Timothy S. Gaginella

Effects of pancreatic polypeptide and vasoactive intestinal polypeptide on rat ileal and colonic water and electrolyte transportin vivo

Two gastrointestinal peptides, vasoactive intestinal polypeptide (VIP) and pancreatic polypeptide, suspected of being associated with symptoms of WDHA syndrome (pancreatic cholera) were tested on the rat small and large intestine for their effects on water and electrolyte transport. Intravenous infusion of VIP (14.3 μg/kg/hr) inhibited net absorption of water and electrolytes in the ileum and reversed net absorption to net secretion in the colon. In contrast, bovine pancreatic polypeptide (52 μg/kg/hr) did not inhibit absorption or stimulate secretion. These data indicate VIP causes colonic secretionin vivo, an effect previously shown onlyin vitro, and that bovine pancreatic polypeptide (at this dose) is not a secretagogue in the small or large intestine of the rat. Thus, while consistent with VIP being a contributory agent to the secretion of pancreatic cholera, the data do not support the notion that pancreatic polypeptide might be a causative agent in this syndrome.

Release of vasoactive intestinal polypeptide by electrical field stimulation of rabbit ileum

The release of vasoactive intestinal polypeptide (VIP) induced by electrical field stimulation (EFS) of rabbit ileum was studied in vitro. EFS parallel to the muscularis propria caused a significant increase in VIP concentration in the buffer bathing the serosal surface of full-thickness ileum. This effect was blocked by 10(-7) M tetrodotoxin. When circular and longitudinal muscle was removed, the amount of measurable VIP in the tissue decreased to about one-half that of full-thickness ileum, and EFS no longer caused release of VIP into the serosal or mucosal buffers. Our data indicate that EFS of rabbit ileum causes release of VIP, presumably form VIP-containing nerves present in the tissue. These results support the idea that VIP may be a physiological neuroregulator of intestinal function.

Octreotide inhibits increases in short-circuit current induced in rat colon by VIP, substance P, serotonin and aminophylline

We investigated the effect of octreotide (OCT), a stable somatostatin analog, (OCT) on changes in short-circuit current (Isc) induced by vasoactive intestinal peptide (VIP), aminophylline, serotonin (5-HT) and substance P. OCT significantly decreased basal Isc at a concentration of 10(-9) M; the maximum decrease in Isc was observed at 10(-6) M. OCT (10(-7) M) significantly inhibited the intestinal secretory response to all the secretagogues studied. The maximum Isc response was reduced when tissues were stimulated with VIP (184.9 +/- 18.0 vs. 119.7 +/- 14.1, P less than 0.05), 5-HT (135.1 +/- 14.4 vs. 79.5 +/- 13.4, P less than 0.05) and substance P (156.0 +/- 19.2 vs. 30.7 +/- 5.4, P less than 0.01). In the case of aminophylline, the concentration-response curve was shifted to the right but the maximum response was not reduced. Because VIP and aminophylline increase cAMP while 5-HT and substance P stimulate intestinal secretion principally by a calcium linked mechanism, we conclude that OCT inhibits Isc in rat colon by more than one mechanism.

Somatostatin and Analogues in the Treatment of VIPoma

Precedent for natural peptide therapy began with the use of insulin in the treatment of insulin-dependent diabetes mellitus in 1922.’ More recently, we have witnessed the use of interferon, interleukin-2, and tumor necrosis factor (TNF), all naturally occurring peptides in man. Most recently, the novel long-acting analogue of natural somatostatin has become available on a compassionate need basis for refractory, symptomatic endocrine tumors of the gastroenteropancreatic (GEP) axis. SMS,,,.,,, (Sandostatin) is a long-acting analogue of the naturally occurring somatostatin,_14.2 Its clinical and therapeutic impact in gut endocrinology and tumors of the GEP system is rapidly being recogni~ed.”~ The rationale for both the development and clinical use of a somatostatin analogue has been best demonstrated in the management of the VIPoma-watery diarrhea syndrome ( WDS).’*6 Somatostatin was initially isolated, sequenced, and synthesized by Brazeau and colleagues in 1973.’ Another form of a naturally occurring somatostatin is somatostatin-28. These two forms of somatostatin are localized in the hypothalamus, pancreas, stomach, and intestinal tract. The potential regulatory importance of these two natural forms of somatostatin has been recently reviewed: Since somatostatin’s discovery, much has been learned regarding its regulatory function. It is indeed a hormone in the classic sense, as it modulates inhibition of growth hormone via the hypothalamopituitary portal circulation. Further, because of its unique location in the pancreatic islet cell and in peptidergic nerve fibers, somatostatin is suggested to have an important role as both a paracrine and neurocrine substance? In addition to its well-known growth hormone inhibition properties, somatostatin inhibits the release or action of thyrotropin, insulin, glucagon, secretin, gastrin, cholecystokinin, gastric inhibitory polypeptide (GIP), pancreatic polypeptide, neurotensin, and motilin. Its nonendocrine actions include inhibition of gastric acid secretion and gastric emptying, pancreatic bicarbonate and enzyme release, gall bladder contraction, and splanchnic blood flow. Further, it attenuates intestinal motility. Both the endocrine and nonendocrine effects of somatostatin have been recently reviewed?.” It is of interest that in addition to its inhibitory action on VIP (discussed below), somatostatin has been shown to release VIP in brain cells by a cyclic AMP-mediated pathway?

Role of peptide radioimmunoassay in understanding peptidepeptide interactions and clinical expression of gastroenteropancreatic endocrine tumors

Peptide radioimmunoassay has become an important clinical and research tool in understanding the role of peptides in the pathophysiology of gut endocrine tumor syndromes. A gut peptide radioimmunoassay laboratory has been established for the diagnosis and clinical monitoring of endocrine tumors of the gastroenteropancreatic (GEP) system. Radioimmunoassay has enhanced our awareness that co-occurring peptide interactions may modify and ultimately influence the clinical expression of these tumors. Furthermore, it has helped develop a rationale for the use of prototype peptides such as somatostatin and its long-acting analogue Sandostatin (SMS 201-995) in the management of GEP tumors. This groups experience, as well as the experience of other investigators, is presented, and the clinical utility of peptide radioimmunoassay in the field of gut endocrinology is demonstrated.

Treatment of endocrine and nonendocrine secretory diarrheal states with Sandostatin

Endocrine tumors of the gastroenteropancreatic (GEP) axis elaborate excessive amounts of peptides that are potent intestinal secretagogues. The actions of these peptides on intestinal transport of water and electrolytes lead to the accumulation of fluid in the intestinal lumen and diarrhea. One of the most clinically relevant secretagogues is vasoactive intestinal polypeptide (VIP). Other relevant secretagogues elaborated from tumors are serotonin, prostaglandins, and kinins. Sandostatin (octreotide, Sandoz, Basle, Switzerland), a long-acting octapeptide analog of somatostatin, inhibits experimentally induced intestinal secretion and has been used successfully to treat patients with secretory diarrhea refractory to other pharmacotherapy. The effective dose is in the range of 50 to 200 micrograms, given subcutaneously two or three times daily. The mechanism for the inhibitory effect on secretion is not clearly understood but it appears to involve inhibition of the adenylate cyclase-cyclic adenosine monophosphate system as well as interference with calcium as an intercellular mediator of enterocyte secretion. A particularly interesting use of this drug has been to treat the watery diarrhea seen in patients with acquired immunodeficiency syndrome. It is also effective in other types of secretory diarrhea not associated with endocrine tumors. These include diabetic diarrhea, idiopathic secretory diarrhea of infancy, and high output ileostomy diarrhea.

In vivo identification of muscarinic receptors on rat colonic epithelial cells binding of [3H]-quinuclidinyl benzilate

Summary(1)We have demonstrated that intravenously administered [3H]-quinuclidinyl benzilate binds to rat colonic and ileal epithelial cells. The binding was prevented by pretreatment with atropine and QNB.(2)Binding was stereoselective in favor of dexetimide, the biologically more active optical isomer of benzetimide.(3)The results with intestinal epithelial cells were qualitatively the same as those obtained using heart and gut muscle as controls.(4)QNB inhibited pilocarpine-induced fluid accumulation in ligated gut segments.(5)The results support the concept that cholinergic receptors, which mediate intestinal secretion, exist on rat intestinal epithelial cell membranes.

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate.

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [3H]-quinuclidinyl benzilate ([3H]-QNB). Receptor related binding of [3H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, KD, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [3H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC50 values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [3H]-QNB binding. The levo isomer of QNB was about seventy times more effective at inhibiting binding than its dextro isomer while dextro benzetimide was greater than two thousand fold more active than levo benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [3H]-QNB binding sites in a stereoselective manner, the levo isomer being eighty-five times more potent than the dextro isomer.

Peptides of human immunodeficiency virus (HIV) evoke rat colonic electrolyte secretion inhibitable by the somatostatin analog octreotide

An integral transmembrane glycoprotein of the Human Immunodeficiency Virus (HIV) is gp 41. Five peptides (P1, P2, P3, P4, and P5) containing a conserved region of the gp 41 molecule have been synthesized. We tested P3, P4 and P5 for their effects on short-circuit current (Isc) across rat colonic mucosa. All three peptides increased the Isc; P5 was the most potent agonist. Serosal pretreatment of tissues with the chloride transport inhibitor, bumetanide (0.1 mM) or chloride replacement with gluconate, inhibited the response, suggesting that the increase in Isc was due to stimulation of active chloride secretion. The synthetic somatostatin analog octreotide (0.1 mM) also inhibited (P less than .05) the response to P5 (1 microM). The data provide a possible rationale for one aspect of the efficacy of octreotide in treating secretory diarrhea in patients with Acquired Immunodeficiency Syndrome (AIDS).

Binding of [3H]quinuclidinyl benzilate to intestinal mucus: An artifact in identification of epithelial cell muscarinic receptors

The widely used muscarinic receptor ligand [3H]quinuclidinyl benzilate ([3H]QNB) was found to bind in a site-specific but artifactual manner to rat intestinal mucus, obscuring specific binding to muscarinic receptors on intestinal epithelial cells. Atropine inhibited [3H]QNB binding to mucus with an apparent IC50 of 2.1 x 10(-7) M, compared to an IC50 of 1.4 x 10(-8) M obtained with a homogenate of intestinal epithelial cells. Unlabeled QNB also inhibited binding of [3H]QNB to mucus but the apparent IC50 (4 x 10(-7) M) was about 300-fold greater than the IC50 determined with a control tissue, heart muscle (IC50, 1.2 x 10(-9) M). [3H]QNB binding was saturable over the concentration range of 1-7 nM in the heart, with an apparent KD of 0.76 nM. As expected from the high IC50 for QNB in the mucus binding experiments, binding to mucus was not saturable over the 1-15 nM concentration range. Based on pH profiles and temperature dependency of binding, it seems unlikely that mucin, the primary component of mucus, was responsible for [3H]QNB binding to the mucus. The findings have implications for studies which involve binding of [3H]QNB in particular and other ligands in general to mucus-secreting epithelial tissues.