Biomedical subjects
G J Dockray
Publications and source records attributed to G J Dockray.
Mechanisms of action of peripherally administered cholecystokinin octapeptide on brain stem neurons in the rat.
We have investigated the pathway and the mechanism by which cholecystokinin octapeptide (CCK-8), given systemically, may influence the discharge of brain stem neurons that have an input from the stomach. Extracellular recordings were made from neurons in the nucleus of the solitary tract (NTS), where vagal afferents terminate, and from neighboring regions of the dorsal medial medulla. Gastric distension and CCK-8 injected intra-aortically close to the stomach evoked either excitatory or inhibitory responses that were abolished by cervical vagal section. In animals from which the celiac/superior mesenteric ganglia were removed, or the gastric antrum resected 2 weeks earlier, responses to gastric distension and CCK-8 were maintained. The effects of CCK-8 are unlikely to be secondary to changes in smooth muscle tone because CCK-8 decreased pressure in the body of the stomach, while distension increased it. Moreover, intravenous noradrenaline and vasoactive intestinal peptide had effects similar to CCK-8 on intragastric pressure, but evoked different patterns of responses from brain stem neurons. The results are consistent with the idea that CCK-8 acts directly on vagal mechanoreceptive endings in the gastric corpus wall. It is well known that peripheral administration of CCK-8 influences short-term regulation of food intake. The effects described here may reflect the pathway by which peripheral CCK influences CNS function.
Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment.
The gene sequence encoding porcine preprogastrin is known; in order to clarify pathways of post-translational processing of the predicted precursor peptide we have characterized material reacting with antibodies to a synthetic peptide corresponding to the expected extreme COOH-terminal portion of the precursor. Radioimmunoassay was used to identify and monitor the purification of peptides in porcine antral mucosa. Two peptides (I and II) were isolated to homogeneity by steps involving gel filtration, ion exchange, and reversed-phase high performance liquid chromatography. The two co-eluted on gel filtration but were separated on anion-exchange chromatography. The more acidic peptide (II) was less hydrophobic on high performance liquid chromatography. Automated gas-phase microsequencing revealed the less acidic peptide (I) to have the sequence of porcine preprogastrin 96-104 (SAEEGDQRP); it would be produced by tryptic-like cleavage of Arg95-Ser96. The second peptide did not yield a phenylthiohydantoin-derivative on the first cycle but thereafter it sequenced as the first peptide (i.e. -AEEGDQRP). Incubation in alkali liberated almost equimolar amounts of phosphate from peptide II but not from I. In addition, alkaline phosphatase liberated phosphate and converted the acidic peptide to the less acidic one. The results suggest that serine in the first position is phosphorylated in peptide II but not I. The tripeptide -Ser(P)-Ala-Glu- also occurs in adrenocorticotropic hormone; this tripeptide is a substrate for physiological casein kinase. Potential phosphorylation sites occur at comparable positions in the precursors of a number of regulatory peptides.
Calcitonin gene-related peptide and substance P in afferents to the upper gastrointestinal tract in the rat.
Combined immunohistochemistry and retrograde tracing was used to investigate the origin of the sensory calcitonin gene-related peptide (CGRP) innervation of the rat stomach. Up to 85% of spinal gastric afferents (T6-L1) contained CGRP immunoreactivity compared with less than 6% of vagal gastric afferents. By comparison substance P immunoreactivity occurred in about 50% of spinal gastric afferents and less than 2% of vagal afferents. The vagal afferents to the oesophagus were 14 and 26% substance P- and CGRP-immunoreactive respectively. The results suggest an important role for CGRP in gastric spinal afferents.
Intramural distribution of Met5-enkephalin-Arg6-Gly7-Leu8 in sphincter regions of the human gut.
The intramural distribution of Met5-enkephalin-Arg6-Gly7-Leu8 (MERGL) was studied in the oesophago-cardiac, pyloric, ileo-caecal and sigmoid-recto-anal regions of the human digestive tract. Serial samples encompassing each area were separated into mucosa, submucosa and muscularis externa and extracted for radioimmunoassay. Comparatively low levels of MERGL immunoreactivity were measured throughout the cardiac junction. Conversely, a remarkable peak of MERGL concentration was detected at the pyloric junction, in both submucosa and muscularis. A progressive decrease in tissue levels of the same peptide, most evident in the submucosa, was detected on the proximal side of the ileo-caecal region. In the distal sigmoid colon and rectum MERGL concentrations showed a rapid decline, down to very low levels in the anal canal. The results may suggest the involvement of an enkephalinergic mechanism in the control of the human pylorus.
Isolation and characterization of the intact gastrin precursor from a gastrinoma.
Antibodies to the extreme C-terminal region of human progastrin have been used to monitor the isolation of high-Mr immunoreactive material in a gastrinoma extract. Microsequence analysis of the product revealed amino acid residues in the first 18 positions corresponding to those predicted from the cDNA sequence for preprogastrin starting at position 22; the sequence and immunochemical data together allow the identification of this material as intact progastrin. Implications for gastrin biosynthesis are discussed.
Segregation of FMRF amide-immunoreactive efferent fibers from NPY-immunoreactive amacrine cells in goldfish retina.
Immunocytochemical studies were conducted on goldfish to determine whether a retinal efferent fiber system, immunoreactive to the tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), might contain instead a substance similar to one of the 36-amino acid pancreatic polypeptides, the C-terminus of which is similar to FMRFamide. Our results demonstrate the presence of two separate peptidergic systems, one containing FMRFamide-like, and the other pancreatic polypeptide-like peptides. Antisera to FMRF amide reveal the efferent fibers, whose axons exit the optic nerve and terminate in layer 1 of the inner plexiform layer, as previously described. Antisera to porcine neuropeptide Y, and to avian and bovine pancreatic polypeptides label a sparse population of putative amacrine cell bodies and a dense fiber plexus in layers 1, 3, and 5 of the inner plexiform layer. Based on intensity of staining, this amacrine cell peptide appears to be most similar to neuropeptide-Y. Radioimmunoassay and immunocytochemical staining of retinas in which the efferent fiber peptide was depleted by optic nerve crush confirm in large part the observation that the two peptide systems are distinct. However, there is some cross-recognition of the FMRF amide-like tissue antigen by pancreatic polypeptide antibodies. Double-label studies with antisera to tyrosine hydroxylase and neuropeptide-Y indicate that the pancreatic polypeptide antigen is not co-localized with catecholamines.
Progastrin in antral mucosa.
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Comparison of the metabolism of sulfated and unsulfated heptadecapeptide gastrin in humans.
The metabolism of synthetic human sulfated heptadecapeptide gastrin (G-17) was studied in normal human volunteers. Plasma concentrations were measured by radioimmunoassay using antibodies specific for intact G-17, and for the C- and N- terminus of G-17, during and after infusion of both sulfated and unsulfated G-17. With all three antibodies, plasma concentrations at a steady state were higher during infusion of sulfated compared with unsulfated G-17. In addition, the half-life in plasma measured by the three antibodies was two to five times higher for sulfated G-17 compared with unsulfated G-17. The half-life measured by N-terminal-specific antibodies was greater than that with antibodies specific for C-terminal or intact G-17. The difference was accounted for by the production during infusion of N-terminal fragments of relatively long half-life. The pattern of fragments generated during infusion of sulfated G-17 resembled that during unsulfated G-17 infusion, but there was no evidence of desulfation in the systemic circulation. The results indicate that in humans, sulfation protects G-17 from metabolism.
Peptides of the gut and brain: the cholecystokinins.
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Reflex decreases in intragastric pressure in response to cholecystokinin in rats.
The actions of intravenous sulfated cholecystokinin octapeptide (CCK-8) on intraluminal pressure in the body of the stomach were studied in urethan-anesthetized rats. There was a dose-related decrease in pressure in response to CCK-8 over the range 0.3-33 pmol. Bilateral cervical vagotomy alone reduced the response to CCK-8 and together with splanchnic section abolished it. Hexamethonium also reduced the response. Vagotomy did not change the response to CCK-8 in hexamethonium-treated rats, but celiac ganglionectomy abolished it. Guanethidine and phentolamine, but not propranolol, significantly decreased the response to CCK-8; subsequent vagotomy abolished the response. Similarly, depletion of tissue catecholamines by pretreatment with 6-OH dopamine, reserpine, or celiac ganglionectomy together with vagal section abolished the effect of CCK-8. It concluded that CCK-8 decreases mean intragastric pressure in the rat by pathways involving both vagal and splanchnic nerves. The splanchnic pathway involves an alpha-adrenergic mechanism but is hexamethonium resistant. The vagal pathway is hexamethonium sensitive and nonadrenergic. Similar pathways may mediate the effect of CCK on gastric emptying.
The biosynthesis of regulatory peptides.
Regulatory peptides are synthesized in their cells of origin as large, usually inactive, precursors. The gene sequences encoding many peptides are now known. Although these indicate the primary structure of the precursor, it remains necessary to define the actual peptide products of cells expressing a particular gene. In several instances, e.g., calcitonin gene-related peptide and preprotachykinin, it is known that there are alternative mRNA splicing mechanisms that generate different precursor molecules. Post-translational processing converts the precursor to smaller active products by cleavage of the chain and by modification to individual residues. The pathways of post-translational processing of the precursors may conveniently be studied using immunochemical methods. These allow detection of products that vary in chain length, or are modified by sulphation, phosphorylation, glycosylation, amidation, or N-terminal acetylation. Patterns of post-translational processing are commonly different between cells expressing the same gene. There are frequently, therefore, multiple active products that can be generated from a precursor, both within a single cell and in different cells expressing the same regulatory peptide gene. Examples are given to indicate the physiologic importance of these mechanisms and how they can be studied.
Biosynthesis of gastrin. Localization of the precursor and peptide products using electron microscopic-immunogold methods.
Antibodies to different peptides produced from the gastrin precursor have been used in light microscopic- and electron microscopic-immunogold studies of gastrinoma tissue and normal antral mucosa. Antibodies to the C terminus of progastrin, which are known to react with the intact precursor, revealed immunoreactive material in the rough endoplasmic reticulum, Golgi region, and electron-dense granules in gastrinoma cells. In normal antrum these antibodies again revealed the Golgi region and a population of electron-dense granules. Other antibodies that react with the products of progastrin processing, but not the precursor, e.g., C-terminal and N-terminal gastrin 17 specific antibodies, revealed only granules. In addition to electron-dense granules already mentioned, the latter antibodies also revealed electron-lucent and intermediate granule populations, which in antrum were the major granule types. It is proposed that the intact precursor occurs in rough endoplasmic reticulum and Golgi; thereafter, in the immature electron-dense granules, and subsequently in electron-lucent granules, biosynthetic processing liberates gastrin 17 and gastrin 34, which are the major active products of gastrin gene expression.
Identification and localization of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis.
Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.
Antibodies to FMRF amide, and the related pentapeptide LPLRF amide, reveal two groups of immunoreactive peptides in chicken brain.
A radioimmunoassay has been developed for the chicken brain peptide, Leu-Pro-Leu-Arg-Phe-amide (LPLRF amide); this peptide was originally discovered because it reacts with antibodies to the molluscan neuropeptide FMRF amide. The present antibody to LPLRF amide reacts about twenty times less well with FMRF amide compared with LPLRF amide. Using radioimmunoassays employing antibodies raised against LPLRF amide and FMRF amide we have separated by gel filtration and HPLC several different immunoreactive peptides in acid alcohol extracts of chicken brain. When LPLRF amide was used as the assay standard one group of peptides reacted similarly with the two types of antibody; the other group, which was represented by a single major component, reacted at least 50 times better with FMRF amide antibodies compared with LPLRF amide antibodies. It seems, therefore, that in the avian central nervous system, and probably other vertebrates, there are several different groups of peptides immunochemically related to FMRF amide.
Isolation, structure and properties of the C-terminal flanking peptide of preprocholecystokinin from rat brain.
The C-terminal flanking peptide of preprocholecystokinin has been isolated from rat brain. Micro-sequence analysis revealed the primary structure: Ser-Ala-Glu-Asp-Tyr-Glu-Tyr-Pro-Ser. Arylsulphatase and mild acid hydrolysis suggested that both tyrosine residues are sulphated. The peptide was not active in bioassay systems that respond to CCK8; the significance of the conserved tripeptide Ser-Ala-Glu is discussed.
Does cholecystokinin-like immunoreactivity in rat primary sensory neurons represent calcitonin gene-related peptide?
Using immunohistochemistry, calcitonin gene-related peptide (CGRP)- and cholecystokinin (CCK)-like immunoreactivity (LI) were found in many of the same spinal and trigeminal ganglion cells and motoneurons in the spinal cord and hypoglossal nucleus, as well as in fibers with an overlapping distribution in the spinal cord (dorsal horn, bundle ventral to the central canal) and in the spinal trigeminal nucleus. CCK-LI in all these structures disappeared after preadsorption of CCK antisera with CGRP at 10(-4) M and almost completely at 10(-5) M. CCK peptide in concentrations up to 10(-4) M, on the other hand, did not influence CGRP staining. The present findings raise the possibility that some CCK-LI in primary sensory neurons in rat may represent CGRP or a similar peptide.
Identification of the C-terminal flanking peptide of preprocholecystokinin in rat brain by a novel radioimmunoassay.
The C-terminal flanking peptide of preprocholecystokinin (preproCCK) has been identified in extracts of rat brain using a novel radioimmunoassay. There is a single form of immunoreactive material on gel filtration, ion exchange and reversed-phase HPLC. The C-terminal preproCCK immunoreactivity had a similar pattern of distribution to CCK8 in different regions of rat brain. This assay should help in studies of neuronal CCK biosynthesis.