Biomedical subjects
Publications and source records attributed to P Robberecht.
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1. The activation of rat pancreatic adenylate cyclase by guanosine 5'-(beta-gamma-imido)triphosphate (p[NH]ppG) and GTP, and by the two gastrointestinal hormones pancreozymin (as C-terminal octapeptide) and secretin was correlated with the binding of [8-3H]guanosine 5'-(beta-gamma-imido)triphosphate to rat pancreatic plasma membranes. 2. The low basal adenylate cyclase activity was stimulated 17-fold by p[NH]ppG (after a 2 min lag period), 3,5-fold only by GTP, 21-fold by C-terminal octapeptide of pancreozymin, and 8-fold by secretin. GTP inhibited competitively the activation of adenylate cyclase by p[NH]ppG with a Ki,app almost identical with the Ka,app (0.3 micron). p[NH]ppG and GTP enhanced the stimulation by secretin more markedly than that by the C-terminal octapeptide of pancreozymin, leading to the same maximal activity. Both hormones suppressed the lag period of activation by p[NH]ppG. 3. The binding of [8-3H]p[NH]ppG was dependent on time, temperature and Mg2+ and it was also a saturable and reversible process. Scatchard plots with a concavity upward were linearized after co-addition of ATP, Mg2+ and an ATP-regenerating system that abolished low-affinity sites for p[NH]ppG without saturating higher affinity sites, GTP, ITP and UTP inhibited [8-3H]p[NH]ppG binding to the high-affinity sites in concentration ranges identical with those found for adenylate cyclase activation. Considerable binding of [8-3H]p[NH]ppG was still evident at 20 degrees C, but enzyme activation was not observed any more, except in the presence of hormones.
The previously described peptide material that reacts with antibodies to gastrin and is found in the central nervous system of various vertebrates is present in only the 100,000 X g pellet of postmortem human cerebral cortical grey matter. This immunoreactive material, extractable in boiling water, is biologically active on rat pancreatic preparations. On the basis of size, charge, immunological specificity, and patterns of biological activity, most of this material is closely related to the COOH-terminal octapeptide of cholecystokinin in its complete, sulfated biologically active form.
Plasma cyclic AMP levels were determined during a 40 minute secretin infusion (1 Cl.U kg-1h-1) followed by a 40 minute combined secretin (1 Cl.U kg-1h-1) caerulein (75 ng kg-1h-1) infusion. In nine healthy subjects, both secretin alone and secretin in combination with caerulein did not affect plasma cyclic AMP levels. The same was observed in six patients with chronic pancreatitis. By contrast, in patients suffering from liver disease (nine cases) or extrahepatic cholestasis (six cases), secretin elicited large increases in plasma cyclic AMP concentration; the mean values attained being, respectively, seven and four times higher than before the infusion. On the other hand, increases in plasma cyclic AMP 10 minutes after a bolus injection of glucagon (1 mg) were four times lower in the liver disease group as compared to the controls. The results reported here suggest that the liver plays a major role in the degradation of plasma cyclic AMP produced by target tissues responding to secretin, and in the release of cyclic AMP under glucagon. Liver disease reduce the capacity of the liver to clear cyclic AMP from the blood. The pancreas does not contribute significantly to the cyclic AMP in the blood.
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Wheat germ agglutinin, but not concanavalin A or soybean lectin, inhibited the basal-and stimulated-adenylate cyclase activity which was present in a plasma membrane preparation from the rat pancreas. The inhibition by wheat germ agglutinin was rapid and sustained. It was of the non-competitive type and never exceeded 20% for Gpp (NH) p- and NaF-stimulated adenylate cyclase activity. The inhibition of secretin-stimulated activity was also non-competitive but more pronounced (57% inhibition at a wheat germ agglutinin concentration of 20 microgram/ml). For the C-terminal octapeptide of cholecystokinin-pancreozymin (OC-PZ)-stimulated cyclase, the inhibition amounted to 68% and was of a mixed type (both competitive and non-competitive). This last observation might be explained by the competitive inhibition exerted by wheat germ agglutinin on the binding of peptides of the OC-PZ family to their membrane specific receptors. The various inhibitory effects of wheat germ agglutinin were completely suppressed by incubating the membranes in the presence of ovomucoid, a N-acetyl-D-glucosamine rich glycoprotein. The possible functional implication of these results is discussed.
The pancreatic secretion of 6 normal human volunteers was collected by endoscopic cannulation of the main pancreatic duct. The appearance of newly synthesized proteins was monitored at 1-min intervals after labeling with [75Se]methionine. The minimum transit time of these proteins from their site of synthesis to the acinar lumen was 36 +/- 8 min. Stimulation of protein secretion by a rapid intravenous injection of caerulein (40 ng per kg), 1 hr after [75Se]methionine administration, greatly decreased (by 73% on an average) the specific radioactivity of the discharged proteins. These data support the concept of a functional heterogeneity of proteins secreted by the human pancreas.
1. The subcellular distribution of adenylate cyclase activity in rat pancreatic homogenates was examined after differential centrifugation. Divalent cations exerted significant effects on this distribution. In addition, the ratio of adenylate cyclase activities in the presence of the C-terminal octapeptide of cholecystokinin-pancreozymin and secretin was lower in the crude 'mitochondrial' fraction than in 'microsomal' fractions. This difference was due to the lability of cholecystokinin-pancreozymin receptors compared to secretin receptors. The Km,app of activation was affected more than the V by this lability. Such a degradation of cholecystokinin-pancreozymin receptors was markedly delayed by isolation and storage in the presence of a phospholipid mixture. 2. A simple, reasonably rapid (6 h), and easily reproducible method was developed to prepare a stable semi-purified plasma membrane fraction, characterized by a 10-fold increase in the specific activity of adenylate cyclase with respect to the whole homogenate. At variance with data obtained on crude subcellular fractions, the V of adenylate cyclase activity observed in this preparation, under maximal concentration of the C-terminal octapeptide of cholecystokinin-pancreozymin, was higher than that obtained with secretin or the vasoactive intestinal polypeptide.
Secretin and vasoactive intestinal peptide (VIP), but not glucagon, stimulate accumulation of cyclic AMP in dispersed guinea pig pancreatic acinar cells. Secretin stimulated cellular accumulation of cyclic AMP by interacting with a single class of high affinity receptors. On the other hand, the dose-response curve for VIP-stimulated cellular cyclic AMP was biphasic and reflected interaction of this peptide with two classes of receptors. Results obtained with synthetic fragments of VIP and secretin indicate that the receptor having a high affinity for VIP has a low affinity for secretin, interacts with, but does not distinguish among, secretin, secretin 5-27 and [6-tyrosine] secretin or among secretin 14-27, VIP 14-28, VIP 15-28, and increases cellular cyclic AMP when occupied by VIP, but not when occupied by secretin, [6-tyrosine] secretin, or secretin 1-14. The receptor having a low affinity for VIP has a high affinity for secretin, interacts with and distinguishes among secretin, secretin 5-27, and [6-tyrosine] secretin, interacts with secretin 14-27 but not with VIP 14-28 or VIP 15-28, and increases cellular cyclic AMP when occupied by VIP, secretin, [6-tyrosine] secretin, or secretin 1-14.
Bombesin (a tetradecapeptide), the C-terminal nonapeptide of bombesin (bombesin-NP), and litorin (a parent nonapeptide), each stimulated amylase secretion from rat pancreatic fragments. These responses were not affected by atropine. The concentrations that produced half-maximal stumulation of secretion were 0.25 nM for bombesin, 0.30 nM for bombesin-NP, and 0.07 nM for litorin, as compared to 0.12 nM for caerulein and 0.80 muM for the cholinergic agent carbamylcholine. When used at maximal concentrations, bombesin, bombesin-NP, and litorin showed no action on cyclic AMP levels in the presence of 5 mM theophylline. By contrast, caerulein and secretin increased cyclic AMP levels by 27 and 208%, respectively. Bombesin, bombesin-NP, and litorin did not activate adenylate cyclase in a purified pancreatic plasma membrane preparation, whereas caerulein and secretin increased this activity 20 and 16-times, respectively...
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The pattern of amylase, lipase, and chymotrpsinogen content found in pancreatic exocrine tissue surrounding the islets of Langerhans (periinsular halos) differs from that of the rest, or teleinsular part, of the pancreas. Such a topographic partition of secretory function may play a role in the regulation of pancreatic juice composition.
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Pancreatic secretion of total protein and of three types of hydrolases (amylase, lipase, and thymotrypsinogen(s) was determined in human subjects free of gastrointestinal disease. Pure pancreatic juice was collected by canulation of Wirsung's duct under duodenoscopy. Rapid intravenous injection of pancreozymin (1 Crick-Harper-Raper U per kg) stimulated secretion of total protein and hydrolases within 1 min. This stimulation was of short duration and had a half-life of 70 to 100 sec. Rapid intravenous injection of caerulein (20) ng per kg) induced a similar response. Parallel variations in total protein, amylase, lipase, and chymotrypsinogen(s) were observed in the three experiments where complete enzyme analyses were performed.
Four-fold increases in cyclic AMP levels were observed 5 to 10 min after rat pancreatic fragments were incubated with 10-7 M secretin or 10-6 M vasoactive intestinal polypeptide (VIP), in addition to 10 mM theophylline. From dose-response curves it appears that, on a molar basis, the potency of secretin was 20 times higher than that of VIP. It is concluded that cyclic AMP is probably the intracellular messenger of both secretin and VIP in centroacinar cells. Pancreozymin, caerulein, and the C-terminal octapeptide of pancreozymin inhibited the production of cyclic AMP observed with secretin of VIP, suggesting that the first three peptides were acting at a binding site different from the agonists, but coupled with the same adenylate cyclase. In acinar cells, secretin was able to exert slight ecbolic effects, and was also able to potentiate the effect of maximal concentrations of pancreozymin, caerulein, or the C-terminal octapeptide of pancreozymin. There was no simple correlation between amylase output and cyclic AMP levels, and copious amylase secretion was elicited even at control levels of cyclic AMP. Glucagon was neither an agonist nor an antagonist of any of the other polypeptides tested.
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