Svend Knuhtsen

Oxyntomodulin (glicentin-(33-69)): pharmacokinetics, binding to liver cell membranes, effects on isolated perfused pig pancreas, and secretion from isolated perfused lower small intestine of pigs

The pharmacokinetics of purified synthetic oxyntomodulin were studied after infusing it into euglycaemic pigs at two rates. The elimination of the peptide from plasma was characterized by two components, a fast one (t1/2 7.2 +/- 0.6 min) and a slow one (t1/2 20.4 +/- 3.8 min) (mean +/- S.E.M., n = 7). The metabolic clearance rate was independent of infusion rate (6.96 +/- 0.99 vs 7.44 +/- 0.98 ml/kg . min (mean +/- S.E.M., n = 7). The synthetic peptide bound to pig hepatic glucagon receptors, but with approximately 2% of the affinity of glucagon, and showed insulinotropic and somatostatinotropic effects when infused into isolated perfused pig pancreases at concentrations higher than 10(-10) M. A dose-dependent increase was also shown for pancreatic glucagon output. A naturally occurring peptide, identified as oxyntomodulin by gel filtration and HPLC, was released into the circulation from the pig lower small intestinal mucosa upon intraluminal administration of glucose, and represented 25 +/- 3.8% of the secreted glucagon-like immunoreactivity. 11 +/- 2.3% of the secreted glucagon-like immunoreactivity was indistinguishable from glucagon itself upon gel filtration; thus at least 36% of the glucagon-like immunoreactivity secreted from the intestinal mucosa is already in an active form.

Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion

Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas.

Gastrin-Releasing Peptide in the Porcine Pancreas

The presence of gastrin-releasing peptide (GRP) was studied in extracts of porcine pancreata. Gel filtration and high-pressure liquid chromatographic profiles of these extracts as monitored with both C-terminally and N-terminally directed radioimmunoassays against GRP showed pancreatic GRP to consist of one main form, namely the 27-amino acid peptide originally extracted from porcine stomach, and small amounts of a C-terminal fragment identical with the C-terminal 10-amino acid peptide. Gastrin-releasing peptide-like immunoreactivity released from the isolated perfused porcine pancreas during electrical vagal stimulation was shown by gel filtration to consist of the same two forms. By use of immunocytochemical techniques employing an antiserum directed against its N terminus, GRP was localized to varicose nerve fibers in close association with the exocrine tissue of the porcine pancreas in particular. Some fibers were found penetrating into pancreatic islets also. Immunoreactive nerve cell bodies as well as fibers were found within intrapancreatic ganglia. The potency of GRP in stimulating exocrine as well as endocrine secretion from the porcine pancreas, its presence in close contact with both acini and islets, and its release during vagal stimulation indicate that GRP may have a role in the parasympathetic regulation of endocrine and exocrine secretion from the pig pancreas.

Autonomic nervous control of the endocrine secretion from the isolated, perfused pig pancreas

The effect of electrical stimulation of the splanchnic and the vagus nerve supply to isolated, perfused pig pancreas on the secretion of insulin, glucagon and pancreatic polypeptide (PP) was investigated. Functional integrity of the autonomic nerve supply was assessed by the effect of nerve stimulation on vascular resistance and exocrine secretion. Splanchnic nerve stimulation increased glucagon and PP output (2 to 3-fold) and inhibited insulin output (by 42%). Propranolol abolished the effect on PP and glucagon secretion, but did not affect the inhibition of insulin secretion. Phenoxybenzamine abolished the inhibition of insulin secretion, reduced the effect on glucagon secretion and enhanced the effect on pancreatic polypeptide secretion. Combined alpha- and beta-adrenergic blockade abolished all effects of splanchnic nerve stimulation. Vagus nerve stimulation increased the secretion of all 3 hormones (PP: up to 30-fold, insulin and glucagon: 3 to 5-fold). The effect on insulin and PP-secretion was mimicked by acetylcholine at 10(-7)-10(-6) M, whereas glucagon secretion was inhibited. The effect of vagus nerve stimulation on insulin and PP secretion was augmented by physostigmine, and inhibited (but not abolished) by atropine at 10(-7)-10(-6) M. The effect on glucagon secretion was inhibited by physostigmine and unaffected by atropine. It is concluded that all of the effects of splanchnic nerve stimulation on insulin and PP secretion can be explained by interactions of norepinephrine with excitatory beta-receptors on PP-cells and inhibitory receptors on the insulin cells. Both cell types are also stimulated via muscarinic cholinoceptors, but the partial atropine resistance suggests that other transmitters participate in vagal activation. The nervous regulation of glucagon secretion is complex and may involve the peptidergic innervation of the pancreatic islets.

The effect of gastrin-releasing peptide on the endocrine pancreas.

The 27-amino acid peptide gastrin releasing peptide (GRP-(1-27] was infused at 4 dose levels (0.01, 0.1, 1.0, and 10 nM) into the arterial line of the isolated perfused porcine pancreas. Infusions were performed at 3 different perfusate glucose levels (3.5, 5.0, and 8.0 mM) and at two levels of amino acids (5 and 15 mM). GRP-(1-27) stimulated insulin and pancreatic polypeptide secretion and inhibited somatostatin secretion in a dose-dependent manner. Glucagon secretion was unaffected by infusion of GRP under all circumstances. The effect of GRP-(1-27) on insulin secretion was enhanced with increasing perfusate glucose levels, whereas the effects upon somatostatin and pancreatic polypeptide secretion were independent of perfusate glucose levels. The responses to GRP were unaffected by elevation of the concentration of amino acids in the perfusate. The effects of GRP were unaffected by atropine at 10(-6) M. The localization of GRP within the porcine pancreas, its release during electrical stimulation of the vagus nerve, and its potent effects upon pancreatic endocrine secretion make it conceivable that the peptide participates in parasympathetic regulation of pancreatic endocrine secretion.

Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

BackgroundThe pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia.MethodsCyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry.ResultsOf four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (pu2009=u20090.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups.ConclusionsOATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE2.

Role of gastrin-releasing peptide in neural control of pancreatic exocrine secretion

We studied the effect of electrical stimulation of the vagus nerves on the exocrine secretion of isolated perfused porcine pancreas before and after procedures that almost completely blocked the effects elicited by infusions of gastrin-releasing peptide (GRP): desensitization of the pancreas for GRP (by perfusion with high concentrations of GRP); administration of an antagonist of GRP action [D-Arg1, D-PrO2, D-Trp7,9, Leu11)-substance PI; and perfusion with Fab fragments of antibodies against GRP. Both desensitization and antagonist administration significantly (p < 0.01) inhibited the effect of vagus stimulation on pancreatic protein secretion (by 42.1 and 33%). The inhibitory effect of anti-GRP perfusion was less pronounced (22% inhibition, 0.05 >p < 0.1). The results support the notion that pancreatic, GRP-producing nerve fibers are involved in the neural control of pancreatic enzyme secretion.

The effect of vagal stimulation on the release of cholecystokinin in anaesthetized pigs.

The effect of electrical vagal stimulation on the release of cholecystokinin (CCK) was studied in nine anaesthetized pigs. Plasma CCK concentrations were measured radioimmunologically by means of an antiserum specific for the sulphated tyrosine region of CCK. Stimulation of both vagal nerves for 10 min induced an increase in CCK concentrations from 1.9 +/- 0.4 pmol/l to a peak value of 3.6 +/- 0.4 pmol/l in portal vein plasma and from 1.5 +/- 0.3 to 2.7 +/- 0.4 pmol/l in arterial plasma. Mean integrated increments during stimulation were 12.0 +/- 2.5 pmol/l/10 min (p less than 0.01) and 8.3 +/- 1.0 pmol/l/10 min (p less than 0.001), respectively. The results suggest a vagal innervation of the CCK cell in the upper small intestine.

Duodenal epithelial transport in functional dyspepsia: Role of serotonin

AIMnTo investigate functional duodenal abnormalities in functional dyspepsia (FD) and the role of serotonin (5-hydroxytryptamine, 5-HT) in mucosal ion transport and signalling.nnnMETHODSnDuodenal mucosal biopsies were obtained from 15 patients with FD and 18 healthy controls. Immunohistochemistry was used to study the number of 5-HT-containing cells and real-time polymerase chain reaction for expression of 5-HT receptors 1A, 1B, 2A, 2B, 3A, 3B, 3C, 3D, 3E, 4 and 7, as well as expression of the serotonin re-uptake transporter (SERT) gene SLC6A4 and tryptophan hydroxylase 1 (TPH1). Biopsies were mounted in Ussing chambers for evaluation of basal and 5-HT-stimulated short-circuit current (SCC).nnnRESULTSnConductance was lower in FD [42.4 ± 4.7 mS/cm(2) (n = 15) vs 62.5 ± 4.5 mS/cm(2) (n = 18), P = 0.005]. 5-HT induced a dose dependent rise in SCC in both FD (n = 8) and controls (n = 9), the rise was lower in FD (P < 0.001). Mean number of 5-HT stained cells per high power field was the same [34.4 ± 8.4 in FD (n = 15) and 30.4 ± 3.7 in controls (n = 18), P = 0.647]. The following genes were highly expressed: 5-HT receptor HTR3E, HTR4, HTR7, SERT gene (SLC6A4) and TPH1. Differences in expression levels were observed for HTR3E (higher expression in FD, P = 0.008), HTR7 (lower expression in FD, P = 0.027), SLC6A4 (higher expression in FD, P = 0.033) and TPH1 (lower expression in FD, P = 0.031).nnnCONCLUSIONnDuodenal ion transport in response to exogenous 5-HT is abnormal in FD patients and associated with high expression of the HTR3E receptor and the serotonin transporter.

The Role of Gastrin‐Releasing Peptide in Pancreatic Exocrine Secretion

Although the pancreas receives a dense supply of nerve fibers, the extent of neural control of pancreatic exocrine secretion has been difficult to assess. For a long time, emphasis has been on the endocrine control of secretion, nourished by the discovery of the rather large number of gut peptides that may influence pancreatic secretion and by the fact that exocrine denervation, e.g., truncal vagotomy, does not seem to bring about pancreatic insufficiency. However, it appears that there is an abundance of mechanisms controlling exocrine secretion,’ which means that seemingly “intact” secretory responses may be obtained even when one mechanism is bypassed. In addition, it appears that the organization of the pancreatic innervation rather closely resembles that of the intestinal innervation, for which the designation “little brain” has been used because of its complexity and because of its ability to generate true reflexes after extrinsic denervatiom2 It follows that long reflexes may not be essential for a certain level of neural control. Finally, the inaccessibility of the gland, its relatively low secretory volume and the viscosity of the juice secreted present technical problems that may have discouraged investigators from attempting to examine the extent of neural control.’ However, as recently reviewed,’.’ there is ample evidence for the importance of the neural control of pancreatic exocrine secretion. In rats, cats, and pigs, electrical stimulation of the vagus nerves elicits pancreatic secretory responses that vary between 40 and 100% of the maximal responses to secretin and/or cholecystokinin.’ Reflex stimulation of pancreatic secretion may be elicited by sham feeding in many mammalian species, including dogs and human^.^ Although pancreatic insufficiency does not result from vagotomy, this operation nevertheless profoundly changes pancreatic ~ecre t ion .~ Similarly, in the dog, the muscarinic cholinergic antagonist, atropine, abolishes important secretory responses to various components of meal ingestion;’ and cholinergic agonists potently stimulate in particular the secretion of pancreatic enzymes.’” It has been known for some time that some of the effects of nerve stimulation are resistant to the inhibitory effect of atropine in cats and pigs?-’ Also, the secretory effects of sham feeding in humans are to a great extent resistant to the action of anticholinergic drugs.4 Hexamethonium, the nicotinic receptor antagonist, effectively blocks the effects of vagal stimulation, suggesting that such effects are transmitted through cholinergic nicotinic receptors in autonomic ganglia? Possibly, therefore, the second neuron is non-cholinergic. It is also believed to be non-adrenergic on the basis of studies with adrenal blockers.’ Thus, a system of non-adrenergic,