Causes of death and prognostic factors in multiple endocrine neoplasia type 1: a prospective study: comparison of 106 MEN1/Zollinger-Ellison syndrome patients with 1613 literature MEN1 patients with or without pancreatic endocrine tumors.

Multiple endocrine neoplasia type 1 (MEN1) is classically characterized by the development of functional or nonfunctional hyperplasia or tumors in endocrine tissues (parathyroid, pancreas, pituitary, adrenal). Because effective treatments have been developed for the hormone excess state, which was a major cause of death in these patients in the past, coupled with the recognition that nonendocrine tumors increasingly develop late in the disease course, the natural history of the disease has changed. An understanding of the current causes of death is important to tailor treatment for these patients and to help identify prognostic factors; however, it is generally lacking.To add to our understanding, we conducted a detailed analysis of the causes of death and prognostic factors from a prospective long-term National Institutes of Health (NIH) study of 106 MEN1 patients with pancreatic endocrine tumors with Zollinger-Ellison syndrome (MEN1/ZES patients) and compared our results to those from the pooled literature data of 227 patients with MEN1 with pancreatic endocrine tumors (MEN1/PET patients) reported in case reports or small series, and to 1386 patients reported in large MEN1 literature series. In the NIH series over a mean follow-up of 24.5 years, 24 (23%) patients died (14 MEN1-related and 10 non-MEN1-related deaths). Comparing the causes of death with the results from the 227 patients in the pooled literature series, we found that no patients died of acute complications due to acid hypersecretion, and 8%-14% died of other hormone excess causes, which is similar to the results in 10 large MEN1 literature series published since 1995. In the 2 series (the NIH and pooled literature series), two-thirds of patients died from an MEN1-related cause and one-third from a non-MEN1-related cause, which agrees with the mean values reported in 10 large MEN1 series in the literature, although in the literature the causes of death varied widely. In the NIH and pooled literature series, the main causes of MEN1-related deaths were due to the malignant nature of the PETs, followed by the malignant nature of thymic carcinoid tumors. These results differ from the results of a number of the literature series, especially those reported before the 1990s. The causes of non-MEN1-related death for the 2 series, in decreasing frequency, were cardiovascular disease, other nonendocrine tumors > lung diseases, cerebrovascular diseases. The most frequent non-MEN1-related tumor deaths were colorectal, renal > lung > breast, oropharyngeal. Although both overall and disease-related survival are better than in the past (30-yr survival of NIH series: 82% overall, 88% disease-related), the mean age at death was 55 years, which is younger than expected for the general population.Detailed analysis of causes of death correlated with clinical, laboratory, and tumor characteristics of patients in the 2 series allowed identification of a number of prognostic factors. Poor prognostic factors included higher fasting gastrin levels, presence of other functional hormonal syndromes, need for >3 parathyroidectomies, presence of liver metastases or distant metastases, aggressive PET growth, large PETs, or the development of new lesions.The results of this study have helped define the causes of death of MEN1 patients at present, and have enabled us to identify a number of prognostic factors that should be helpful in tailoring treatment for these patients for both short- and long-term management, as well as in directing research efforts to better define the natural history of the disease and the most important factors determining long-term survival at present.

Endothelial progenitor cells and plasma vascular endothelial growth factor and stromal cell-derived factor-1 during ranibizumab treatment for neovascular age-related macular degeneration.

PURPOSE: To evaluate endothelial progenitor cell [late outgrowth endothelial progenitor cells (OECs)], vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1α (SDF-1α) plasma levels as potential biomarkers before and during ranibizumab (Lucentis(®)) treatment for neovascular age-related macular degeneration (nvAMD). METHODS: Thirty-one patients with untreated nvAMD presenting for 3 consecutive intravitreal ranibizumab injections and a follow-up visit at 4 weeks intervals were enrolled. Peripheral blood was collected before each injection and at the follow-up visit and OEC clusters were cultured and evaluated according to previously published protocols. VEGF and SDF-1α plasma levels were measured by enzyme-linked immunosorbent assay and compared to values from healthy young and old control. RESULTS: Patients with a high OEC count before treatment presented significantly more often with a short symptom duration and a smaller choroidal neovascularization size. VEGF plasma levels were significantly higher in nvAMD (282.4±195.2 pg/mL) compared to young (45.5±6.8 pg/mL) and old control (46.1±8.5 pg/mL). OEC levels decreased nonsignificantly during ranibizumab treatment, returning to baseline levels after the third injection. VEGF and SDF-1α plasma levels decreased significantly during treatment toward control values. Patients needing retreatment after 3 ranibizumab injections had significantly higher VEGF plasma levels at pretreatment compared to patients not needing further treatment. CONCLUSIONS: The results presented here suggest that VEGF plasma levels may warrant further evaluation regarding biological, therapeutical, and predictive implications in nvAMD.

PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades.

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/ß, 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).

Molecular basis of P2-receptor-mediated calcium signaling in activated pancreatic stellate cells.

OBJECTIVES: There is growing evidence that extracellular nucleotide-induced signaling confers to fibrogenesis in liver and pancreas. Pancreatic stellate cells (PSC) are the most important cell type in pancreatic fibrosis. P2 purine and pyrimidine receptors, again, are pivotal mediators of inflammatory and profibrogenic signals. Our aim was to elucidate the underlying signaling components in activated PSC. METHODS: We performed expression analysis of calcium ion (Ca(2+)) signaling components and monitored real-time intracellular Ca(2+) responses to nucleotides in rat PSC. RESULTS: Adenosine monophosphate, adenosine diphosphate, and adenosine-5'-triphosphate elicited detectable rises in intracellular Ca(2+) concentrations. Stimulation of PSC by ATP led to intracellular Ca signals mediated through both P2X and P2Y receptors. Whereas uridine triphosphate-mediated Ca(2+) signals were generated by activation of P2Y receptors only, uridine diphosphate stimulated P2X receptors as well. Of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate pathway, all PLC-facilitating Gα subunits were present in activated cells as were all 3 inositol-1,4,5-trisphosphate receptor isoforms. In addition, transcripts of PLC-ß and PLC-δ isoforms were also strongly detectable. CONCLUSIONS: Activated PSC feature a plethora of elements from the Ca signaling toolkit and functionally express a subset of P2 nucleotide receptors. Purines and pyrimidines elicit robust intracellular Ca(2+) signals likely contributing to the fibrogenetic potential of these cells.

SU5416 induces premature senescence in endothelial progenitor cells from patients with age-related macular degeneration.

PURPOSE: We recently demonstrated increased frequency and growth potential of late outgrowth endothelial progenitor cells (OECs) in patients with neovascular age-related macular degeneration (nvAMD). This study investigated the effects of short- and long-term in vitro inhibition of vascular endothelial growth factor (VEGF) Receptor-2 (VEGFR-2) signaling by SU5416 and other inhibitors of the VEGF signaling pathway in OECs. METHODS: OECs, from the peripheral blood of patients with nvAMD, and human umbilical vein endothelial cells were grown in the presence of SU5416, other VEGFR-2 tyrosine kinase inhibitors (TKIs), and inhibitors of phosphatidylinositol 3'-Kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) in complete angiogenic medium. Apotosis was assessed after 48 h using the fluorescein isothiocyanate Annexin V method. Cell counts were performed for 10 days, and features of senescence were analyzed using senescence-associated ß-galactosidase staining, the telomeric repeat amplification protocol for telomerase activity, Southern blot analysis for mean telomere length, flow cytometric analysis for cell-cycle arrest, and western blot for p53 and p21. Control OECs, cells treated for 7 days with inhibitors, as well as naturally senescent OECs were analyzed for expression of different endothelial antigens, including VEGFR-2 and the receptor for stromal cell-derived factor 1, chemokine receptor 4 (CXCR-4). Migration in vitro to VEGF and stromal cell-derived factor 1 of OECs was assessed. RESULTS: SU5416, other VEGFR-2 TKIs, and inhibitors of PI3K, Akt, and PKC induced apoptosis, inhibited long-term proliferation, reduced telomerase activity, and induced premature senescence and cell-cycle arrest in OECs as well as in human umbilical vein endothelial cells. Naturally senescent cells and cells rendered senescent by VEGFR-2 TKIs had reduced VEGFR-2 and CXCR-4 expression and demonstrated reduced migratory ability to VEGF. CONCLUSIONS: This study demonstrates apoptosis upon short-term inhibition and inhibition of long-term survival of OECs from patients with nvAMD by SU5416, presumably via PI3K/Akt and/or PKC-mediated reduction in telomerase activity and subsequent induction of premature senescence, which is accompanied by impaired endothelial activity. Therefore, induction of premature senescence in endothelial cells may represent a potential therapeutic target in nvAMD.

CCK1 and CCK2 receptors are expressed on pancreatic stellate cells and induce collagen production.

The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-ß-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-ß, both CCK and gastrin inhibit proliferation in PSC.

Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells.

Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr(1068) of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)alpha antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species.

Gastrointestinal growth factors and hormones have divergent effects on Akt activation.

Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigated in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters [CCK, bombesin, carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations (pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations (nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.

Inherited pancreatic endocrine tumor syndromes: advances in molecular pathogenesis, diagnosis, management, and controversies.

Pancreatic endocrine tumors (PETs) can occur as part of 4 inherited disorders, including Multiple Endocrine Neoplasia type 1 (MEN1), von Hippel-Lindau disease (VHL), neurofibromatosis 1 (NF-1) (von Recklinghausen disease), and the tuberous sclerosis complex (TSC). The relative frequency with which patients who have these disorders develop PETs is MEN1>VHL>NF-1>TSC. Over the last few years, there have been major advances in the understanding of the genetics and molecular pathogenesis of these disorders as well in the localization and the medical and surgical treatment of PETs in such patients. The study of PETs in these disorders not only has provided insights into the possible pathogenesis of sporadic PETs but also has presented several unique management and treatment issues, some of which are applicable to patients with sporadic PETs. Therefore, the study of PETs in these uncommon disorders has provided valuable insights that, in many cases, are applicable to the general group of patients with sporadic PETs. In this article, these areas are reviewed briefly along with the current state of knowledge of the PETs in these disorders, and the controversies that exist in their management are summarized briefly and discussed.

Late outgrowth endothelial progenitor cells in patients with age-related macular degeneration.

PURPOSE: To evaluate the feasibility of isolating and expanding endothelial progenitor cells (EPCs), in the form of late outgrowth endothelial progenitor cells (OECs), from the peripheral blood of an aged population, particularly patients affected by different forms of AMD. METHODS: Peripheral blood mononuclear cells were collected from young control subjects (n = 18) and from elderly subjects with non-AMD/low-risk dry AMD (n = 15), high-risk dry AMD (n = 6), or neovascular AMD (nvAMD; n = 32); cultured in established conditions; and observed for appearance of OEC clusters and growth characteristics on expansion. Expression of VEGF receptor-2 (KDR) in OECs after expansion was determined by Western blot. Plasma samples of study subjects were analyzed for CRP and VEGF levels. RESULTS: OEC cultures were successfully generated from a similar number of subjects in each group. After adjustment for all other variables, subjects with high-risk dry AMD had a 5.6-fold higher number of OEC clusters per 20 mL blood, and subjects with nvAMD had a 5.1-fold high number than did subjects with non-AMD/low-risk dry AMD (P < 0.05). High-risk dry AMD generated 63 times more (NS) and nvAMD 32-times more (P < 0.05) OECs on expansion of clusters than did non-AMD/low-risk dry AMD. Population doubling occurred significantly faster in cultures from nvAMD eyes compared to non-AMD/low-risk dry AMD eyes. In addition, a significant correlation between the number of OEC clusters, expanded OECs and levels of KDR was demonstrated. CONCLUSIONS: An OEC population was isolated and expanded from the blood of elderly control and AMD-affected patients and demonstrated significantly higher number of initial OEC clusters and expansion potential of OECs in patients at risk for or already affected by nvAMD. OECs may be used for further phenotypic, genetic, and functional analyses in patients with nvAMD.

Prospective study of surgery for primary hyperparathyroidism (HPT) in multiple endocrine neoplasia-type 1 and Zollinger-Ellison syndrome: long-term outcome of a more virulent form of HPT.

BACKGROUND: Primary hyperparathyroidism (HPT) in multiple endocrine neoplasia type 1 (MEN1) patients with Zollinger-Ellison syndrome (ZES) is caused by parathyroid hyperplasia. Surgery for parathyroid hyperplasia is tricky and difficult. Long-term outcome in ZES/MEN1/HPT is not well known. METHODS: Eighty-four consecutive patients (49 F/35 M) with ZES/MEN1/HPT underwent initial parathyroidectomy (PTX) and were followed at 1- to 3-year intervals. RESULTS: Age at PTX was 36 +/- 2 years. Mean follow-up was 17 +/- 1 years. Before PTX, mean Ca = 2.8 mmol/L (normal level (nl <2.5), PTH i = 243 pg/mL (nl <65), and gastrin = 6950 pg/mL (nl < 100). Sixty-one percent had nephrolithiasis. Each patient had parathyroid hyperplasia. Fifty-eight percent of patients had 4 parathyroid glands identified. Nine of 84 (11%) had 4 glands removed with immediate autograft, 40/84 (47%) 3 to 3.5 glands, whereas 35/84 (42%) <3 glands removed. Persistent/recurrent HPT occurred in 42%/48% of patients with <3 glands, 12%/44% with 3 to 3.5 glands, and 0%/55% with 4 glands removed. Hypoparathyroidism occurred in 3%, 10%, and 22%, respectively. The disease-free interval after surgery was significantly longer if >3 glands were removed. After surgery to correct the HPT, each biochemical parameter of ZES was improved and 20% of patients no longer had laboratory evidence of ZES. CONCLUSIONS: HPT/MEN1/ZES is a severe form of parathyroid hyperplasia with a high rate of nephrolithiasis, persistent and recurrent HPT. Surgery to correct the hypercalcemia significantly ameliorates the ZES. Removal of less than 3.5 glands has an unacceptably high incidence of persistent HPT (42%), whereas 4-gland resection and transplant has a high rate of permanent hypoparathyroidism (22%). More than 3-gland resection has a longer disease-free interval. The surgical procedure of choice for patients with HPT/MEN1/ZES is 3.5-gland parathyroidectomy. Careful long-term follow-up is necessary as a significant proportion will develop recurrent HPT.

A prospective study of gastric carcinoids and enterochromaffin-like cell changes in multiple endocrine neoplasia type 1 and Zollinger-Ellison syndrome: identification of risk factors.

CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) patients frequently develop Zollinger-Ellison syndrome (ZES). These patients can develop proliferative changes of gastric enterochromaffin-like (ECL) cells and gastric carcinoids (ECL-cell tumors). ECL-cell changes have been extensively studied in sporadic ZES patients and can be precursor lesions of gastric carcinoids, but little is known about factors influencing their severity or development of carcinoids in MEN1/ZES patients. OBJECTIVES: Our objective was to prospectively analyze ECL-cell changes and gastric carcinoids (ECL-cell tumors) in a large series of MEN1/ZES patients to detect risk factors and deduct clinical guidelines. SETTING AND PATIENTS: Fifty-seven consecutive MEN1/ZES patients participated in this prospective study at two tertiary-care research centers. INTERVENTIONS AND OUTCOME MEASURES: Assessment of MEN1, gastric hypersecretion, and gastroscopy with multiple biopsies was done according to a fixed protocol and tumor status. ECL-cell changes and alpha-human chorionic gonadotropin staining were assessed in each biopsy and correlated with clinical, laboratory, and MEN1 features. RESULTS: ECL-cell proliferative changes were universally present, advanced changes in 53% and carcinoids in 23%. Gastric nodules are common and are frequently associated with carcinoids. Patients with high fasting serum gastrin levels, long disease duration, or a strong alpha-human chorionic gonadotropin staining in a biopsy are at higher risk for an advanced ECL-cell lesion and/or gastric carcinoid. CONCLUSIONS: Gastric carcinoids and/or advanced ECL-cell changes are frequent in MEN1/ZES patients, and therefore, regular surveillance gastroscopy with multiple routine biopsies and biopsies of all mucosal lesions are essential. Clinical/laboratory data and biopsy results can be used to identify a subgroup of MEN1/ZES patients with a significantly increased risk for developing gastric carcinoids, allowing development of better surveillance strategies.

Progress in developing cholecystokinin (CCK)/gastrin receptor ligands that have therapeutic potential.

Gastrin and cholecystokinin (CCK) are two of the oldest hormones and within the past 15 years there has been an exponential increase in knowledge of their pharmacology, cell biology, receptors (CCK1R and CCK2R), and roles in physiology and pathological conditions. Despite these advances there is no approved disease indication for CCK receptor antagonists and only a minor use of agonists. In this review, the important factors determining this slow therapeutic development are reviewed. To assess this it is necessary to briefly review what is known about the roles of CCK receptors (CCK1R and CCK2R) in normal human physiology, their role in pathologic conditions, the selectivity of available potent CCKR agonists/antagonists as well as to review their use in human conditions to date and the results. Despite extensive studies in animals and in humans, recent studies suggest that monotherapy with CCK1R agonists will not be effective in obesity, nor CCK2R antagonists in panic disorders or CCK2R antagonists to inhibit growth of pancreatic cancer. Areas that require more study include the use of CCK2R agonists for imaging tumors and radiotherapy, CCK2R antagonists in hypergastrinemic states especially with long-term PPI use and for potentiation of analgesia as well as use of CCK1R antagonists for a number of gastrointestinal disorders [motility disorders (irritable bowel syndrome, dyspepsia, and constipation) and pancreatitis (acute and chronic)].

Secretin-receptor and secretin-receptor-variant expression in gastrinomas: correlation with clinical and tumoral features and secretin and calcium provocative test results.

CONTEXT/OBJECTIVES: The diagnosis of Zollinger-Ellison syndrome requires secretin testing in 60% of patients. Even with secretin, the diagnosis may be difficult because variable responses occur, and 6-30% have negative testing. The basis for variability or negative responses is unclear. It is unknown whether the tumor density of secretin receptors or the presence of a secretin-receptor-variant, which can act as a dominant negative, is important. The aim of this study was to investigate these possibilities. PATIENTS/METHODS: Secretin-receptor and variant mRNA expression was determined in gastrinomas using real-time PCR from 54 Zollinger-Ellison syndrome patients. Results were correlated with Western blotting, secretin-receptor immunohistochemistry, with gastrin-provocative test results and tumoral/clinical/laboratory features. RESULTS: Secretin-receptor mRNA was detectible in all gastrinomas but varied 132-fold with a mean of 0.89 +/- 0.12 molecules per beta-actin. Secretin-receptor PCR results correlated closely with Western blotting (r = 0.95; P < 0.0001) and receptor immunohistochemistry (P = 0.0015; r = 0.71). The variant was detected in all gastrinomas, but levels varied 102-fold and were 72-fold lower than the total. Secretin-receptor levels correlated with variant levels, Deltasecretin, but not Deltacalcium and with tumor location, but not growth, extent, or clinical responses. Variant levels did not correlate with the Deltasecretin. Detailed analysis provides no evidence that variant expression modified the secretin-receptor response or accounted for negative tests. CONCLUSIONS: Secretin-receptor and secretin-receptor-variant expressions occur in all gastrinomas. Because the expression of the total, but not variant, correlated with the secretin results and no evidence for dominant negative activity of the variant was found, our results suggest that the total secretin-receptor density is an important determinant of the secretin test response.

Role of CCK/gastrin receptors in gastrointestinal/metabolic diseases and results of human studies using gastrin/CCK receptor agonists/antagonists in these diseases.

In this paper, the established and possible roles of CCK1 and CCK2 receptors in gastrointestinal (GI) and metabolic diseases are reviewed and available results from human agonist/antagonist studies are discussed. While there is evidence for the involvement of CCK1R in numerous diseases including pancreatic disorders, motility disorders, tumor growth, regulation of satiety and a number of CCK-deficient states, the role of CCK1R in these conditions is not clearly defined. There are encouraging data from several clinical studies of CCK1R antagonists in some of these conditions, but their role as therapeutic agents remains unclear. The role of CCK2R in physiological (atrophic gastritis, pernicious anemia) and pathological (Zollinger-Ellison syndrome) hypergastrinemic states, its effects on the gastric mucosa (ECL cell hyperplasia, carcinoids, parietal cell mass) and its role in acid-peptic disorders are clearly defined. Furthermore, recent studies point to a possible role for CCK2R in a number of GI malignancies. Current data from human studies of CCK2R antagonists are presented and their potential role in the treatment of these conditions reviewed. Furthermore, the role of CCK2 receptors as targets for medical imaging is discussed.

CCK causes PKD1 activation in pancreatic acini by signaling through PKC-delta and PKC-independent pathways.

Protein kinase D1 (PKD1) is involved in cellular processes including protein secretion, proliferation and apoptosis. Studies suggest PKD1 is activated by various stimulants including gastrointestinal (GI) hormones/neurotransmitters and growth factors in a protein kinase C (PKC)-dependent pathway. However, little is known about the mechanisms of PKD1 activation in physiologic GI tissues. We explored PKD1 activation by GI hormones/neurotransmitters and growth factors and the mediators involved in rat pancreatic acini. Only hormones/neurotransmitters activating phospholipase C caused PKD1 phosphorylation (S916, S744/748). CCK activated PKD1 and caused a time- and dose-dependent increase in serine phosphorylation by activation of high- and low-affinity CCK(A) receptor states. Inhibition of CCK-stimulated increases in phospholipase C, PKC activity or intracellular calcium decreased PKD1 S916 phosphorylation by 56%, 62% and 96%, respectively. PKC inhibitors GF109203X/Go6976/Go6983/PKC-zeta pseudosubstrate caused a 62/43/49/0% inhibition of PKD1 S916 phosphorylation and an 87/13/82/0% inhibition of PKD1 S744/748 phosphorylation. Expression of dominant negative PKC-delta, but not PKC-epsilon, or treatment with PKC-delta translocation inhibitor caused marked inhibition of PKD phosphorylation. Inhibition of Src/PI3K/MAPK/tyrosine phosphorylation had no effect. In unstimulated cells, PKD1 was mostly located in the cytoplasm. CCK stimulated translocation of total and phosphorylated PKD1 to the membrane. These results demonstrate that CCK(A) receptor activation leads to PKD activation by signaling through PKC-dependent and PKC-independent pathways.

Serum gastrin in Zollinger-Ellison syndrome: II. Prospective study of gastrin provocative testing in 293 patients from the National Institutes of Health and comparison with 537 cases from the literature. evaluation of diagnostic criteria, proposal of new criteria, and correlations with clinical and tumoral features.

In two-thirds of patients with Zollinger-Ellison syndrome (ZES), fasting serum gastrin (FSG) levels overlap with values seen in other conditions. In these patients, gastrin provocative tests are needed to establish the diagnosis of ZES. Whereas numerous gastrin provocative tests have been proposed, only the secretin, calcium, and meal tests are widely used today. Many studies have analyzed gastrin provocative test results in ZES, but they are limited by small patient numbers and methodologic differences. To address this issue, we report the results of a prospective National Institutes of Health (NIH) study of gastrin provocative tests in 293 patients with ZES and compare these data with those from 537 ZES and 462 non-ZES patients from the literature. In 97%-99% of gastrinoma patients, an increase in serum gastrin post secretin (Delta secretin) or post calcium (Delta calcium) occurred. In NIH ZES patients with <10-fold increase in FSG, the sensitivity/specificity of the widely used criteria were as follows: Delta secretin > or =200 pg/mL (83%/100%), Delta secretin >50% (86%/93%), Delta calcium > or =395 pg/mL (54%/100%), and Delta calcium >50% (78%/83%). A systematic analysis of the sensitivity and specificity of other possible criteria for a positive secretin or calcium test allowed us to identify a new criterion for secretin testing (Delta > or =120 pg/mL) with the highest sensitivity/specificity (94%/100%) and to confirm the commonly used criterion for calcium tests (Delta > or =395 pg/mL) (62%/100%). This analysis further showed that the secretin test was more sensitive than the calcium test (94% vs. 62%). Our results suggest that secretin stimulation should be used as the first-line provocative test because of its greater sensitivity and simplicity and lack of side effects. In ZES patients with a negative secretin test, 38%-50% have a positive calcium test. Therefore the calcium test should be considered in patients with a strong clinical suspicion of ZES but a negative secretin test. Furthermore, we found that some clinical (diarrhea, duration of medical treatment), laboratory (basal acid output), and tumoral (size, extent) characteristics correlate with the serum gastrin increase post secretin and post calcium. However, using the proposed criteria, the result of these provocative tests (that is, positive or negative) is minimally influenced by these factors, so secretin and calcium provocative tests are reliable in patients with different clinical, laboratory, and tumor characteristics. A systematic analysis of meal testing showed that 54%-77% of ZES patients have a <50% postprandial serum gastrin increase. However, 9%-20% of ZES patients had a >100% increase post meal, causing significant overlap with antral syndromes. Furthermore, we could not confirm the usefulness of meal tests for localization of duodenal gastrinomas. We conclude that the secretin test is a crucial element in the diagnosis of most ZES patients, the calcium test may be useful in selected patients, but the meal test is not helpful in the management of ZES. For secretin testing, the criterion with the highest sensitivity and specificity is an increase of > or =120 pg/mL, which should replace other criteria commonly used today.

Serum gastrin in Zollinger-Ellison syndrome: I. Prospective study of fasting serum gastrin in 309 patients from the National Institutes of Health and comparison with 2229 cases from the literature.

The assessment of fasting serum gastrin (FSG) is essential for the diagnosis and management of patients with the Zollinger-Ellison syndrome (ZES). Although many studies have analyzed FSG levels in patients with gastrinoma, limited information has resulted from these studies because of their small size, different methodologies, and lack of correlations of FSG levels with clinical, laboratory, or tumor features in ZES patients. To address this issue, we report the results of a prospective National Institutes of Health (NIH) study of 309 patients with ZES and compare our results with those of 2229 ZES patients in 513 small series and case reports in the literature. In the NIH and literature ZES patients, normal FSG values were uncommon (0.3%-3%), as were very high FSG levels >100-fold normal (4.9%-9%). Two-thirds of gastrinoma patients had FSG values <10-fold normal that overlap with gastrin levels seen in more common conditions, like Helicobacter pylori infection or antral G-cell hyperplasia/hyperfunction. In these patients, FSG levels are not diagnostic of ZES, and gastrin provocative tests are needed to establish the diagnosis. Most clinical variables (multiple endocrine neoplasia type 1 status, presence or absence of the most common symptoms, prior medical treatment) are not correlated with FSG levels, while a good correlation of FSG values was found with other clinical features (prior gastric surgery, diarrhea, duration from onset to diagnosis). Increasing basal acid output, but not maximal acid output correlated closely with increasing FSG. Numerous tumoral features correlated with the magnitude of FSG in our study, including tumor location (pancreatic > duodenal), primary size (larger > smaller) and extent (liver metastases > local disease). In conclusion, this detailed analysis of FSG in a large number of patients with ZES allowed us to identify important clinical guidelines that should contribute to improved diagnosis and management of patients with ZES.

Gastrointestinal hormones cause rapid c-Met receptor down-regulation by a novel mechanism involving clathrin-mediated endocytosis and a lysosome-dependent mechanism.

The activated c-Met receptor has potent effects on normal tissues and tumors. c-Met levels are regulated by hepatocyte growth factor (HGF); however, it is unknown if they can be regulated by gastrointestinal (GI) hormones. c-Met is found in many GI tissues/tumors that possess GI hormone receptors. We studied the effect of GI hormones on c-Met in rat pancreatic acini, which possess both receptors. CCK-8, carbachol, and bombesin, but not VIP/secretin, decreased c-Met. CCK-8 caused rapid and potent c-Met down-regulation and abolished HGF-induced c-Met and Gab1 tyrosine phosphorylation, while stimulating c-Met serine phosphorylation. The effect of cholecystokinin (CCK) was also seen in intact acini using immunofluorescence, in a biotinylated fraction representing membrane proteins, in single acinar cells, in Panc-1 tumor cells, and in vivo in rats injected with CCK. CCK-8 did not decrease cell viability or overall responsiveness. GF109203X, thapsigargin, or their combination partially reversed the effect of CCK-8. In contrast to HGF-induced c-Met down-regulation, the effect of CCK was decreased by a lysosome inhibitor (concanamycin) but not the proteasome inhibitor lactacystin. Inhibitors of clathrin-mediated endocytosis blocked the effect of CCK. HGF but not CCK-8 caused c-Met ubiquitination. These results show CCK and other GI hormones can cause rapid c-Met down-regulation, which occurs by a novel mechanism. These results could be important for c-Met regulation in normal as well as in neoplastic tissue in the GI tract.