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Background and Aims: Long-term treatment with H(+)-K(+)-adenotriphosphatase (ATPase) inhibitors, such as omeprazole or lansoprazole, for severe gastroesophageal reflux disease is now widely used. Whether such treatment will result in vitamin B12 deficiency is controversial. We studied whether long-term treatment with omeprazole alters serum vitamin B12 levels in patients with Zollinger-Ellison syndrome.Methods: In 131 consecutive patients treated with either omeprazole (n = 111) or histamine H2-receptor antagonists (n = 20), serum vitamin B12 and folate levels and complete blood counts were determined after acid secretion had been controlled for at least 6 months. These studies were repeated yearly. Serum vitamin B12 and folate levels were correlated with the type of antisecretory drug and the extent of inhibition of acid secretion.Results: The mean duration of omeprazole treatment was 4.5 years, and for H2-receptor antagonists 10 years. Vitamin B12 levels, but not serum folate levels or any hematological parameter, were significantly (P = 0.03) lower in patients treated with omeprazole, especially those with omeprazole-induced sustained hyposecretion (P = 0.0014) or complete achlorhydria (P < 0.0001). In 68 patients with two determinations at least 5 years apart, vitamin B12 levels decreased significantly (30%; P = 0.001) only in patients rendered achlorhydric. The duration of omeprazole treatment was inversely correlated with vitamin B12 levels (P = 0.013), but not folate levels. Eight patients (6%) developed subnormal B12 levels during follow-up.Conclusions: Long-term omeprazole treatment leads to significant decreases in serum vitamin B12 but not folate levels. These results suggest patients with Zollinger-Ellison syndrome treated with H(+)-K(+)-ATPase inhibitors should have serum vitamin B12 levels monitored. Furthermore, these results raise the possibility that other patients treated chronically with H(+)-K(+)-ATPase inhibitors may develop B12 deficiency. [ABSTRACT FROM AUTHOR]

Rat pancreatic acinar cells possess only the p21-activated kinase (PAKs), PAK4 of the group II PAK, and it is activated by gastrointestinal hormones/neurotransmitters stimulating PLC and by a number of growth factors. However, little is known generally of cAMP agents causing PAK4 activation, and there are no studies with gastrointestinal hormones/neurotransmitters activating cAMP cascades. In the present study, we examined the ability of VIP and secretin, which stimulate cAMP generation in pancreatic acini, to stimulate PAK4 activation, the signaling cascades involved, and their possible role in activating sodium-potassium adenosine triphosphatase (Na + ,K + -ATPase). PAK4 activation was compared with activation of the well-established cAMP target, cyclic AMP response element binding protein (CREB). Secretin-stimulated PAK4 activation was inhibited by KT-5720 and PKA Type II inhibitor (PKI), protein kinase A (PKA) inhibitors, whereas VIP activation was inhibited by ESI-09 and HJC0197, exchange protein directly activated by cAMP (EPAC) inhibitors. In contrast, both VIP/secretin-stimulated phosphorylation of CREB (pCREB) via EPAC activation; however, it was inhibited by the p44/42 inhibitor PD98059 and the p38 inhibitor SB202190. The specific EPAC agonist 8-CPT-2- O-Me-cAMP as well 8-Br-cAMP and forskolin stimulated PAK4 activation. Secretin/VIP activation of Na + ,K + -ATPase, was inhibited by PAK4 inhibitors (PF-3758309, LCH-7749944). These results demonstrate PAK4 is activated in pancreatic acini by stimulation of both VIP-/secretin-preferring receptors, as is CREB. However, they differ in their signaling cascades. Furthermore, PAK4 activation is needed for Na + ,K + ATPase activation, which mediates pancreatic fluid secretion. These results, coupled with recent studies reporting PAKs are involved in both pancreatitis/pancreatic cancer growth/enzyme secretion, show that PAK4, similar to PAK2, likely plays an important role in both pancreatic physiological/pathological responses. NEW & NOTEWORTHY Pancreatic acini possess only the group II p21-activated kinase, PAK4, which is activated by PLC-stimulating agents/growth factors and is important in enzyme-secretion/growth/pancreatitis. Little information exists on cAMP-activating agents stimulating group II PAKs. We studied ability/effect of cyclic AMP-stimulating agents [vasoactive intestinal polypeptide (VIP), secretin] on PAK4 activity in rat pancreatic-acini. Both VIP/secretin activated PAK4/CREB, but the cAMP signaling cascades differed for EPAC, MAPK, and PKA pathways. Both hormones require PAK4 activation to stimulate sodium-potassium adenosine triphosphatase activity. This study shows PAK4 plays an important role in VIP-/secretin-stimulated pancreatic fluid secretion and suggests it plays important roles in pancreatic acinar physiological/pathophysiological responses mediated by cAMP-activating agents.

p21-activated kinases (PAKs) are highly conserved serine/threonine protein kinases, which are divided into two groups: group-I (PAKs1-3) and group-II (PAKs4-6). In various tissues, Group-II PAKs play important roles in cytoskeletal dynamics and cell growth as well as neoplastic development/progression. However, little is known about Group-II PAK's role in a number of physiological events, including their ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth factors (GFs). We used rat pancreatic acini to explore the ability of GI hormones/neurotransmitters/GFs to activate Group-II-PAKs and the signaling cascades involved. Only PAK4 was detected in pancreatic acini. PAK4 was activated by endothelin, secretagogues-stimulating phospholipase C (bombesin, CCK-8, and carbachol), by pancreatic GFs (insulin, insulin-like growth factor 1, hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor), and by postreceptor stimulants (12-O-tetradecanoylphobol-13-acetate and A23187 ). CCK-8 activation of PAK4 required both high- and low-affinity CCK 1 -receptor state activation. It was reduced by PKC-, Src-, p44/42-, or p38-inhibition but not with phosphatidylinositol 3-kinase-inhibitors and only minimally by thapsigargin. A protein kinase D (PKD)-inhibitor completely inhibited CCK-8-stimulated PKD-activation; however, stimulated PAK4 phosphorylation was only inhibited by 60%, demonstrating that it is both PKD-dependent and PKD-independent. PF-3758309 and LCH-7749944, inhibitors of PAK4, decreased CCK-8-stimulated PAK4 activation but not PAK2 activation. Each inhibited ERK1/2 activation and amylase release induced by CCK-8 or bombesin. These results show that PAK4 has an important role in modulating signal cascades activated by a number of GI hormones/neurotransmitters/GFs that have been shown to mediate both physiological/pathological responses in acinar cells. Therefore, in addition to the extensive studies on PAK4 in pancreatic cancer, PAK4 should also be considered an important signaling molecule for pancreatic acinar physiological responses and, in the future, should be investigated for a possible role in pancreatic acinar pathophysiological responses, such as in pancreatitis. NEW & NOTEWORTHY This study demonstrates that the only Group-II p21-activated kinase (PAK) in rat pancreatic acinar cells is PAK4, and thus differs from islets/pancreatic cancer. Both gastrointestinal hormones/neurotransmitters stimulating PLC and pancreatic growth factors activate PAK4. With cholecystokinin (CCK), activation is PKC-dependent/-independent, requires both CCK 1 -R affinity states, Src, p42/44, and p38 activation. PAK4 activation is required for CCK-mediated p42/44 activation/amylase release. These results show PAK4 plays an important role in mediating CCK physiological signal cascades and suggest it may be a target in pancreatic acinar diseases besides cancer.

Bombesin-receptor-subtype-3 (BB3 receptor) is a G-protein-coupled-orphan-receptor classified in the mammalian Bombesin-family because of high homology to gastrin-releasing peptide (BB2 receptor)/neuromedin-B receptors (BB1 receptor). There is increased interest in BB3 receptor because studies primarily from knockout-mice suggest it plays roles in energy/glucose metabolism, insulin-secretion, as well as motility and tumor-growth. Investigations into its roles in physiological/pathophysiological processes are limited because of lack of selective ligands. Recently, a selective, peptide-antagonist, Bantag-1, was described. However, because BB3 receptor has low-affinity for all natural, Bn-related peptides, little is known of the molecular basis of its high-affinity/selectivity. This was systematically investigated in this study for Bantag-1 using a chimeric-approach making both Bantag-1 loss-/gain-of-affinity-chimeras, by exchanging extracellular (EC) domains of BB3/BB2 receptor, and using site-directed-mutagenesis. Receptors were transiently expressed and affinities determined by binding studies. Bantag-1 had >5000-fold selectivity for BB3 receptor over BB2/BB1 receptors and substitution of the first EC-domain (EC1) in loss-/gain-of affinity-chimeras greatly affected affinity. Mutagenesis of each amino acid difference in EC1 between BB3 receptor/BB2 receptor showed replacement of His(107) in BB3 receptor by Lys(107) (H107K-BB3 receptor-mutant) from BB2 receptor, decreased affinity 60-fold, and three replacements [H107K, E11D, G112R] decreased affinity 500-fold. Mutagenesis in EC1's surrounding transmembrane-regions (TMs) demonstrated TM2 differences were not important, but R127Q in TM3 alone decreased affinity 400-fold. Additional mutants in EC1/TM3 explored the molecular basis for these changes demonstrated in EC1, particularly important is the presence of aromatic-interactions by His(107), rather than hydrogen-bonding or charge-charge interactions, for determining Bantag-1 high affinity/selectivity. In regard to Arg(127) in TM3, both hydrogen-bonding and charge-charge interactions contribute to the high-affinity/selectivity for Bantag-1., (Published by Elsevier Inc.)

In pancreatic acinar cells, the Src family of kinases (SFK) is involved in the activation of several signaling cascades that are implicated in mediating cellular processes (growth, cytoskeletal changes, apoptosis). However, the role of SFKs in various physiological responses such as enzyme secretion or in pathophysiological processes such as acute pancreatitis is either controversial, unknown, or incompletely understood. To address this, in this study, we investigated the role/mechanisms of SFKs in acute pancreatitis and enzyme release. Enzyme secretion was studied in rat dispersed pancreatic acini, in vitro acute-pancreatitis-like changes induced by supramaximal COOH-terminal octapeptide of cholecystokinin (CCK). SFK involvement assessed using the chemical SFK inhibitor (PP2) with its inactive control, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3), under experimental conditions, markedly inhibiting SFK activation. In CCK-stimulated pancreatic acinar cells, activation occurred of trypsinogen, various MAP kinases (p42/44, JNK), transcription factors (signal transducer and activator of transcription-3, nuclear factor-κB, activator protein-1), caspases (3, 8, and 9) inducing apoptosis, LDH release reflective of necrosis, and various chemokines secreted (monocyte chemotactic protein-1, macrophage inflammatory protein-1α, regulated on activation, normal T cell expressed and secreted). All were inhibited by PP2, not by PP3, except caspase activation leading to apoptosis, which was increased, and trypsin activation, which was unaffected, as was CCK-induced amylase release. These results demonstrate SFK activation is playing a dual role in acute pancreatitis, inhibiting apoptosis and promoting necrosis as well as chemokine/cytokine release inducing inflammation, leading to more severe disease, as well as not affecting secretion. Thus, our studies indicate that SFK is a key mediator of inflammation and pancreatic acinar cell death in acute pancreatitis, suggesting it could be a potential therapeutic target in acute pancreatitis., (Copyright © 2016 the American Physiological Society.)

In a recent study we explored Group-1-p21-activated kinases (GP.1-PAKs) in rat pancreatic acini. Only PAK2 was present; it was activated by gastrointestinal-hormones/neurotransmitters and growth factors in a PKC-, Src- and small-GTPase-mediated manner. PAK2 was required for enzyme-secretion and ERK/1-2-activation. In the present study we examined PAK2's role in CCK and TPA-activation of important distal signaling cascades mediating their physiological/pathophysiological effects and analyzed its role in pathophysiological processes important in early pancreatitis. In rat pancreatic acini, PAK2-inhibition by the specific, GP.1.PAK-inhibitor, IPA-3-suppressed cholecystokinin (CCK)/TPA-stimulated activation of focal-adhesion kinases and mitogen-activated protein-kinases. PAK2-inhibition reversed the dual stimulatory/inhibitory effect of CCK/TPA on the PI3K/Akt/GSK-3β pathway. However, its inhibition did not affect PKC activation. PAK2-inhibition protected acini from CCK-induced ROS-generation; caspase/trypsin-activation, important in early pancreatitis; as well as from cell-necrosis. Furthermore, PAK2-inhibition reduced proteolytic-activation of PAK-2p34, which is involved in programmed-cell-death. To ensure that the study did not only rely in the specificity of IPA-3 as a PAK inhibitor, we used two other approaches for PAK inhibition, FRAX597 a ATP-competitive-GP.1-PAKs-inhibitor and infection with a PAK2-dominant negative(DN)-Advirus. Those two approaches confirmed the results obtained with IPA-3. This study demonstrates that PAK2 is important in mediating CCK's effect on the activation of signaling-pathways known to mediate its physiological/pathophysiological responses including several cellular processes linked to the onset of pancreatitis. Our results suggest that PAK2 could be a new, important therapeutic target to consider for the treatment of diseases involving deregulation of pancreatic acinar cells., (Published by Elsevier B.V.)

P-21-activated kinases (PAKs) are serine/threonine kinases comprising six isoforms divided in two groups, group-I (PAK1-3)/group-II (PAK4-6) which play important roles in cell cytoskeletal dynamics, survival, secretion and proliferation and are activated by diverse stimuli. However, little is known about PAKs ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth-factors. We used rat pancreatic acini to explore the ability of GI-hormones/neurotransmitters/growth-factors to activate Group-I-PAKs and the signaling cascades involved. Only PAK2 was present in acini. PAK2 was activated by some pancreatic growth-factors [EGF, PDGF, bFGF], by secretagogues activating phospholipase-C (PLC) [CCK, carbachol, bombesin] and by post-receptor stimulants activating PKC [TPA], but not agents only mobilizing cellular calcium or increasing cyclic AMP. CCK-activation of PAK2 required both high- and low-affinity-CCK1-receptor-state activation. It was partially reduced by PKC- or Src-inhibition, but not with PI3K-inhibitors (wortmannin, LY294002) or thapsigargin. IPA-3, which prevents PAK2 binding to small-GTPases partially inhibited PAK2-activation, as well as reduced CCK-induced ERK1/2 activation and amylase release induced by CCK or bombesin. This study demonstrates pancreatic acini, possess only one Group-I-PAK, PAK2. CCK and other GI-hormones/neurotransmitters/growth-factors activate PAK2 via small GTPases (CDC42/Rac1), PKC and SFK but not cytosolic calcium or PI3K. CCK-activation of PAK2 showed several novel features being dependent on both receptor-activation states, having PLC- and PKC-dependent/independent components and small-GTPase-dependent/independent components. These results show that PAK2 is important in signaling cascades activated by numerous pancreatic stimuli which mediate their various physiological/pathophysiological responses and thus could be a promising target for the development of therapies in some pancreatic disorders such as pancreatitis., (Copyright © 2015. Published by Elsevier B.V.)

For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects., (Published by Elsevier B.V.)

The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCK(A)-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125(FAK) and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth)., (Published by Elsevier B.V.)

Well differentiated neuroendocrine tumors (NETs) of the stomach (gastric carcinoid tumors) are observed more often, with a tenfold increase in the US in the last 30 - 35 years, and the prognosis has improved greatly in that time. Nowadays most carcinoids of the stomach are diagnosed at an early stage. Four types of gastric NETs have been proposed and recognition of the type is important for defining the diagnostic approach and treatment. Often gastric NETs (especially type 1) are found incidentally during a gastroscopy performed for other reasons; most of these NETs are smaller than 20 mm in size. Conservative management and endoscopic surveillance is adequate for well differentiated, multifocal gastric carcinoids (type 1 or type 2 gastric NETs) that are less than 10 - 20 mm in diameter, unless they show angioinvasion, infiltrate the muscular wall, or have a proliferation rate above 2 %. Endoscopic ultrasound is the method of choice to determine tumor size and depth of infiltration. It is essential to distinguish between multifocal (types 1 and 2) and unifocal type 3 or type 4 gastric NETs, since surgery is indicated for type 3 gastric NETs larger than 10 mm in diameter and for poorly differentiated (localized) neuroendocrine gastric carcinomas (type 4 gastric NET). For optimal management, the type, biology, and stage of the tumor as well as the individual situation of the patient must be considered. Most patients with well differentiated gastric NETs can be treated conservatively and be followed up with endoscopic surveillance., ((c) Georg Thieme Verlag KG Stuttgart . New York.)

We have previously demonstrated that pregnant ovine endometrium expresses the gastrin-releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP 1-46, the primary product of this gene in the pregnant endometrium. Full thickness 125-140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 muBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP 1-46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP(1-46) would require preferential cleavage at the Glu-Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP(18-27) and GRP(1-27) in other tissues. GRP 1-46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP 1-46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP 1-46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR., ((c) 2009 Elsevier Inc. All rights reserved.)

Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.

Numerous studies have established that gastroenteropancreatic endocrine tumours (carcinoids and pancreatic endocrine tumours) resemble a number of other tumours in overexpressing somatostatin receptors that can bind octreotide or lanreotide with high affinity (i.e. possess sst2/sst5 receptors). Recent studies report that radiolabelled somatostatin analogues can be used to image these tumours (somatostatin receptor scintigraphy) and may be useful for peptide-directed radionuclide therapy. In this paper the evidence is reviewed that has led to establishing somatostatin receptor scintigraphy as the initial imaging modality of choice in patients with gastroenteropancreatic tumours. This conclusion is based on an understanding of the results with conventional imaging modalities (ultrasound, computed tomographic scan, magnetic resonance imaging, angiography) available prior to somatostatin receptor scintigraphy and the results of studies demonstrating the sensitivity and specificity of somatostatin receptor scintigraphy. Most important in this regard are the results of studies that have assessed the use of somatostatin receptor scintigraphy on clinical management. Each of these areas is reviewed in this paper.

The receptor of hepatocyte growth factor (HGF), c-met induces different physiological responses in several cell types. Little is known about the role of HGF in exocrine pancreas. However, abnormal HGF signaling has been strongly implicated in pancreatic tumorigenesis and association of HGF with pancreatitis has been demonstrated. We have studied the presence of c-met and activation of their intracellular pathways associated in rat pancreatic acini in comparison with cholecystokinin (CCK) and epidermal growth factor (EGF). C-met expression in rat exocrine pancreas was identified by immunohistochemistry and immunoprecipitation followed by Western analysis. Tyrosine phosphorylation of c-met is strongly stimulated as well as kinase pathways leading to ERK1/2 cascade. HGF, but not CCK or EGF, selectively caused a consistent increase in the amount of p85 regulatory subunit of PI3-K present in anti-phosphotyrosine immunoprecipitates. Downstream of PI3-K, HGF increased Ser473 phosphorylation of Akt selectively, as CCK or EGF did not affect it. HGF selectively stimulated tyrosine phosphorylation of phosphatase PTP1D. HGF failed to promote the well-known CCK effects in pancreatic acini such as amylase secretion and intracellular calcium mobilization. Although HGF shares activation of ERK1/2 with CCK, we demonstrate that it promotes the selective activation of intracellular pathways not regulated by CCK or EGF. Our results suggest that HGF is an in vivo stimulus of pancreatic acini and provide novel insight into the transduction pathways and effects of c-met/HGF in normal pancreatic acinar cells.

Until recently, the association of primary hyperparathyroidism and gastric carcinoid, with or without hypergastrinaemia, had been considered an incomplete form of multiple endocrine neoplasia type 1. This is because it seemed unlikely that the rare joint appearance of these diseases could occur only by chance. It is now possible to evaluate the pathogenetic involvement of the multiple endocrine neoplasia type 1 gene in many, apparently sporadic, clinical conditions. This is a case report of a female mimicking multiple endocrine neoplasia type 1 due to the presence of hyperparathyroidism, gastric carcinoid, and hypergastrinaemia. However, involvement of the MEN-1 gene (exons 2-10) was not detected, whereas hypergastrinaemia was attributed to a chronic atrophic gastritis.

A subset of gastrointestinal neuroendocrine tumours (carcinoids and pancreatic endocrine tumours) show aggressive growth. Early identification of this subset is essential for management; however, clinical, laboratory and histologic features frequently fail to achieve this. Currently, there is an increased understanding of the molecular pathogenesis/changes in neuroendocrine tumours and this may identify important prognostic factors and possibly, new treatments. Recent findings and progress in this area are briefly reviewed in this article.

Background: Enterochromaffin-like cell hyperplasia of the gastric body mucosa occurs in hypergastrinaemic conditions such as atrophic body gastritis and Zollinger-Ellison syndrome. However, the time course of change or factors involved are not known., Aims: To compare the rate of change of enterochromaffin-like cell proliferation in patients with atrophic body gastritis and Zollinger-Ellison syndrome., Patients: From a consecutive series of atrophic body gastritis and Zollinger-Ellison syndrome patients, studied at the time of first diagnosis, 10 atrophic body gastritis (4 with pernicious anaemia) and 14 Zollinger-Ellison syndrome (4 with multiple endocrine neoplasia type 1) patients were followed-up for a median time of 48 months., Methods: At entry and during follow-up patients underwent: plasma gastrin determination, endoscopic sampling of body mucosa for qualitative assessment of enterochromaffin-like cell hyperplasia pattern and degree of glandular atrophy, qualitative and morphometric analyses of body mucosa endocrine cells., Results: At time of diagnosis, enterochromaffin-like cell lesions were more severe in atrophic body gastritis than in Zollinger-Ellison syndrome. During follow-up, no significant variations were observed in gastrin values, enterochromaffin-like cell patterns and grade of body mucosa atrophy in atrophic body gastritis. In contrast, gastrin levels were significantly increased [median 1200 (235-2625) vs 1947 (225-5200) pg/ml; p<0.001)] as was total volume density of enterochromaffin-like cells [median 1.60 (0.53-4.06) vs 3.18 (1.35-21.13)% of mucosal epithelial component; (p<0.005)] in Zollinger-Ellison syndrome. Micronodular hyperplasia of enterochromaffin-like cells, present in only one patient at diagnosis, was observed in 8 Zollinger-Ellison syndrome patients at follow-up., Conclusions: These data suggest that the progression of enterochromaffin-like cell growth in human gastric mucosa requires an increase of and/or a prolonged exposure to severe hypergastrinaemia.

The effects of cholecystokinin (CCK) antagonists on small cell lung cancer (SCLC) cells were investigated. CI-988, L-365,260, and L-364,718 inhibited specific (125)I-CCK-8 binding to NCI-H209 cells with IC(50) values of 5, 2, and 200 nM. ([R-(R*,R*)]-4[[2-[[3-(1H-Indole-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3.3.1.1(3,7)]- dec-2-yloxy)carbonyl[amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid) (CI-988; 100 nM) inhibited the ability of 10 nM CCK-8 to elevate cytosolic Ca(2+) in 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester-loaded NCI-H209 cells. By Western blot, CI-988 inhibited tyrosine phosphorylation of focal adhesion kinase and paxillin stimulated by CCK-8. Also, CI-988 inhibited tyrosine phosphorylation of mitogen-activated protein kinase stimulated by CCK-8. By Northern blot, CI-988 antagonized the ability of 10 nM CCK-8 to increase c-fos mRNA in NCI-H209 cells. Also, CI-988 inhibited the ability of CCK-8 to increase vascular endothelial cell growth factor mRNA. Using a [3-(4,5 dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide] and clonogenic assay, CI-988 inhibited the proliferation of NCI-H209 cells in vitro. Using nude mice, CI-988 inhibited the proliferation of NCI-H209 xenografts. These results suggest that CI-988 is a CCK(2) receptor antagonist that inhibits the proliferation of SCLC cells.

Background: Patients with multiple endocrine neoplasia type 1 and hyperparathyroidism often undergo multiple operations because of inadequate initial surgery, presence of supernumerary and ectopic glands, regrowth of remnant glands, or autograft hyperfunction. Management of this patient population is complex., Methods: From January 1975 to December 2000 we performed 94 reoperative parathyroidectomies consisting of 79 neck reexplorations, 12 autograft removals, and 3 median sternotomies in 75 patients. Data were gathered by retrospective chart review and follow-up telephone interviews., Results: Excluding autograft excision, reoperative surgery was successful (normocalcemia longer than 6 months) in 91%; autograft removal was successful in only 58%. With a median follow-up of 59 months, 64% of patients are currently free from hypercalcemia, and this outcome was not influenced by the total number of glands resected. The median time to recurrent hypercalcemia was 125 months. Thirty patients received an autograft after reoperation. The complication rate for all reoperations was 12%, including permanent recurrent laryngeal nerve injury in 2 patients (2.1%)., Conclusions: Reoperative parathyroidectomy in patients with multiple endocrine neoplasia type 1 was safe and successful in the majority of patients; however, recurrent hyperparathyroidism is likely to develop in most individuals beyond 10 years of follow-up. The total number of glands accounted for after reoperation is not associated with successful outcome.

The natural history of pancreatic endocrine tumors (PETs) in patients with MEN1 is largely unknown. Recent studies in patients with sporadic PETs show that in a subset, tumor growth is aggressive. To determine whether PETs in patients with MEN1 show similar growth behavior, we report results from a long-term prospective study of 57 patients with MEN1 and Zollinger-Ellison syndrome. All patients had tumor imaging studies yearly, and the mean follow-up was 8 yr. Only patients with PETs 2.5 cm or larger underwent abdominal surgical exploration. Hepatic metastases occurred in 23%, and in 14% tumors demonstrated aggressive growth. Three tumor-related deaths occurred, each due to liver metastases, and in each, aggressive tumor growth was present. Overall, 4% of the study group, 23% with liver metastases and 38% with aggressive disease, died. Aggressive growth was associated with higher gastrins and larger tumors. Patients with liver metastases with aggressive growth differed from those with liver metastases without aggressive growth in age at MEN1 onset or diagnosis and primary tumor size. Survival was decreased (P = 0.0012) in patients with aggressive tumor growth compared with those with liver metastases without aggressive growth or with no liver metastases without aggressive growth. Based on these results a number of factors were identified that may be clinically useful in determining in which patients aggressive tumor growth may occur. These results demonstrate in a significant subset of patients with MEN1 and Zollinger-Ellison syndrome, aggressive tumor growth occurs and can lead to decreased survival. The identification of prognostic factors that identify this group will be important clinically in allowing more aggressive treatment options to be instituted earlier.

Objective: To determine the role of surgery in patients with Zollinger-Ellison syndrome (ZES) and multiple endocrine neoplasia type 1 (MEN1) with either limited or advanced pancreatic endocrine tumors (PETs)., Summary Background Data: The role of surgery in patients with MEN1 and ZES is controversial. There have been numerous previous studies of surgery in patients with PETs; however, there are no prospective studies on the results of surgery in patients with advanced disease., Methods: Eighty-one consecutive patients with MEN1 and ZES were assigned to one of four groups depending on the results of imaging studies. Group 1 (n = 17) (all PETs smaller than 2.5 cm) and group 3 (n = 8) (diffuse liver metastases) did not undergo surgery. All patients in group 2A (n = 17; single PET 2.5-6 cm [limited disease]) and group 2B (n = 31; two or more lesions, 2.5 cm in diameter or larger, or one lesion larger than 6 cm) underwent laparotomy. Tumors were preferably removed by simple enucleation, or if not feasible resection. Patients were reevaluated yearly., Results: Pancreatic endocrine tumors were found in all patients at surgery, with groups 2A and 2B having 1.7 +/- 0.4 and 4.8 +/- 1 PETs, respectively. Further, 35% of the patients in group 2A and 88% of the patients in group 2B had multiple PETs, 53% and 84% had a pancreatic PET, 53% and 68% had a duodenal gastrinoma, 65% and 71% had lymph node metastases, and 0% and 12% had liver metastases. Of the patients in groups 2A and 2B, 24% and 58% had a distal pancreatectomy, 0% and 13% had a hepatic resection, 0% and 6% had a Whipple operation, and 53% and 68% had a duodenal resection. No patient was cured at 5 years. There were no deaths. The early complication rate, 29%, was similar for groups 2A and 2B. Mean follow-up from surgery was 6.9 +/- 0.8 years, and during follow-up liver metastases developed in 6% of the patients in groups 2A and 2B. Groups 1, 2A, and 2B had similar 15-year survival rates (89-100%); they were significantly better than the survival rate for group 3 (52%)., Conclusions: Almost 40% of patients with MEN1 and ZES have advanced disease without diffuse distant metastases. Despite multiple primaries and a 70% incidence of lymph node metastases, tumor can be removed with no deaths and complication rates similar to those in patients with limited disease. Further, despite previous studies showing that patients with advanced disease have decreased survival rates, in this study the patients with advanced tumor who underwent surgical resection had the same survival as patients with limited disease and patients without identifiable tumor. This suggests that surgical resection should be performed in patients with MEN1 who have ZES and advanced localized PET.

Background: Proton pump inhibitors are potent acid suppressants which, at normal doses, can result in hypergastrinaemia in patients with idiopathic oesophageal reflux disease and in the control of symptoms in most patients with gastrinomas. Therefore, their use could delay or mask the diagnosis of gastrinoma., Aim: To investigate whether the widespread use of proton pump inhibitors masks or complicates the diagnosis of gastrinoma., Subjects and Methods: Data from two centres with different referral criteria for suspected gastrinomas were analysed (Gastroenterology Unit, Rome, Italy and National Institutes of Health, Bethesda, MD, USA). The number of referrals and the number of new patients with gastrinoma diagnosed in the years prior to the widespread use of proton pump inhibitors (1986-1992) were compared with the numbers since proton pump inhibitors became widely available (1993-1998)., Results: The decrease in referral rate (P=0.0009) and the decrease in the annual rate of gastrinoma diagnosis (P=0.0020) at both centres correlated with the increased use of proton pump inhibitors. At the Italian centre, there was a 62% decrease in annual referrals (P < 0.0001) in the post-proton pump inhibitor period, relative to the pre-proton pump inhibitor period, whereas there was an increase in the rate of referral of other gastrointestinal endocrine tumours. The number of new cases of gastrinoma diagnosed decreased by 40%. At the US centre, the referral rate decreased by 28% (P=0.024) in the post-proton pump inhibitor period. There was also a 43% decrease in the number of new cases diagnosed annually in the post-proton pump inhibitor period (P=0.0012). There was a 2.6-fold increase in the post-proton pump inhibitor period in the percentage of referrals with a false diagnosis of gastrinoma as the cause of hypergastrinaemia (P=0.0040)., Conclusions: In both referral centres, less patients have been referred with a possible diagnosis of gastrinoma and fewer new patients with gastrinoma have been diagnosed since proton pump inhibitors became widely available. These data support the conclusion that, since proton pump inhibitors have been released, the diagnosis of gastrinoma has been masked and will probably be delayed, with the result that patients with gastrinoma will be diagnosed at more advanced stages in their disease course.

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of their selectivity or affinity. The receptor mediating the action of the mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. To investigate the molecular basis for their high affinity for the GRPR, two classes of peptide antagonists, a statine analogue, JMV594 ([d-Phe(6),Stat(13)]Bn(6-14)), and a pseudopeptide analogue, JMV641 (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi(CHOH-CH(2))-(CH(2))(2)-CH(3)), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. The substitutions of Thr(297) in GRPR by Pro from the comparable position in NMBR, Phe(302) by Met, and Ser(305) by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr(297), Phe(302), and Ser(305) in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 A of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr(297), Phe(302), and Ser(305) of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.

Growth hormone (GH) is used or is being evaluated for efficacy in treatment of short stature, aspects of aging, cardiac disorders, Crohn's disease, and short bowel syndrome. Therefore, we synthesized several stable growth hormone-releasing factor (GRF) analogues that could be therapeutically useful. One potent analog, [D-Ala(2),Aib(8, 18,)Ala(9, 15, 16, 22, 24-26,)Gab(27)]hGRF(1-27)NH(2) (GRF-6), with prolonged infusion caused severe diarrhea in monkeys; however, it had no side-effects in rats. Because GRF has similarity to VIP/PACAP and VIPomas cause diarrhea, this study investigated the ability of this and other GRF analogues to interact with the VIP/PACAP receptors. Rat VPAC(1)-R (rVPAC(1)-R), human VPAC(1)-R (hVPAC(1)-R), rVPAC(2)-R and hVPAC(2)-R stably transfected CHO and PANC 1 cells were made and T47D breast cancer cells containing native human VPAC(1)-R and AR4-2J cells containing PAC(1)-R were used. hGRF(1-29)NH(2) had low affinity for both rVPAC(1)-R and rVPAC(2)-R while VIP had a high affinity for both receptors. GRF-6 had a low affinity for both rVPAC(1)-R and rVPAC(2)-R and very low affinity for the rPAC(1)-R. VIP had a high affinity, whereas hGRF(1-29)NH(2) had a low affinity for both hVPAC(1)-R and hVPAC(2)-R. In contrast GRF-6, while having a low affinity for hVPAC(2)-R, had relatively higher affinity for the hVPAC(1)-R. In guinea pig pancreatic acini, all GRF analogues were full agonists at the VPAC(1)-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(1-29)NH(2,) GRF-6 has a relatively high affinity for the human VPAC(1)-R but not for the human VPAC(2)-R, rat VPAC(1)-R, rat VPAC(2)-R or rat PAC(1)-R. These results suggest that the substituted GRF analog, GRF-6, likely causes the diarrheal side-effects in monkeys by interacting with the VPAC(1)-R. Furthermore, they demonstrate significant species differences can exist for possible therapeutic peptide agonists of the VIP/PACAP/GRF receptor family and that it is essential that receptor affinity assessments be performed in human cells or from a closely related species.

In the presence of somatostatin-14 or some of its receptorial agonists, the uptake of large neutral amino acids by isolated brain microvessels was found to be inhibited up to 50%, no other transport system being affected. Although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only uptake from the abluminal side appears to be inhibited by somatostatin. The involvement of a type-2 somatostatin receptor was suggested by assays with a series of receptor-specific somatostatin agonists, and was confirmed by the release of inhibition caused by a specific type-2 receptor antagonist. A type-2-specific mRNA was indeed shown to be present in both bovine brain microvessels ex vivo and primary cultures of endothelial cells from rat brain microvessels.

Purpose: To assess the value of the initial fasting serum gastrin (FSG) at presentation in patients with Zollinger-Ellison Syndrome (ZES) in predicting primary tumor characteristics and survival., Patients and Methods: A total of 239 patients were treated for ZES between December 1981 and September 1998, with a mean follow-up of 9.1 +/- 0.6 years. At initial evaluation, 86 patients (36%) had mild (0 to 499 pg/mL), 61 (25.5%) had moderate (500 to 1,000 pg/mL), and 92 (38.5%) had severe (> 1,000 pg/mL) elevations in FSG. Primary tumor location and size, presence of lymph node or hepatic metastases, and survival were analyzed based on the level of initial FSG., Results: In patients with sporadic ZES, but not in those with multiple endocrine neoplasia type 1 (MEN-1) and ZES, there was a significant relationship between the level of initial FSG and tumor size and location of primary tumor, frequency of lymph node and liver metastases, and survival. The median 5- and 10-year survival decreased with increasing initial FSG (P <.001) in patients with sporadic ZES; MEN-1 patients lived longer than sporadic ZES patients (P =.012), and survival in this group was not associated with the level of initial FSG. Multivariate analysis showed that factors independently associated with death from disease in patients with sporadic ZES were liver metastases (P =.0001), a pancreatic site (P =.0027), and primary tumor size (P =.011) but not initial FSG (P >.30)., Conclusion: The severity of FSG at presentation is associated with size and site of tumor and the presence of hepatic metastases, factors that are significant independent predictors of outcome. The level of FSG at presentation may be useful in planning the nature and extent of the initial evaluation and management in patients with sporadic ZES.

Carcinoid tumors were identified in the antro-pyloric mucosa of four patients with multiple endocrine neoplasia type 1 (MEN-1)/Zollinger-Ellison syndrome, accounting for 8.7% of 46 patients with this condition examined by endoscopy and histology. In contrast, no tumors were found in the antral biopsies from 124 cases of sporadic Zollinger-Ellison syndrome (P < 0.001), indicating a prominent role for the MEN-1 gene defects in tumor development. Immunohistochemically the tumors did not express the hormones produced by antral endocrine cells (gastrin, somatostatin, serotonin). In contrast, two of them were diffusely immunoreactive for the isoform 2 of the vesicular monoamine transporter (VMAT-2), a marker specific for the gastric nonantral enterochromaffin-like (ECL) cells. In one of these patients a second antral VMAT-2-positive carcinoid was seen 21 months after the first diagnosis. The other two antral carcinoids were unreactive for VMAT-2. Multiple ECL cell tumors were found in the gastric body-fundus mucosa of the two patients with VMAT-2-positive, but not in those with VMAT-2-negative, antral carcinoids. In one case, the former tumors were diagnosed 22 months after the detection of the antral tumor. We conclude that the antral mucosa is an additional tissue that may harbor endocrine tumors in MEN-1 syndrome. These tumors did not express the phenotype of normal antral endocrine cells and, in at least two cases, were identified as ectopic ECL cell carcinoids.

We prospectively studied 235 patients with Zollinger-Ellison syndrome (ZES) (205 without and 30 with prior acid-reducing surgery) and compared the results with 984 patients from 182 reports in the literature. The aims of the study were to evaluate the sensitivity of proposed acid secretory criteria for the diagnosis of ZES, propose new criteria, evaluate the variability and methodology of gastric secretory testing, and correlate the symptoms and signs of ZES, tumor extent, and primary tumor size and location with the degree of gastric acid hypersecretion. Multiple endocrine neoplasia-type 1 (MEN1) occurred in 22% of patients. The mean basal acid output (BAO) in patients without and with prior acid-reducing surgery was 41.2 +/- 1.7 mEq/hr (range, 1.6-118.3 mEq/hr) and 27.6 +/- 3.5 mEq/hr (range 5.9-102.9 mEq/hr), respectively. In patients with MEN1, those with female gender, Hispanic, or Asian race had lower BAOs. Diarrhea, esophageal stricture, and pyloric scarring were associated with a higher BAO. Neither other symptoms nor the tumor extent, primary tumor location, or size correlated with the magnitude of acid hypersecretion. ZES diagnosis was delayed a mean of 5.5 +/- 0.4 yr. Patients who were misdiagnosed as having either Crohn or celiac disease had higher BAOs. The sensitivities from our study and the literature review of the proposed BAO criteria for the diagnosis of ZES in patients without previous gastric acid-reducing surgery were 91% and 90% for BAO > or = 15 mEq/hr, 86% and 82% for BAO > or = 18 mEq/hr, 69% and 67% for BAO > 25 mEq/hr, and < 60% for BAO > 31 mEq/hr, respectively. The specificities of all the proposed BAO criteria were high. Both the criterion of BAO > or = 15 mEq/hr and BAO > or = 18 mEq/hr had good specificities and equal sensitivity. With prior acid-reducing surgery, the sensitivities in our study and from the literature review were 100% and 81% for BAO > or = 5 mEq/hr, 73% and 45% for BAO > 14.4 mEq/hr, and 37% and 31% for BAO > 19.2 mEq/hr, respectively. The reported mean specificity for the criterion of BAO > or = 5 mEq/hr was 85%, while it was 100% for the other 2 criteria. The maximal acid output (MAO) criterion of > 70 mEq/hr had sensitivities in the present National Institutes of Health (NIH) study and the literature review of 39% and 31%, respectively, and the criterion of MAO > 100 mEq/hr had a sensitivity of < 15% in patients with no prior acid-reducing surgery. The proposed criterion of BAO/MAO ratio > 0.6 had a low sensitivity. The proposed criterion of the ratio of basal and maximal acid H+ concentration (BAC/MAC ratio) > or = 0.6 had an excellent sensitivity-- > or = 89% in patients with or without previous acid-reducing surgery. The reported specificity for both the BAO/MAO criterion and the BAC/MAC criterion were similar, but BAC/MAC had a better sensitivity. Combination criteria of BAO generally did not improve sensitivity. The criterion of pH < or = 1 was met by only 27% of patients, and pH < or = 0.96 by 21% of patients with previous acid-reducing surgery. For patients with MEN1 with no prior acid-reducing surgery, the sensitivities were lower compared with patients with the sporadic form of ZES. The mean gastric volume in patients without prior acid-reducing surgery was 314 +/- 10 mL/hr and 247 +/- 25 mL/hr in patients with prior acid-reducing surgery. A basal volume criteria of > 160 mL/hr in patients without prior acid-reducing surgery occurred in > 86% of patients, and > 140 mL/hr in 87% of patients with prior acid-reducing surgery; these, thus, are neglected findings that have good sensitivities. Our analysis shows criteria based on MAO, pH, and BAO/MAO ratio do not have high sensitivities and thus are not useful. In patients without prior acid-reducing surgery, the criteria of BAO > or = 15 mEq/hr, BAC/MAC ratio > or = 0.6, and basal gastric volume > 160 mL/hr are useful for the diagnosis of ZES and have good specificities. In patients with prior acid-reducing surgery, the criteria of BAO > or = 5 mEq/hr, BAC/MAC ratio > or = 0.6, and basal gastric volume > 140 mL/hr have high sensitivities. In patients with sporadic ZES without acid-reducing surgery, the criterion of BAO > or = 18 mEq/hr is recommended as it has a similar sensitivity but higher specificity than the criterion of BAO > or = 15 mEq/hr. Only 1 patient in either data set (NIH or the literature) with or without previous acid-reducing surgery had a basal gastric pH > 2, therefore this finding essentially excludes the diagnosis of ZES. Gastric secretory measurements for 30 minutes, but not 15 minutes, give results comparable to those for a full hour. On the basis of these results, a number of gastric secretory criteria are proposed, including some for the first time, and alterations in methodology are proposed that should prove useful in the diagnosis of ZES.

Numerous epidemiologic studies suggest a relationship between lung cancer and peptic ulcer disease. Furthermore, various lung cancers synthesize and release a number of peptides such as gastrin and gastrin-releasing peptide that could cause acid hypersecretion; however, Zollinger-Ellison syndrome (ZES), because of a lung tumor, has never been described. We report such a patient for the first time. A 60-year-old man with a non-small cell lung carcinoma (large cell type) presented with diarrhea, heartburn, abdominal pain, and duodenal ulcers. Evaluation showed ZES was present (fasting hypergastrinemia, hyperchlorhydria) and control of all symptoms by omeprazole. No abdominal or cardiac tumor, the other known locations of gastrinomas causing ZES, was found on detailed tumor imaging studies. Resection of the lung tumor resulted in a decrease in gastrin levels to normal values. Plasma radioimmunoassays showed elevated gastrin, chromogranin A and normal levels of gastrin-releasing peptide, and 9 other hormones. The tumor showed similar immunocytochemical results. The characteristics of this case are compared with 100 cases of sporadic abdominal gastrinomas, and the evidence reviewed suggests why ZES should be considered in patients with lung cancer with peptic symptoms.

The orphan receptor, bombesin (Bn) receptor subtype 3 (BRS-3), shares high homology with bombesin receptors (neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R)). This receptor is widely distributed in the central nervous system and gastrointestinal tract; target disruption leads to obesity, diabetes, and hypertension, however, its role in physiological and pathological processes remain unknown due to lack of selective ligands or identification of its natural ligand. We have recently discovered (Mantey, S. A., Weber, H. C., Sainz, E., Akeson, M., Ryan, R. R. Pradhan, T. K., Searles, R. P., Spindel, E. R., Battey, J. F., Coy, D. H., and Jensen, R. T. (1997) J. Biol. Chem. 272, 26062-26071) that [d-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]Bn-(6-14) has high affinity for BRS-3 and using this ligand showed BRS-3 has a unique pharmacology with high affinity for no known natural Bn peptides. However, use of this ligand is limited because it has high affinity for all known Bn receptors. In the present study we have attempted to identify BRS-3 selective ligands using a strategy of rational peptide design with the substitution of conformationally restricted amino acids into the prototype ligand [d-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]Bn-(6-14) or its d-Phe(6) analogue. Each of the 22 peptides synthesized had binding affinities determined for hBRS-3, hGRPR, and hNMBR, and hBRS-3 selective ligands were tested for their ability to activate phospholipase C and increase inositol phosphates ([(3)H]inositol phosphate). Using this approach we have identified a number of BRS-3 selective ligands. These ligands functioned as receptor agonists and their binding affinities were reflected in their potencies for altering [(3)H]inositol phosphate. Two peptides with an (R)- or (S)-amino-3-phenylpropionic acid substitution for beta-Ala(11) in the prototype ligand had the highest selectivity for the hBRS-3 over the mammalian Bn receptors and did not interact with receptors for other gastrointestinal hormones/neurotransmitters. Molecular modeling demonstrated these two selective BRS-3 ligands had a unique conformation of the position 11 beta-amino acid. This selectivity was of sufficient magnitude that these should be useful in explaining the role of hBRS-3 activation in obesity, glucose homeostasis, hypertension, and other physiological or pathological processes.

A synthetic peptide, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was used to investigate the signal transduction mechanisms of bombesin receptor subtype-3. Using NCI-1299#5 human lung cancer cells stably transfected with bombesin receptor subtype-3, 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) elevated the cytosolic Ca2+ from 150 to 250 nM within 10 s. Addition of (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused phosphorylation of mitogen activated protein kinase in a time- and concentration-dependent manner. The mitogen activated protein kinase phosphorylation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by 2'-amino-3'-methyoxyflavone (PD98059), a mitogen activated protein kinase kinase (MEK-1) inhibitor. Using a luciferase reporter gene construct, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused Elk-1 activation after 10 min and the increase in Elk-1 activation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059 as well as a dominant-negative MEK-1. (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased c-fos as well as c-jun mRNAs 1 h after addition to NCI-H1299#5 cells. The 47-fold increase in c-fos mRNA caused by 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059, a dominant-negative MEK-1 and a substance P antagonist but not (3-phenylpropanoyl-D-Ala(24), Pro(26), Psi(26,27), Phe(27))GRP-(20-27) (BW2258U89), a GRP receptor antagonist. These results indicate that (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased nuclear oncogene expression and upstream events include mitogen activated protein kinase phosphorylation and Elk-1 activation.

The effects of some oncogenes, growth factors and neuropeptides are mediated by tyrosine phosphorylation of focal adhesion kinase (p125(FAK)) and paxillin cytoskeletal proteins. In this study the ability of bombesin/gastrin releasing peptide (BB/GRP) to stimulate tyrosine phosphorylation of p125(FAK) and paxillin in non-small cell lung cancer (NSCLC) H1299 cells was investigated. BB, 100 nM caused increased p125(FAK) and paxillin tyrosine phosphorylation maximally after 1 min. The effect of BB on p125(FAK) and paxillin tyrosine phosphorylation was concentration-dependent, being half maximal at 4-8 nM. Also, 100 nM GRP, GRP(14-27) but not GRP(1-16) increased p125(FAK) and paxillin tyrosine phosphorylation indicating that the C-terminal of GRP is essential. BW2258U89, a GRP receptor antagonist, caused a dose-dependent inhibition of BB-stimulated p125(FAK) and paxillin tyrosine phosphorylation with an IC50 value of 3 microM. Cytochalasin D (0.3 microM), which inhibits actin polymerization, reduced the ability of BB to stimulate tyrosine phosphorylation of p125(FAK) and paxillin. Genistein (50 microM) and H-7 (50 microM), which are kinase inhibitors, reduced the tyrosine phosphorylation of p125(FAK) and paxillin stimulated by BB. Also, treatment of NCI-H1299 cells with FAK antisense resulted in decreased FAK tyrosine kinase activity and proliferation. These results suggest that p125(FAK) is an important enzyme for NSCLC proliferation.

Peptoid antagonists are increasingly being described for G protein-coupled receptors; however, little is known about the molecular basis of their binding. Recently, the peptoid PD168368 was found to be a potent selective neuromedin B receptor (NMBR) antagonist. To investigate the molecular basis for its selectivity for the NMBR over the closely related receptor for gastrin-releasing peptide (GRPR), we used a chimeric receptor approach and a site-directed mutagenesis approach. Mutated receptors were transiently expressed in Balb 3T3. The extracellular domains of the NMBR were not important for the selectivity of PD168368. However, substitution of the 5th upper transmembrane domain (uTM5) of the NMBR by the comparable GRPR domains decreased the affinity 16-fold. When the reverse study was performed by substituting the uTM5 of NMBR into the GRPR, a 9-fold increase in affinity occurred. Each of the 4 amino acids that differed between NMBR and GRPR in the uTM5 region were exchanged, but only the substitution of Phe(220) for Tyr in the NMBR caused a decrease in affinity. When the reverse study was performed to attempt to demonstrate a gain of affinity in the GRPR, the substitution of Tyr(219) for Phe caused an increase in affinity. These results suggest that the hydroxyl group of Tyr(220) in uTM5 of NMBR plays a critical role for high selectivity of PD168368 for NMBR over GRPR. Receptor and ligand modeling suggests that the hydroxyl of the Tyr(220) interacts with nitrophenyl group of PD168368 likely primarily by hydrogen bonding. This result shows the selectivity of the peptoid PD168368, similar to that reported for numerous non-peptide analogues with other G protein-coupled receptors, is primarily dependent on interaction with transmembrane amino acids.