Pancreatic polypeptide: identification of target tissues using an in vivo radioreceptor assay.

The definitive function of pancreatic polypeptide in mammalian physiology remains unknown. The identification of specific PP target tissues should be helpful to further investigations into the possible regulatory actions of this peptide. An in vivo radioreceptor assay was used in the rat to locate potential binding sites of I(125) bovine PP. In vitro, high concentrations of unlabeled hormone competitively inhibit binding to receptors by low concentrations of labeled hormone. In vivo studies showed that, in the presence of concentrated unlabeled pancreatic polypeptide, labeled PP distributes between the plasma and interstitial fluid. When excess unlabeled PP is replaced with saline in the companion animals, the labeled peptide appears to distribute in a volume that exceeds the combined plasma volume and interstitial fluid volume of the tissue. Using this in vivo receptor assay, the distribution volume that exceeds the anatomic extracellular volume has been identified as the receptor compartment. With this assay we demonstrated in the rat specific and displaceable PP binding to the ductus choledochus, duodenum, ileum, and adrenal gland. The NVV determined in the adrenal gland of experimental animals was 3.9 times greater than that found in the control group. Binding was rapid and was displaced only by excess unlabeled pancreatic polypeptide. Neither excess insulin nor excess neuropeptide Y significantly reduced this binding.

Neurohumoral control of the exocrine pancreas.

Recent advances in the study of pancreatic exocrine secretion are reviewed, with an emphasis on neurohumoral mechanisms. In the past year, cDNA for the human pancreatic sodium-bicarbonate cotransporter was cloned, and the expressed protein was localized to pancreatic acini and ductal cells. Recent information suggests that the cholecystokinin B receptor has a role in pancreatic amylase release. Further evidence supports the concept of a protease-sensitive negative feedback mechanism regulating pancreatic exocrine secretion. Study of the expression of the receptors responsible for the regulation of pancreatic function has proven fruitful in the determination of the molecular mechanisms of hormone signal transduction and desensitization. Studies of peptide 1, pituitary adenylate cyclase-activating peptide, and gastrin-releasing peptide have shown how these peptides participate in the regulation of pancreatic secretion and have provided information on intracellular signaling pathways obtained using rat pancreatic tumor cells. Neural regulation via cholinergic receptors in isolated pancreatic acini and the mechanisms responsible for other neurotransmitters, such as calcitonin gene-related peptide, histamine, and dopamine, are reviewed. This review highlights recent discoveries in the neurohumoral regulation of pancreatic exocrine secretion.

Endoscopic therapy of acute diverticular hemorrhage.

OBJECTIVE: Diverticular hemorrhage is a common cause of lower GI bleeding and can be diagnosed acutely during colonoscopy. However, whether early diagnosis leads to effective intervention remains controversial. The aim of this study was to evaluate whether urgent colonoscopic therapy is effective as acute and long term treatment for diverticular bleeding with stigmata of hemorrhage. METHODS: We reviewed the medical records of all patients who underwent endoscopic therapy for diverticular bleeding from January, 1994 to June, 2000 at Duke University Medical Center. Patients or their families were contacted to obtain complete follow-up including data on subsequent bleeding. RESULTS: We identified 13 patients who underwent colonoscopic hemostatic management for the treatment of acute diverticular bleeding. Therapy consisted of epinephrine injection and/or multipolar electrocoagulation. Five patients (38%) experienced early rebleeding, within 30 days of the index bleed, four of whom required surgery, and three patients (23%) had late rebleeding. There were no complications of endoscopic therapy. CONCLUSIONS: Endoscopic therapy can provide early hemostasis in some cases of acute diverticular hemorrhage. However, its value in preventing subsequent diverticular bleeding is unclear.

Direct percutaneous endoscopic jejunostomy with small bowel enteroscopy and fluoroscopy.

BACKGROUND: Approaches to the creation of a percutaneous jejunostomy (PEJ) include enteroscopy with jejunal transillumination, fluoroscopy with small bowel distension and tract dilation, and jejunal enteral tube placement through a percutaneous endoscopic gastrostomy. Although all have been successful, the combination of enteroscopy and fluoroscopy may improve visualization and the success of PEJ placement. This is a description of such a technique and its successful use in 7 patients. METHODS: The procedure was performed with the patient under conscious sedation in a manner similar to standard PEG placement. The proximal jejunum was visualized and a standard snare was passed though the enteroscope and was opened. A needle and guidewire were directed percutaneously though the snare by using fluoroscopic guidance. Under direct endoscopic visualization the snare was closed around the guidewire. A standard 20F push-type "gastrostomy" tube was passed over the guidewire and through the mouth and the dome seated in the jejunum. A bumper was passed externally over the tube and tightened at the skin. RESULTS: PEJ placement was successful in all 7 patients. The average length of the procedure was 40 minutes (range 22-64 minutes). There were no major complications. Mean follow-up was 124 days (range 28-308 days). Feeding tubes remained functional until removal (2), death (1), or surgical removal for an unrelated reason (1). Three tubes are still in use. CONCLUSIONS: Percutaneous endoscopic jejunostomy tube placement can be performed successfully with enteroscopy and fluoroscopy. This technique is safe and efficient and provides distal enteral nutritional support for patients in whom PEG cannot be used.

Provocative angiography in obscure gastrointestinal bleeding.

Obscure gastrointestinal (GI) bleeding is relatively common but difficult to manage. By definition, diagnosis of a specific etiology is particularly challenging. We report the diagnostic use of provocative angiography in a patient with recurrent obscure GI bleeding. Although provocative angiography led to localization of bleeding and allowed specific treatment (placement of a 2-mm long, 0.010-inch diameter platinum coil, resulting in cessation of bleeding for 2 months), ultimately, the use of provocative angiography delayed specific diagnosis in our patient. We conclude that provocative angiography is a potentially powerful adjunct in the management of obscure GI bleeding, but that caution is required when using it. Provocative angiography should be reserved for patients who have had adequate imaging studies with negative results.

Identification of tissue insulin receptors: use of a unique in vivo radioreceptor assay.

An in vivo radioreceptor assay for polypeptide hormones has been developed and applied to the identification of tissue insulin receptors. The theoretical basis for this assay is presented elsewhere in this issue. 125I-insulin and 131I-albumin were infused into male rats with increasing amounts of unlabeled insulin. Plasma samples were taken at 1-min intervals until the animals were killed at 5 min. Tissue samples were excised and weighed and the activity due to each isotope counted. By comparing the differential distribution of the labeled tracers and applying the results to a compartment model, the specific, displaceable binding of insulin to tissue receptors could be demonstrated. Binding was detected in the liver, muscle, fat, adrenal glands, pancreas, small intestines, and spleen.

Mice lacking the dopamine transporter display altered regulation of distal colonic motility.

The mechanisms by which dopamine (DA) influences gastrointestinal (GI) tract motility are incompletely understood and complicated by tissue- and species-specific differences in dopaminergic function. To improve the understanding of DA action on GI motility, we used an organ tissue bath system to characterize motor function of distal colonic smooth muscle segments from wild-type and DA transporter knockout (DAT -/-) mice. In wild-type mice, combined blockade of D(1) and D(2) receptors resulted in significant increases in tone (62 +/- 9%), amplitude of spontaneous phasic contractions (167 +/- 24%), and electric field stimulation (EFS)-induced (40 +/- 8%) contractions, suggesting that endogenous DA is inhibitory to mouse distal colonic motility. The amplitudes of spontaneous phasic and EFS-induced contractions were lower in DAT -/- mice relative to wild-type mice. These differences were eliminated by combined D(1) and D(2) receptor blockade, indicating that the inhibitory effects of DA on distal colonic motility are potentiated in DAT -/- mice. Motility index was decreased but spontaneous phasic contraction frequency was enhanced in DAT -/- mice relative to wild-type mice. The fact that spontaneous phasic and EFS-induced contractile activity were altered by the lack of the DA transporter suggests an important role for endogenous DA in modulating motility of mouse distal colon.

Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor.

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.

A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor.

The secretin receptor is a member of a structurally distinct class of G protein-coupled receptors designated as Class II. The molecular mechanisms of secretin receptor signal termination are unknown. Using transiently transfected HEK 293 cells expressing the secretin receptor, we investigated its mechanisms of desensitization. Binding of [125I]-secretin to plasma membranes of receptor-expressing cells was specific, with a Kd of 2 nM. Secretin evoked an increase in cellular cAMP with an EC50 of 0.4 nM. The response was maximal by 20 min and desensitized rapidly and completely. Immunoprecipitation of a functional, N-terminal epitope-tagged secretin receptor was used to demonstrate agonist-dependent receptor phosphorylation, with an EC50 of 14 nM. Pretreatment with protein kinase A or C inhibitors failed to alter secretin-stimulated cAMP accumulation. G protein-coupled receptor kinases (GRKs) are known to be involved in the desensitization of Class I G protein-coupled receptors; therefore, the effect of cotransfection of GRKs on secretin-stimulated cAMP signaling and phosphorylation was evaluated. GRKs 2 and 5 were the most potent at augmenting desensitization, causing a 40% reduction in the maximal cAMP response to secretin. GRK 5 also caused a shift in the EC50 to the right (p < 0.05). GRK 4 and GRK 6 did not alter dose-dependent signaling, and GRK 3 was intermediate in effect. Receptor phosphorylation correlated with desensitization for each GRK studied, whereas second messenger-dependent kinase phosphorylation appeared to be less important in secretin receptor signal termination. We demonstrate agonist-dependent secretin receptor phosphorylation coincident with profound receptor desensitization of the signaling function in HEK 293 cells, suggesting a role for receptor phosphorylation in this paradigm. Although GRK activity appears important in secretin receptor desensitization in HEK 293 cells, protein kinases A and C appear to play only a minor role. These results demonstrate that the GRK-arrestin system regulates Class II G protein-coupled receptors.