O6-Methylguanine-DNA methyltransferase status in neuroendocrine tumours: prognostic relevance and association with response to alkylating agents.

BACKGROUND: O(6)-Methylguanine-DNA methyltransferase (MGMT) loss of expression has been suggested to be predictive of response to temozolomide in neuroendocrine tumours (NETs), but so far, only limited data are available. We evaluated the prognostic and predictive value of MGMT status, assessed by two molecular methods and immunohistochemistry, in a large series of NETs of different origins. METHODS: A total of 107 patients, including 53 treated by alkylants (temozolomide, dacarbazine or streptozotocin), were retrospectively studied. In each case, we used methyl-specific PCR (MS-PCR) and pyrosequencing for evaluation of promoter methylation and immunohistochemistry for evaluation of protein status. RESULTS: MGMT promoter methylation was detected in 12 out of 99 (12%) interpretable cases by MS-PCR and in 24 out of 99 (24%) by pyrosequencing. O(6)-Methylguanine-DNA methyltransferase loss of expression was observed in 29 out of 89 (33%) interpretable cases. Status of MGMT was not correlated with overall survival (OS) from diagnosis. Progression-free survival and OS from first alkylant use (temozolomide, dacarbazine and streptozotocin) were higher in patients with MGMT protein loss (respectively, 20.2 vs 7.6 months, P<0.001 and 105 vs 34 months, P=0.006) or MGMT promoter methylation assessed by pyrosequencing (respectively, 26.4 vs 10.8 months, P=0.002 and 77 vs 43 months, P=0.026). CONCLUSIONS: Our results suggest that MGMT status is associated with response to alkylant-based chemotherapy in NETs.

Contrast-enhanced harmonic endoscopic ultrasound in solid lesions of the pancreas: results of a pilot study.

BACKGROUND AND STUDY AIMS: Distinguishing pancreatic adenocarcinoma from other pancreatic masses remains challenging with current imaging techniques. This prospective study aimed to evaluate the accuracy of a new procedure, imaging the microcirculation pattern of the pancreas by contrast-enhanced harmonic endoscopic ultrasound (CEH-EUS) with a new Olympus prototype echo endoscope. PATIENTS AND METHODS: 35 patients presenting with solid pancreatic lesions were prospectively enrolled. All patients had conventional B mode and power Doppler EUS. After an intravenous bolus injection of 2.4 ml of a second-generation ultrasound contrast agent (SonoVue) CEH-EUS was then performed with a new Olympus prototype echo endoscope (xGF-UCT 180). The microvascular pattern was compared with the final diagnosis based on the pathological examination of specimens from surgery or EUS-guided fine-needle aspiration (EUS-FNA) or on follow-up for at least 12 months. RESULTS: The final diagnoses were: 18 adenocarcinomas, 9 neuroendocrine tumors, 7 chronic pancreatitis, and 1 stromal tumor. Power Doppler failed to display microcirculation, whereas harmonic imaging demonstrated it in all cases. Out of 18 lesions with a hypointense signal on CEH-EUS, 16 were adenocarcinomas. The sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and accuracy of hypointensity for diagnosing pancreatic adenocarcinoma were 89 %, 88 %, 88 %, 89 %, and 88.5 %, compared with corresponding values of 72 %, 100 %, 77 %, 100 %, and 86 % for EUS-FNA. Of five adenocarcinomas with false-negative results at EUS-FNA, four had a hypointense echo signal at CEH-EUS. CONCLUSIONS: CEH-EUS with the new Olympus prototype device successfully visualizes the microvascular pattern in pancreatic solid lesions, and may be useful for distinguishing adenocarcinomas from other pancreatic masses.

[Gastrointestinal stromal tumors (GIST)<5 cm in size: review of the literature and expert propositions for clinical management]. / Tumeurs stromales gastro-intestinales (GIST) de taille limitée (inférieure à 5cm): revue de la littérature et propositions pour la prise en charge.

Clinical recommendations for diagnosis, treatment and follow-up of GIST have been established. However, management of tumors limited in size, more often diagnosed by gastroenterologists, remains controversial. The aim of this work was in a first part to analyze the literature on GIST less than 5cm in size and in a second part to elaborate propositions for the clinical management based on an expert panel opinion.

Thirteen-month registration of patients with gastroenteropancreatic endocrine tumours in France.

The prevalence, clinical profiles and management of gastroenteropancreatic endocrine tumours (GEP) in France are not known. From August 1, 2001 to September 1, 2002, standardized records on patients with GEP were prospectively completed in 87 participating centres. The total group amounted to 668 patients (median age: 56 years, range: 12-89). WHO performance status was 0/1 for 80.2% of patients. The primary sites were the small bowel and colon (288), pancreas (211), unknown (77), stomach (33), non-digestive primary sites (24), appendix (20), rectum-anus (12), and oesophagus or cardia (3). GEP were functional in 260 patients (39%). Most pancreatic tumours were non-functional (72%). Metastatic disease was observed in 73.4% of cases. Most tumours (85.8%) were well or moderately differentiated. Somatostatin receptor scintigraphy was performed in only 55% of patients. The following treatment modalities were employed: resection of primary tumour: 66%; systemic chemotherapy: 41%; somatostatin analogues: 44 and 26% for GEP of small intestine and pancreas, respectively; interferon: 12%, and intra-arterial hepatic (chemo)embolization in 23 and 15% of GEP arising from the midgut and pancreas, respectively. Despite their low prevalence, well-differentiated GEP represent a significant and heterogeneous clinical group, which warrants improved medical education, referral to expert centres at an early stage, and the design of prospective therapeutic trials.

Expression of beta-catenin in gastroenteropancreatic endocrine tumours: a study of 229 cases.

AIMS: To clarify the role of beta-catenin in digestive endocrine carcinogenesis, a large and representative series of gastroenteropancreatic endocrine tumours was analysed in order to determine the incidence and pattern of beta-catenin changes and to analyse the clinical and histological characteristics of the tumours presenting immunohistochemically detectable changes in beta-catenin expression. METHODS: 229 cases of gastroenteropancreatic endocrine tumours (stomach, 11; duodenum and ampulla, 29; jejunum and ileum, 51; appendix, 13; colon and rectum, 17; and pancreas, 108) were studied by immunohistochemistry to assess the pattern of distribution of beta-catenin (membranous, cytoplasmic or nuclear). DNA was analysed to detect mutations in exon 3 of the CTNNB1 gene. RESULTS: The distribution of immunoreactive beta-catenin protein was membranous in 164 cases, cytoplasmic in 58 cases and nuclear in seven cases. No mutation was detected in exon 3 of the CTNNB1 gene in any case. The seven cases with nuclear accumulation of beta-catenin were large tumours (mean size 44 (standard deviation (SD) 18.5) mm) with metastases, including liver metastases in five cases, high Ki-67 index (mean 34% (SD 16.5%)) and cyclin D1 overexpression; p53 accumulation was detected in six cases. Five patients died of disease; the mean (SD) survival was 13.6 (4.8) months. CONCLUSIONS: Immunohistochemically detectable nuclear accumulation of beta-catenin is infrequent in gastroenteropancreatic endocrine tumours and is usually not associated with mutations in CNNTB1 exon 3. Changes in beta-catenin expression are late events in digestive endocrine carcinogenesis, associated with tumour progression and dissemination.

Bombesin stimulates adhesion, spreading, lamellipodia formation, and proliferation in the human colon carcinoma Isreco1 cell line.

The neuropeptide bombesin and its mammalian homologue, gastrin-releasing peptide (GRP), enhance proliferation in some but not all human tumor cell lines. The pathophysiological relevance of the bombesin/GRP receptor (GRP-R), which is expressed in 30% of human colon tumor cell lines and in 24-40% of native tumors, has not been clearly assessed at this time. We studied the effects of bombesin in the recently characterized human colon carcinoma Isreco1 cell line. Competitive reverse transcription-PCR showed a high GRP-R mRNA level in Isreco1 cells, and binding studies confirmed the expression of bombesin/GRP-subtype receptors (Kd = 0.42 nM; Bmax = 18,000 sites/cell). Exposure to bombesin resulted in an increase of intracellular calcium concentrations. Bombesin (1 nM) induced cell spreading at 24 h (21.7+/-1.6% versus 6.4+/-0.8% in control cells; P<0.01) and markedly increased the formation of lamellipodia. In addition, adhesion of Isreco1 cells to collagen I-coated culture dishes was stimulated in the presence of 1 nM bombesin (69+/-6% versus 42+/-1% in control cells; P<0.01). Finally, bombesin significantly increased [3H]thymidine uptake by Isreco1 cells in a dose-dependent manner, with a first significant response at 0.1 nM and a maximal effect at 100 nM bombesin (192.2+/-9.7% of control). These results clearly indicate that bombesin exerts morphological, adhesive, and proliferative effects on Isreco1 cells, suggesting that expression of the bombesin/GRP-R may contribute to the malignant properties of colon carcinoma cells.

Peptones stimulate both the secretion of the incretin hormone glucagon-like peptide 1 and the transcription of the proglucagon gene.

Truncated glucagon-like peptide (GLP)-1 is a potent incretin. Its synthesis and secretion are modulated by food, but the influence of individual nutrients remains to be established. The hypothesis that protein hydrolysates (peptones) can directly regulate both GLP-1 secretion and proglucagon (PG) gene transcription was tested in this study, ex vivo in the isolated vascularly perfused rat intestine and in vitro in the murine enteroendocrine cell line STC-1. Peptones were albumin egg hydrolysate (AEH) and meat hydrolysate (MH). We demonstrate in these two models that peptones dose-dependently stimulate GLP-1 release, whereas isocaloric quantities of bovine serum albumin or of an amino acid mixture had no stimulatory effect. A strong and rapid increase of PG RNA level was observed in STC-1 cells treated with peptones (14-fold and 7-fold increase after 4 h of incubation with 3% wt/vol MH and AEH, respectively). Peptones also increased the PG RNA level in the colonic PG-expressing cell line GLUTag. In contrast, peptones did not modify the PG RNA level in two pancreatic glucagon-producing cell lines, namely, the RINm5F and INR1G9 cells. The peptone effect in STC-1 cells was completely abolished by blocking transcription before MH treatment. The stability of proglugacon transcripts was not modified by MH treatment, but nascent transcripts were more abundant in STC-1 cells preincubated with MH. Finally, MH treatment strongly stimulated (15-fold stimulation) the transcriptional activity of two PG gene promoter fragments (-1100 and -350 base pair) linked to the CAT reporter gene transiently transfected in STC-1 cells. Overall, peptones evoke an as yet undescribed release of GLP-1 when brought into contact with native intestinal L-cells or with STC-1 enteroendocrine cells. The increased transcription of the glucagon gene in the latter system suggests an important role of protein hydrolysates in the control of not only the secretion but also the synthesis of the incretin hormone.

Regulation of glucagon-like peptide-1-(7-36) amide secretion by intestinal neurotransmitters and hormones in the isolated vascularly perfused rat colon.

Glucagon-like peptide-1 (GLP-1) is promptly released from endocrine cells of the distal part of the gut after oral ingestion of a meal. To test the possibility that hormones produced by the proximal small intestine or transmitters of the enteric nervous system may be involved in the early phase of meal-induced GLP-1 secretion, various intestinal regulatory peptides and neurotransmitters of the gut were administered intraarterially in the isolated vascularly perfused rat colon preparation. The release of GLP-1 in the portal effluent was measured by a specific RIA. Intraarterial infusion of glucose-dependent insulinotropic peptide (GIP) over the concentration range 0.25-1 nM evoked a dose-dependent release of GLP-1, with a maximal response of 350% of the basal value. Tetrodotoxin did not modify the GIP-induced release of GLP-1. Secretin or cholecystokinin did not stimulate the secretion of GLP-1. Bombesin (10(-9)-10(-7) M) provoked a dose-dependent release of GLP-1, consisting of an early peak, followed by a sustained response. Calcitonin gene-related peptide (5 x 10(-8) M) induced a dramatic rise of GLP-1 immunoreactivity in the portal effluent (peak at 800% of the basal value 10 min after the start of infusion). Similarly, the beta-adrenergic agonist isoproterenol at concentrations of 10(-7) and 10(-6) M provoked a pronounced release of GLP-1 (peak at 500% of the basal value with 10(-6) M isoproterenol). Finally, the muscarinic cholinergic agonist bethanechol at a concentration of 10(-4) M evoked a gradual increase in GLP-1 immunoreactivity, which reached a maximal value (900% over basal) at the end of the 30-min infusion period. The lowest concentration of bethanechol used in the present study (10(-5) M) did not increase portal GLP-1 immunoreactivity over the basal value. Tetrodotoxin did not modify the bethanechol-, isoproterenol-, calcitonin gene-related peptide-, or bombesin-induced GLP-1 release. In conclusion, the present study conducted with the isolated vascularly perfused rat colon shows that there are interactions between the two most potent incretins, GIP and GLP-1, probably through an enteroendocrine pathway. Additionally, several transmitters of the gut are potent stimulants of GLP-1 release and, therefore, represent potential tools in the treatment of the noninsulin-dependent diabetes mellitus.

Luminal bile salts and neurotensin release in the isolated vascularly perfused rat jejuno-ileum.

Bile salts in the distal small intestine are strong stimulants of neurotensin (NT) release, but the underlying mechanisms are not known. They were investigated using an isolated vascularly perfused rat jejuno-ileum preparation. Luminal administration of crude ox bile extract (0.25-1.5%, wt/vol) produced a dose-dependent release of NT-like immunoreactivity (NT-LI), with a maximal effect after infusion of 1% bile extract (500% of basal). Pretreatment of the 1% bile extract with the bile salt-sequestering resin cholestyramine (2%, wt/vol) abolished NT-LI release. Taurocholate (TC), the major bile salt in rats, dose dependently increased the release of NT. The maximal secretion of NT-LI was observed after infusion of 20 mM TC (400% of basal). Taurodeoxycholate (20 mM) was as potent as TC in stimulating NT-LI release, but the threshold concentration of taurodeoxycholate for NT-LI secretion was lower than that of TC. Glycocholate and cholate were 2- to 3-fold less potent than TC in releasing NT-LI over the concentration range 5-20 mM. Luminal infusion of oleic acid (sodium salt; 100 mM) increased by 100% the level of NT-LI in the portal effluent, whereas 20 mM oleate had no effect. In contrast, the micellar form of oleic acid (20 and 100 mM) in bile extract (1%) or TC (20 mM) dose dependently reduced the release of NT-LI induced by bile extract or TC alone. Neither intraarterial tetrodotoxin (10(-6) M), EGTA (2 mM), verapamil (5 x 10(-5) M), nor atropine (10(-5) M) had any effect on TC-induced NT-LI release. These results show that the tauro-conjugated forms of cholic and deoxycholic acid are strong stimulants of NT-LI release. The N-cell response is blunted when bile salts are complexed in the lumen by oleic acid. Finally, bile salt-induced NT-LI release is not mediated by intramural nerves and is not dependent on the activation of calcium channels.

Regulation of glucagon-like peptide-1-(7-36) amide, peptide YY, and neurotensin secretion by neurotransmitters and gut hormones in the isolated vascularly perfused rat ileum.

Neurotensin (NT), peptide YY (PYY), and several peptides derived from proglucagon are promptly released from endocrine cells of the distal part of the gut after oral ingestion of a meal, thus suggesting that release of these peptides is partly under neural and/or hormonal control. Our previous studies conducted with a model of isolated vascularly perfused rat colon showed that colonic L cells are highly responsive to several transmitters of the gut and to the hormonal peptide GIP. To test the possibility that hormones produced by the proximal small intestine or transmitters of the enteric nervous system may also modulate the secretory activity of the ileal L cells, various intestinal regulatory peptides and neurotransmitters were administered intraarterially for 30 min in the isolated vascularly perfused rat ileum preparation. The secretory activity of the ileal N cells was comparatively assessed. The release of NT, PYY, and glucagon-like peptide-1 (GLP-1) in the portal effluent was measured with specific RIAs. The muscarinic cholinergic agonist bethanechol at a concentration of 10(-4) M provoked a biphasic release of PYY, GLP-1, and NT, consisting of an early peak followed by a sustained response. Similarly, bombesin (10(-7) M) induced a marked biphasic release of PYY and GLP-1. In contrast, the NT response was essentially monophasic, characterized by an early peak secretion. Tetrodotoxin did not modify the bombesin-induced release of PYY, GLP-1, and NT. The beta-adrenergic agonist isoproterenol at a concentration of 10(-6) M induced a transient rise in portal PYY and GLP-1 concentrations, whereas the effect on NT release was clearly biphasic. Calcitonin gene-related peptide (5 x 10(-8) M) induced a dramatic rise in PYY, GLP-1, and NT immunoreactivities in the portal effluent (peaks at 600%, 500%, and 550% of the basal values, respectively, 4 mi n after the start of infusion). Intraarterial infusion of GIP over the concentration range (0.5-3 nM) evoked a significant increase in portal concentration of the three peptides only at the threshold concentration of 3 nM. Secretin (50 pM) or cholecystokinin (50 pM) did not affect the release of ileal hormones. In conclusion, ileal L and N cells respond to a variety of transmitters of the gut. The pattern of peptide release depends on the cell type studied. The two cosynthesized peptides, PYY and GLP-1, appear to be cosecreted in the conditions of the present study.

Effects of gastrin on circulating levels of somatostatin, pancreatic polypeptide, and vasoactive intestinal peptide in dogs.

The effect of synthetic human gastrin I, infused at two doses, on the concentrations of somatostatin, pancreatic polypeptide, and vasoactive intestinal peptide in portal and systemic blood was studied in six anesthetized dogs. Intragastric pH was maintained at 5.5, and acid output was measured by intragastric titration. Significant increases in somatostatin and pancreatic polypeptide concentrations in portal blood were found with the lower dose of gastrin (0.5 microgram/kg . h) infused for 40 min. When gastrin was infused at 1.5 microgram/kg . h for 100 min, both portal and systemic blood concentrations of somatostatin and pancreatic polypeptide rose significantly. Cimetidine (300 mg, as an iv bolus), given 40 min after the beginning of the second infusion, did not affect gastrin-stimulated release of somatostatin and pancreatic polypeptide, whereas acid output was completely abolished. Vasoactive intestinal peptide concentrations did not change with the infusion of either dose of gastrin. This daily shows that gastrin releases somatostatin and pancreatic polypeptide in a dose-dependent fashion, and since this release was not acid mediated, it appears likely to be a direct action of gastrin.

Regulation of cholecystokinin secretion by peptones and peptidomimetic antibiotics in STC-1 cells.

Peptones are potent stimulants of cholecystokinin (CCK) release in rats, both in vivo and ex vivo in a model of isolated vascularly perfused duodeno-jejunum preparation and in vitro in the intestinal CCK-producing cell line STC-1. The underlying mechanisms were here investigated with this cell line. Protein hydrolysates from various origins (meat, casein, soybean, and ovalbumin; 0.5-1%, wt/vol) dose dependently increased CCK release. Cephalosporin antibiotics, which mimic tripeptides, also stimulated the release of CCK over the concentration range 1-20 mM. The study of concentration dependence of cephalosporin uptake indicated a passive diffusion process at either pH 7.4 or pH 6.0, thus arguing against the involvement of a peptide transporter in CCK secretion. After pertussis toxin treatment (200 ng/ml; 5 h), the peptone- and cephalexin-induced CCK secretion was significantly reduced, suggesting the involvement of pertussis toxin-sensitive heterotrimeric G protein(s) in the secretory activity of STC-1 cells. Consistent with this was the identification by Western blot of G(i2)alpha, G(i3)alpha, and G(o)alpha immunoreactivities in STC-1 cell extracts. Additionally, peptones and cephalexin increased the cellular content in inositol phosphates, whereas a mild increase in cAMP content was restricted to peptone-treated cells. Protein kinase A or C inhibition did not modify peptone- or antibiotic drug-evoked CCK release. The extracellular Ca2+ chelator EGTA (500 microM) and the intracellular Ca2+ chelator BAPTA-AM [1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; 20 microM] abolished the peptone- and antibiotic drug-induced CCK release. Nifedipine and verapamil (10 microM) reduced by about 50% the CCK secretion evoked by these two secretagogues. In conclusion, peptones and some cephalosporins are potent stimulants of CCK release in the STC-1 cell line. The cellular mechanisms involve pertussis toxin-sensitive G protein(s) and are dependent on Ca2+ availability. We suggest that the STC-1 cell line is a useful model to study the molecular basis of peptone-induced CCK secretion.

Bombesin potentiates taurocholic acid-induced neurotensin release in rats.

The hypothesis of synergistic effects between luminal stimulants and the intramural neural network in the control of intestinal neurotensin (NT) release was investigated with an isolated vascularly perfused rat jejuno-ileum. Luminal administration of low doses of taurocholic acid (TC; 1, 5, and 10 mM) provoked only a small increase in NT-like immunoreactivity (NT-LI) in the portal effluent (50%, 100%, and 130%, respectively, above basal). Increasing the concentration of TC to 20 mM induced a strong and sustained release of NT-LI (700% above basal). The arterial infusion of bombesin (BOM; 10(-9) M) induced only a transient rise in NT-LI levels (200% above basal), with a rapid return to basal values. In contrast, 10(-9) M BOM synergistically enhanced NT-LI responses induced by 10 mM TC; the integrated response of NT-LI release was 3.2-fold higher than the sum of responses to 10(-9) M BOM and 10 mM TC alone. These cooperative effects were apparent with 10(-10) M BOM and TC concentrations over the physiological range 1-10 mM. Neither tetrodotoxin (10(-6) M) nor atropine (10(-5) M) was able to modify this synergy. Substance-P and methacholine, two other NT secretagogues, did not potentiate the TC-induced NT responses. In conclusion, BOM potentiated the release of NT induced by TC, thus suggesting that bombesinergic neurons, among other stimulatory neurons of the enteric nervous system, may modulate the sensitivity of N-cells to luminal stimulants.

Stimulation of glucagon-like peptide-1 secretion by muscarinic agonist in a murine intestinal endocrine cell line.

Studies on the cholinergic regulation of intestinal L-cells have been focused on the release of enteroglucagon, but the signal transduction pathways were not defined. These were here investigated by using as index the release of immunoreactive glucagon-like peptide-1 (GLP-1) from the endocrine cell line STC-1, that has been shown to contain proglucagon mRNA transcripts. Abundant GLP-1 immunoreactivity was revealed in STC-1 cells at immunocytochemistry and by RIA. The cell content was 4927 +/- 689 pg/10(6) cells, as measured with antiserum 199D that recognizes specifically the C-terminal amidated forms of GLP-1. The secretion of GLP-1 over a 2-h incubation period amounted to 1.4 +/- 0.3% of the total GLP-1 cell content and was significantly increased by 10 microM forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate to 206% and 574% of control values, respectively. The cholinergic agonist carbachol stimulated GLP-1 secretion in a concentration-dependent manner, maximal release was observed at 1 mM carbachol (228% of the control value). Binding of the muscarinic antagonist [N-methyl-]scopolamine ([3H]NMS) on cell homogenates was time dependent, specific, and saturable. Scatchard analysis revealed one class of receptors (Kd, 14 pM; binding capacity, 20 fmol/mg protein). Carbachol (0.1 microM to 1 mM) dose dependently displaced [3H] NMS binding and increased the intracellular calcium concentration without modification of adenylate cyclase activity. The order of potency of different antagonists, showing a preferential affinity for M1, M2, and M3 muscarinic receptor subtypes, to inhibit [3H]NMS binding, the carbachol-induced increase in intracellular calcium, and carbachol-stimulated GLP-1 secretion, was as follows: atropine (nonselective) > 4-diphenylacetoxy-N-methylpiperidine methiodide (M3) > pirenzepine (M1) > AF-DX 116 (M2). The results of the present study, therefore, demonstrate that secretion of GLP-1 induced by cholinergic agonist depends on muscarinic M3-subtype receptors in the endocrine intestinal cell line STC-1. This system may prove useful to study the cellular mechanisms of GLP-1 secretion.

Effects of somatostatin-14 and somatostatin-28 on bombesin-stimulated release of gastrin, insulin, and glucagon in the dog.

The effects of somatostatin-14 (S14) and somatostatin-28 (S28), a novel intestinal peptide containing somatostatin tetradecapeptide in its C-terminal position, on the bombesin-stimulated release of gastrin, insulin, and glucagon were tested. On iv infusion of bombesin, the increase in the level of glucagon was seen to be twice that of gastrin, and the insulin increase was 8 times that of gastrin. Plasma concentrations of somatostatin were not modified. S14 and S28 inhibited the release of gastrin, insulin, and glucagon; insulin release was inhibited most effectively, with glucagon release next, and gastrin release least inhibited. Based on the exogenous dose needed to produce equal effects, S28 was more potent than S14 on a molar basis, but based on the plasma concentrations needed to produce equal effects, S14 and S28 were equipotent.

Neuromedin-N is not released with neurotensin from rat ileum.

Neuromedin-N (NN) and neurotensin (NT) were shown recently to be encoded in the same precursor molecule. Colocalization and corelease of ileal NT and NN have not yet been demonstrated and were investigated in the rat using antisera that separately recognized intact NT and NN in ileal extracts. Immunofluorescence labeling of full thickness ileal wall revealed that NN-positive fluorescence was only found in the N-cells. However, only 50% of the N-cells also contained NN-like immunoreactivity (NN-LI). This was associated with a level of extractable NN that was 5-fold lower than that of NT. Corelease of NN- and NT-LI was investigated with the isolated, vascularly perfused jejunoileum model by using various substances that were described as potent stimulants of NT release in vivo. Luminal infusion of mixed nutrients, oleic acid (100 mM), glucose (5%), and taurocholic acid (1%) induced a well sustained release of NT, with plateau secretion of about 200%, 120%, 300%, and 700% above basal, respectively. Vascular bombesin (10(-7) M) and carbachol (10(-5) M) provoked a biphasic release of NT, consisting of a transient rise (approximately 600% above basal) followed by a less pronounced but sustained response. HPLC analysis of portal effluent revealed that 70-80% of NT-LI was intact NT. NN-LI was not coreleased with NT even upon vascular coinfusion of phenanthroline, which markedly protected exogenously infused NN. The coexistence but lack of corelease to any significant degree of NN with NT suggests different fates of these two precursor-related peptides within the ileal mucosa.

Peptones stimulate cholecystokinin secretion and gene transcription in the intestinal cell line STC-1.

In rats, protein hydrolysates (peptones) stimulate cholecystokinin (CCK) release both in vivo and in a model of isolated vascularly perfused duodeno-jejunum. However, the mechanisms involved in peptone-induced stimulation of CCK cells are not well understood. In particular, the possibility that peptones may directly interact with CCK-producing cells to stimulate CCK release and gene transcription has not yet been examined. To test this hypothesis, we used the enteroendocrine cell line STC-1. Incubation of STC-1 cells for 2 h with albumin egg hydrolysate over the concentration range 0.01-1% (wt/ vol) caused a dose-dependent release of CCK, with a maximal increase at 1420% of the control value. In contrast, BSA (1%, wt/vol) or a mixture of amino acids (1%, wt/vol) induced a modest rise in CCK secretion. A dose-dependent, hydrolysate-specific, increase in the CCK steady state RNA level was also observed. It was detectable by 2-4 h of peptone treatment and sustained until 24-48 h. Peptones did not increase the CCK RNA level in the colonic CCK-producing cell line GLUTag or in nonintestinal CCK-expressing cell lines, namely the pancreatic cell line RINm5F and the medullar thyroid carcinoma cell line CA77. The peptone-induced increase in the CCK RNA level resulted from enhanced gene transcription, because labeled CCK transcripts from nuclear run-on incubations increased 3-fold when cells were incubated with peptones, whereas the level of beta-actin transcripts was not modified. Finally, peptones dose-dependently stimulated the transcriptional activity of an 800-bp fragment of CCK gene promoter transfected in STC-1 cells. These studies indicate that peptones specifically stimulate CCK secretion and gene transcription in the intestinal cell line STC-1, and that cis-acting elements conferring peptone inducibility are located in the first 800 bp of the 5'-flanking region of the CCK gene.

Immunoreactive gastrin-releasing peptide in medullary thyroid carcinoma.

We have previously shown that immunoassayable concentration of somatostatin (SRIH) was elevated in 70% of 34 consecutive medullary thyroid carcinoma (MTC) tissue samples. In the present study gastrin releasing peptide (GRP)-like immunoreactivity was measured in tissue extracts from these 34 MTC (25 inherited, 7 sporadic, 2 unclassified) and in 7 normal thyroid tissue. Plasma SRIH, calcitonin (CT) and carcinoembryonic antigen were assayed in all patients. Normal thyroid tissue contained less than 61 pmol GRP per g wet weight; in contrast GRP concentration was elevated (62-7800 pmol/g) in 32/34 tumor extracts. The distribution of tissue GRP values were similar in sporadic as well as in familial MTC. We found no significant correlation between tumor GRP concentration and plasma SRIH (r = -0.05), plasma CT (r = -0.24), or plasma carcinoembryonic antigen levels (r = -0.21). Tumor concentrations of immunoreactive GRP and SRIH were positively correlated when logarithmic transformation was used (P less than 0.01). Thus GRP, as well as SRIH, is a major product of tumoral C cells in human MTC when systematically evaluated in a large number of cases.

Effects of the somatostatin analog BIM 23014 on the secretion of growth hormone, thyrotropin, and digestive peptides in normal men.

BIM 23014 (BIM) is a long-acting octapeptide somatostatin analog. We studied the effects of this analog on the secretion of GH, TSH, and gastroenteropancreatic hormones [secretin, motilin, and pancreatic polypeptide (PP)] in normal men. In the first protocol three BIM doses (125, 250, and 500 micrograms) and vehicle were administered sc in random order at 2000 h to eight normal young men. Plasma GH concentrations decreased during the first part of the night only after the highest dose (P less than 0.05). Plasma secretin levels did not change, while plasma motilin decreased after the 250- and 500-micrograms doses (P = 0.05 and P = 0.02, respectively), and plasma PP decreased after all three doses (P less than 0.05, P less than 0.01, and P less than 0.01, respectively) during the first part of the night. In the second protocol, eight men received BIM, administered by constant sc infusion during the night in a dose of 2 mg/12 h, or vehicle, either alone or in association with a 10 ng/kg.min iv GHRH or vehicle infusion. Nocturnal GH secretion was suppressed by the BIM infusion (P less than 0.001). GH secretion, stimulated by GHRH infusion (P less than 0.001), was reduced by concomitant BIM infusion (P less than 0.001) and was pulsatile during the combined infusions. BIM infusion suppressed the physiological nighttime rise in plasma TSH levels. Plasma motilin and PP levels were reduced by BIM, when administered either alone or in combination with GHRH. We conclude that: 1) BIM is capable of reducing GH secretion when administered sc in a dose of 500 micrograms and of abolishing nocturnal GH secretion when constantly infused at a dose of 2 mg/12 h; 2) BIM, constantly infused, reduces the nocturnal rise in TSH secretion; and 3) motilin and PP secretion are more sensitive than that of GH to BIM, as they are reduced by a lower dose.

Effects of human pancreatic tumor growth hormone-releasing hormone (hpGRH1-44-NH2) on immunoreactive and bioactive plasma growth hormone in normal young men.

Synthetic human pancreatic tumor GH-releasing hormone (hpGRH 1-44-NH2) was given by iv bolus injection to 10 normal men at doses of 75, 150, 300, and 600 micrograms. At all doses the plasma GH responses were similar in an individual subject. Among subjects, however, the responses were significantly different, with peak GH concentrations ranging between 9.0 micrograms/liter and 54.9 micrograms/liter. The GH released in response to GRH was bioactive in the Nb2 lymphoma cell multiplication assay. The circulating GH 30 and 60 min after GRH was detected in 3 molecular forms corresponding to little, big, and big-big GH. These forms averaged 50%, 30%, and 20% of the total immunoreactive GH, respectively. The mean rise of plasma somatomedin-C, from 1.86 U/ml to 2.21 U/ml 24 h after GRH, was not statistically significant. A small but statistically significant GRH dose-dependent rise in plasma PRL (mean PRL concentrations 10 min after 600 micrograms GRH, 11.13 micrograms/liter occurred consistently after GRH injection. The evidence that the GH released by GRH is bioactive supports the potential use of GRH for therapeutic applications.