Arachidonic-acid-selective cytosolic phospholipase A2 is involved in gastrin-induced AR4-2J-cell proliferation.

Gastrin/CCK(B) G protein-coupled receptors have been shown to mediate proliferation stimulated by their endogenous ligands. The present study demonstrates the proliferative effect of arachidonic acid on AR4-2J cells. Gastrin induces an [3H]arachidonic-acid release in a dose-dependent manner. The use of a specific inhibitor of cPLA2, AACOCF3 established the involvement of a cPLA2 in the proliferative effect of gastrin. The results also demonstrate that a cytosolic high-molecular-weight PLA2 is activated by gastrin in AR4-2J cells.

Autocrine stimulation of AR4-2J rat pancreatic tumor cell growth by glycine-extended gastrin.

Glycine-extended gastrin (gastrin-Gly) stimulates proliferation of AR4-2J pancreatic tumor cell line through a specific receptor, different from the gastrin-cholecystokinin B receptor. Our purpose was to determine whether AR4-2J cells produced gastrin-Gly and then whether the peptide was involved in an autocrine loop. First, proliferation of AR4-2J cells in serum-free medium was inhibited by a gastrin anti-sense oligodeoxynucleotide phosphorothioate and by antibodies specific for gastrin-Gly. In contrast, antibodies specific for alpha-amidated gastrin were without effect. By using RT-PCR, we have shown that AR4-2J cells expressed gastrin mRNA. The presence of gastrin-Gly, but not alpha-amidated gastrin, in serum-free media was detected by radioimmunoanalysis. Gel chromatography revealed that the predominant molecular forms secreted were glycine-extended gastrin-34 and gastrin- 17. Furthermore, epidermal growth factor (EGF), a stimulator of gastrin gene transcription, modulates gastrin-Gly secretion by AR4-2J. These data together suggest that gastrin-Gly is an autocrine growth factor for AR4-2J cells and that it participates with EGF in the regulation of AR4-2J-cell proliferation.

Silent human pancreatic glucagonoma and "A" nesidioblastosis.

Surgical fragments of healthy and tumor-bearing pancreas from a patient with pancreatic tumor were studied by electron or light microscopy, histochemistry, and immunocytochemistry (human insulin, glucagon, somatostatin, gastrin, and bovine pancreatic polypeptide). Histological results were compared to those obtained by radioimmunoassay, both in tumor and serum. The tumor was identified as a glucagonoma because reactions for Grimelius' silver impregnation and immunoreaction with an antiserum against glucagon were positive and because a very high level of glucagon in the tumor was observed. Insulin, somatostatin, and gastrin levels remained normal, both in tumor and serum, but the glucagon level was normal in serum. Associated with this silent glucagonoma, an uncommon nesidioblastosis was also diagnosed with many A cells irregularly mixed with acinar cells, isolated or clustered in small groups. Acinar "intermediate" cells of "A" type were also observed. Such associative histopathological processes evoked possible development of an endocrine tumor from nesidioblastic-like tissue. Its embryogenic origin remained uncertain.

VIP regulation of a human pancreatic cancer cell line: Capan-1.

VIP and secretin control the secretory function of the normal pancreas. We analysed their regulatory functions in a human pancreatic cancer cell line: Capan-1. Saturation binding experiments with 125I-VIP showed the existence of one class of binding sites of very high affinity: KD 6.4 +/- 3.0 X 10(-11) M and a low Bmax: 12 fmoles/10(6) cells, in both intact cells and membrane preparations. This site has not yet been described in normal or tumorous digestive cells. Competition binding experiments let us characterize two more binding sites, KD: 2.1 +/- 0.7 X 10(-9) M and 5.0 +/- 0.6 X 10(-8) M and the corresponding Bmax: 120 and 500 fmoles/10(6) cells. These sites are similar to those found on cells of the digestive tract. Competition binding experiments gave the following IC50: 3.0 +/- 0.9 X 10(-9) M for VIP; 2 +/- 0.6 X 10(-6) M for PHI; and 1 +/- 0.7 X 10(-5) M for secretin. VIP elicited a cAMP rise, the half maximal response being obtained at 1.2 X 10(-10) M. Secretin induced a cAMP response but only for concentrations higher than 10(-8) M. VIP receptors were found to be modulated by two factors: cell ageing and cell density. Cells chronically treated with VIP showed a slight decrease of their proliferation; insulin exerted an opposite effect. It is concluded that at the difference of normal pancreatic cells, the present cell line lacks secretin-preferring receptors and acquires some of the properties of intestinal cells.

Interaction of [125I]-Tyr4-bombesin with specific receptors on normal human pancreatic membranes.

The binding of bombesin to its receptors on normal human pancreatic membranes was investigated using high specific activity, radioiodinated bombesin ([125I]-Tyr4-bombesin), prepared by an oxidative method with chloramine-T. Binding was specific, temperature-dependent, saturable, reversible and linearly related to membranes protein concentration. After a 30 min period of incubation with membranes the degradation of the tracer has never been found superior to 20%. Scatchard analysis of binding data was compatible with a single class of binding sites with a high affinity (0.96 nM) and a Bmax of 753 fmol/mg protein. [125I]-Tyr4-bombesin binding to human pancreatic membranes was competitively inhibited by (1-Tyr4-)bombesin, GRP, the nonapeptide of bombesin and litorin but not by unrelated hormones such as somatostatin, CCK, human gastrin, etc. These results describe for the first time the presence of specific receptors for bombesin on human pancreatic membranes. The binding characteristics obtained are comparable with those found in other species.

Analysis of somatostatin peptides produced by an endocrine pancreatic cell line.

Biological active forms of somatostatin are produced by cleavage of large precursors. If the sequence of the pre-proform of somatostatin has been deduced from cDNA structure in several species, little is known about the processing of these large precursors. For this purpose, the analysis of immunoreactive components secreted by the R.I.N. cell line was investigated. After selection of a cell population and culture conditions providing the optimal production of these peptides, analysis of their molecular forms was done by molecular gel filtration. The results show that mainly pro-forms accumulate in the culture medium while besides the pre-proform the smaller immunoreactive species behaving like S-28 and S-14 were found in cell extracts. Incorporation studies in serum free medium showed rapid formation of an intermediate compound eluted at 1.87 V0.

[Presence of hormonal polypeptides in the pure pancreatic secretion in man under stimulation by cerulein and secretin]. / Présence de polypeptides hormonaux dans la sécrétion pancréatique pure chez l'homme sous stimulation par sécrétine et céruléine.

We have investigated in stimulated human pancreatic juice the presence of the following peptides: insulin, glucagon, gastrin, somatostatin, VIP and secretin. Collection of pancreatic juice (3 periods: 20 min each) was completed by endoscopic cannulation of the pancreatic duct during the infusion of secretin (0.5 U/kg/h) and cerulein (75 ng/kg/h) in 6 healthy volunteers. Pure pancreatic juice was recovered in the presence of kallikrein inhibitor (iniprol 8,000 U/ml) in refrigerated collection tubes (4 degrees C). The material was acidified, boiled for 5 min and centrifuged. Radioimmunoassays were performed on the supernatant solutions. The elution profiles on Sephadex G 25 gel filtration of the immunoreactivities were compared with standard samples of hormones, immuno-reactive insulin, glucagon and somatostatin were found in every sample: insulin was present at a constant level (50 microU/ml) during the three periods of collection; glucagon was encountered in large amounts in the first sample and decreased significantly during the subsequent periods; somatostatin which occurred at a low level during the first period was significantly increased in the following periods. Gastrin, VIP and secretin were undetectable or only inconstantly found in very small amounts. These results are in agreement with a two-directional secretion of the human pancreatic endocrine cells. The cellular origin and function of these exocrine secreted peptides need further studies.

Purification of radioiodinated somatostatin-related peptides by reversed-phase high-performance liquid chromatography.

In order to set up specific radioimmunoassays for the two N- and C-terminal tetradecapeptides of somatostatin-28 the peptides somatostatin SS14 and SS28 and the somatostatin by-products 1-Tyr-SS14, 11-Tyr-14 and desaminotyrosyl-beta-alanine fragment (1----14) of SS28 were radioiodinated by the chloramine-T or Bolton-Hunter techniques. Reversed-phase high-performance liquid chromatography was shown to be a very efficient and reliable method for the purification of different radioactive reaction media. The corresponding labelled peptides were tested for their relative immunological (radioimmunoassay) and biological (binding studies) properties.