several forms can be distinguished on the basis of HPLC retention times. Concentrations are highest (200 pmol X g-1) in hypothalamus, and lowest (less than 10 pmol X g-1) in cerebellum, spinal cord and gut. There are two common residues in FMRFamide and gastrin/CCK related peptides (-Met-X-Phe-amide). However, LPLRFamide is readily distinguishable from avian CCK and gastrin. The latter, like the mammalian gastrins, is a relatively strong stimulant of acid, and a weak stimulant of pancreatic enzyme secretion, in the chicken. The physiological roles of the vertebrate peptides with FMRFamide and LPLRFamide immunoreactivity remain to be elucidated, although evidence is emerging to suggest that they might act as transmitters in CNS regions associated with the input of sensory information (dorsal spinal cord and nucleus tractus solitarius).
Amino Acid Sequence↗
Elevated serum and antral gastrin in obese (ob/ob) mice.
Elevations in serum gastrin concentration of six to seven times were found in postabsorptive 5- and 10-wk-old, but not 30- and 40-wk-old, obese mice in comparison with the appropriate lean controls. At 10 wk of age a fourfold hypergastrinemia was also evident in (ob/ob) mice denied food for 48 h. In 10-wk-old (ob/ob) mice that had eaten the same amount of food as lean mice from weaning, serum gastrin was six times that of lean controls. Antral gastrin concentration was 54% higher in fed 10-wk-old (ob/ob) mice than in lean mice. No relationship was found between alterations in serum gastrin and measures of gastrointestinal size or proliferative status. Maximal gastric acid output, expressed with respect to oxyntic mucosal dry weight, was reduced by 52% in 10-wk-old (ob/ob) mice compared with lean controls. It is concluded that the hypergastrinemia of 10-wk-old (ob/ob) mice is not caused by hyperphagia, but may be a consequence of reduced acid inhibition of gastrin release.
Animals↗
Characterization of FMRF amide-like immunoreactivity in rat spinal cord by region-specific antibodies in radioimmunoassay and HPLC.
Material in rat spinal cord extracts that reacts with antibodies to the molluscan tetrapeptide FMRF amide (Phe-Met-Arg-Phe-NH2) has been characterized by HPLC and radioimmunoassay using region specific antibodies. An antibody to the N-terminally extended analogue, Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 (YGGFMRF amide), did not react with the rat material. Two antibodies to FMRF amide were characterized that differed markedly in their affinities for analogues with substitutions in the second and third positions from the C-terminus; both required the C-terminal amide, and neither showed appreciable sensitivity to substitutions in the fourth position from the C-terminus. With both antibodies the relative potency of the avian brain peptide, LPLRF amide, was about 0.1. Both antibodies revealed similar concentrations of immunoreactive material in rat spinal cord extracts. On reversed-phase HPLC using Techsil C18 and Spherisorb-phenyl columns, two peaks were separated that could be distinguished in retention times from FMRF amide, Leu-Pro-Leu-Arg-Phe-NH2 (LPLRF amide), and YGGFMRF amide. The results suggest that the rat spinal cord peptides are structurally related to the C-terminal tripeptide of FMRF amide and are probably extended at the N-terminus by sequences immunochemically distinct from other known peptides.
Animals↗
Cholecystokinin and gastrin forms in the nervous system.
Cholecystokinin octapeptide is the predominant representative of the gastrin-CCK family in the central nervous system. Other forms occur in low concentrations, or restricted locations, as do the gastrins. The pathways of biosynthetic processing can now be studied in detail, following the elucidation of the cDNA sequence for the two peptides. In the vagus both CCK and gastrin can be found. Brain stem neurons receiving an input from gastric mechanoreceptors respond to CCK-8, but most do not respond to gastrin given intravenously or intra-arterially. This system, which may well be involved in mediating the peripheral satiety effect of CCK, is therefore able to distinguish between the two peptides.
Amino Acid Sequence↗
Acid and gastrin responses during intragastric titration in normal subjects and duodenal ulcer patients with G-cell hyperfunction.
Amino acid induced acid and gastrin responses during intragastric titration at pH 2.5 and 5.5 were compared in normal subjects and duodenal ulcer patients with G-cell hyperfunction. The latter were identified on the basis of raised basal or maximal acid outputs and increased gastrin responses to feeding. In normal subjects the mixed amino acid meal stimulated only modest increases in serum gastrin, and the highest observed increase was about 30% that after a standard meal. In contrast, in the G-cell hyperfunction group the highest gastrin concentrations were similar to those after a standard meal. In the G-cell hyperfunction group the increment in serum gastrin at pH 2.5 expressed as a proportion of that at pH 5.5 was 0.29 indicating that the capacity of acid to inhibit gastrin release was well established in these patients. Acid secretory rates were close to maximal at both pH 2.5 and 5.5 during intragastric titration in the ulcer patients, but in normal subjects acid output was about 50% maximal at 2.5 and close to maximal at 5.5. The results suggest that the enhanced gastrin response to feeding in G-cell hyperfunction patients is because of increased sensitivity to amino acid stimulation rather than to diminished acid-inhibitory mechanisms.
Adult↗
Metabolism of heptadecapeptide gastrin in humans studied by region-specific antisera.
The metabolism of synthetic human heptadecapeptide gastrin (G17) in vivo, and in serum in vitro, was studied by radioimmunoassay using region specific antisera, gel filtration, ion exchange chromatography, and high performance liquid chromatography. After infusion of G17 intravenously in normal human volunteers, COOH-terminal and NH2-terminal immunoreactive G17 fragments were generated. At a steady state, approximately 15% of COOH-terminal immunoreactivity was attributable to G14-like material and up to 25% of total NH2-terminal immunoreactivity was attributable to two NH2-terminal fragments; one had the chromatographic properties of 1-13 G17, and the other was less acidic and less hydrophobic. After stopping the infusion of G17, the latter fragments accounted for progressively greater proportions of total gastrin activity. When G17 was incubated in serum in vitro, there was time-dependent and temperature-dependent loss of immunoreactivity, and again COOH-terminal and NH2-terminal immunoreactive fragments were formed. Removal of the NH2-terminal pyroglutamic acid was probably the rate limiting step because synthetic 2-17 G17 was degraded more rapidly in serum (t1/2, 2-3 h) than G17 (t1/2, 3-5 h). EDTA blocked degradation at the COOH-terminus of both 2-17 G17 and G17 but cleavage at the NH2-terminus still occurred, giving rise to a G14-like peptide. The rate of conversion of G17 in serum was not enough to account for the production of fragments in vivo, and it is proposed that these are formed when G17 encounters enzymes on cell surfaces, perhaps during passage through the capillary circulation. The production of these fragments needs to be considered in interpreting studies of the identity, metabolism, and release of gastrin in health and disease.
Adult↗
Peripheral projections and neuropeptide coexistence in a subpopulation of fluoride-resistant acid phosphatase reactive spinal primary sensory neurons.
Combined retrograde axonal tracing with the fluorescent dye Fast Blue and fluoride-resistant acid phosphatase (FRAP) histochemistry revealed that the FRAP-containing sensory neurons project to both somatic and autonomic peripheral nerves. Furthermore, the combination of indirect immunohistochemistry after colchicine treatment and FRAP histochemistry showed that a population of FRAP-containing sensory neurons are also substance P, cholecystokinin or somatostatin positive.
Acid Phosphatase↗
Effects of cysteamine on pain behaviour and on somatostatin- and substance P-like immunoreactivity in the substantia gelatinosa of the rat.
Immunohistochemical studies on rats showed cysteamine to deplete immunoreactive somatostatin, but not substance P, in the substantia gelatinosa of the spinal trigeminal nucleus and spinal cord. The effect of cysteamine treatment on chemical pain was investigated using capsaicin as the pain-producing stimulus. Thermal pain was assessed with a hot plate test after cysteamine treatment. It was found that cysteamine did not alter the hind paw lick latency in the hot plate test or the number of forepaw wipes after application of a drop of capsaicin into the cornea, compared to normal animals. In capsaicin-treated animals a significantly lower number of forepaw wipes were seen after corneal capsaicin application. However, in cysteamine-treated animals slower wiping was observed compared to untreated and capsaicin-treated animals. This suggests that somatostatin may be of importance for the modulation of nociceptive information, although it is not a major pain transmitter.
Animals↗
Evidence for different patterns of post-translational processing of pro-enkephalin in the bovine adrenal, colon and striatum indicated by radioimmunoassay using region-specific antisera to Met-Enk-Arg6-Phe7 and Met-Enk-Arg6-Gly7-Leu8.
Specific antisera to two opioid peptides, Met-Enk-Arg6-Phe7 and Met-Enk-Arg6-Gly7-Leu8, were used in radioimmunoassay to study molecular forms in extracts of bovine adrenal, striatum and colon. The antisera to Met-Enk-Arg6-Phe7 and Met-Enk-Arg6-Gly7-Leu8 were each highly specific for the C-terminus of their respective haptens and showed less than 1% cross-reactivity with related enkephalins and their variants. In adrenal extracts, gel filtration on Sephadex G-50 indicated N-terminally extended forms of Met-Enk-Arg6-Phe7 and Met-Enk-Arg6-Gly7-Leu8 as the predominating immunoreactive species, whereas in the striatum and colon the main immunoreactive forms were compatible with the hepta- and octapeptides. The results suggest different patterns of post-translational processing of precursors in the adrenal medulla compared with striatal and colonic neurones.
Adrenal Glands↗
Immunohistochemical localization to pyloric antral G cells of peptides derived from porcine preprogastrin.
Antibodies to the peptides (designated cryptic A and B) that flank the G34 region of pig progastrin were used in immunohistochemical studies of the gastrointestinal tract. In elution and restaining experiments, the same cells were revealed by the cryptic peptide antibodies, and by antibodies specific for C-terminus of G17 and N-terminus of G34. The cells reacting with the cryptic peptide antibodies were localized predominantly to antral mucosa. They were found in pig, ferret, dog and cat but not in man, guinea pig, rat or mouse; presumably in the latter species there are amino acid substitutions in the cryptic peptides that influence immunoreactivity with the present antibodies. The results indicate that progastrin production occurs only in G cells in the gut, and that a single population of cells produces all the predicted regions of progastrin.
Animals↗
Increases in arterial blood pressure in the rat in response to a new vertebrate neuropeptide, LPLRFamide, and a related molluscan peptide, FMRFamide.
Vasopressor responses in urethane-anaesthetized rats were evoked by a new peptide from chicken brain, LPLRFamide, and the immunochemically related molluscan neuropeptide, FMRFamide. In doses of 50 to 200 nmol X kg-1, i.v., both peptides produced a rapid increase in arterial pressure that returned to basal in 1-2 min. When given intracisternally in similar doses the two peptides again increased arterial pressure, but the time to peak response (1-2 min) and the duration of the responses (5-8 min) were prolonged. There was a marked, reversible, specific, tachyphylaxis following intracisternal but not intravenous injection of LPLRFamide, indicating separate sites of action after administration by these routes. Guanethidine and phentolamine blocked the responses to both intravenous and intracisternal administration, and hexamethonium significantly reduced the response to intracisternal administration. Both by intravenous and intracisternal routes it is therefore likely that responses are mediated by noradrenaline release from sympathetic endings. In addition, following adrenalectomy and propranolol the response to intravenous (but not intracisternal) LPLRFamide was increased, suggesting that adrenaline released from the adrenal medulla might act at beta-adrenoreceptors causing vasodilatation. The physiological significance of these observations remains to be established, but it is significant that peptides immunochemically related to FMRFamide and LPLRFamide occur in areas of rat brain concerned with autonomic control.
Animals↗
Dopamine and cholecystokinin immunoreactive neurons in mesencephalic grafts reinnervating the neostriatum: evidence for selective growth regulation.
Pieces of embryonic mesencephalic tissue rich in dopamine and cholecystokinin immunoreactive neurones were grafted to the dorsal surface of the caudate-putamen of adult host rats subjected to unilateral dopamine depleting lesions. After 3 months, neuronal survival in the graft and fibre outgrowth into the host brain were studied by tyrosine hydroxylase and cholecystokinin immunohistochemistry, both in serial sections and by elution and restaining of the same sections. Both dopamine- and cholecystokinin-containing neurones as well as neurons containing both compounds survived the transplantation process. The ratio of neurones in which dopamine and cholecystokinin-like immunoreactivity occurred independently and in coexistence was similar in the grafts to that seen in the intact ventral mesencephalon. This suggests that the grafted cells maintain and express at least some of their normal chemical characteristics in the ectopic cortical location. Only those fibres which contained tyrosine hydroxylase but apparently lacked the cholecystokinin-like peptide showed extensive reinnervation of the host neostriatum. The cholecystokinin-positive fibres were found in a narrow zone immediately adjoining the graft. These results indicate that the dopaminergic reinnervation of the denervated neostriatum is preferentially carried out by the population of grafted mesencephalic dopamine neurones apparently lacking the cholecystokinin-like peptide. This suggests the presence of growth regulating mechanisms in the denervated neostriatum which selectively favour the ingrowth of fibres from the appropriate dopaminergic neuronal subset. The transplantation technique may therefore provide a powerful tool for the study of neurone-target interactions in the establishment of neuronal connections, and of the possible role of peptidergic coexistence in the development and organization of monoaminergic pathways and their innervation patterns.
Animals↗