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Objective Secretin is a key regulator of pancreatic secretion, but the molecular basis of its action is not well understood, especially in the calf pancreas. Our study investigated the expression and functional competence of secretin receptors (SEC-R) in calf pancreatic membranes. Methods We used reverse transcriptase-polymerase chain reaction, sequencing, and Northern blot to assess the expression of the SEC-R gene. The functional characterization of SEC-R was accomplished using adenylate cyclase (AC) assay. Results We successfully amplified, by reverse transcriptase-polymerase chain reaction, a fragment of the SEC-R gene from 119-day-old calf pancreas. This sequence shows higher homology with SEC-R than with vasoactive intestinal polypeptide (VIP)-1 and VIP-2 receptors from other species. Northern blot analysis detected a 1.8-kb transcript. Accordingly, secretin stimulates AC activity in calf pancreatic membranes isolated from 28- and 119-day-old animals with a potency (Ka) of 1.9 to 2.7 nmol/L. Maximal AC stimulation induced by secretin represented a 3- to 4-fold increase of basal activity. AC activation by secretin was inhibited by the 2 SEC-R antagonists, [psi4,5] secretin (l micromol/L) and [5-27] secretin (10 micromol/L). Interestingly, [psi4,5] secretin was ineffective against VIP-induced AC stimulation. Conclusion Our data indicate that secretin exerts a direct action on pancreatic secretion through specific SEC-R coupled to the AC system. Calf pancreatic SEC-Rs are coexpressed with VIP-2 receptors that we previously identified by ligand binding and cross-linking experiments.

The molecular and functional expression of serpentine membrane receptors for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and calcitonin (CT) were characterized in human thymus and thymomas from myasthenia gravis (MG) patients and thymic epithelial cells either in primary culture (PTEC) or transformed by the siman virus 40 large T (SV40LT) oncogene (LT-TEC). Using RT-PCR combined with Southern analysis, we identified the PCR products corresponding to the receptor (-R) transcripts for VIP, CGRP, and CT in thymus from control subjects and MG patients with either hyperplasia or thymoma. Similar expressions of the VIP- and CGRP-R transcripts were observed in PTEC, whereas the CT-R message was not detected. In LT-TEC, the signals for VIP-R, CGRP-R, and CT-R transcripts were seen with a lower intensity than those in control and MG thymus. In agreement with our molecular analysis, 1) VIP was the most potent peptide among VIP-related peptides (VIP > PACAP > PHM > PHV) to stimulate cAMP production through specific type 1 VIP receptors in both PTEC and LT-TEC; 2) cAMP generation was induced by CGRP in PTEC and by CT in LT-TEC; 3) in frozen thymic sections and by flow cytometry, type 1 VIP-R, CGRP-R, and CT-R were localized in epithelial cells; and 4) in parallel, the transcription of the acetylcholine receptor α subunit (the main autoantigen in MG) was induced by CGRP and CT in PTEC and LT-TEC, respectively. Our data suggest that the neuroendocrine peptides VIP, CGRP, and CT may exert functional roles during MG and malignant transformation of the human thymus.

Attempts at altering plasma glucose and, as a consequence, food intake were performed in fed broiler chickens by single i.v. injection of des-His1(Glu9) glucagon amide (a glucagon antagonist) or a non-stimulating anti-insulin serum. Plasma glucose level was not altered by des-His1(Glu9) glucagon amide but was rapidly and largely increased (for at least 2 h) by the injection of the insulin-immune serum. Hour and cumulative food intake were unaltered up to 10 h post injection. These results strongly suggest that in fed chickens, plasma glucose is mainly, if not exclusively, controlled by plasma insulin, and that the transient and heavy hyperglycemia evoked by inhibiting insulin action does not alter food intake.

Molecular markers can improve staging and predict aggressive clinical behavior in esophageal cancer, thus helping to define appropriate therapeutic protocols and to identify patients who will benefit from surgery. We therefore characterized, by Northern blot and/or immunohistochemistry, the relative expression of three effectors involved in the invasion, angiogenesis, and dissemination of tumor cells in esophageal cancer versus nontumoral mucosae: (a) stromelysin-3 (ST3), a member of the metalloproteinase family; (b) basement membrane 40/secreted protein acidic and rich in cysteine (BM-40/SPARC), an extracellular matrix-associated protein involved in angiogenesis; and (c) the hepatocyte growth factor receptor MET, which triggers the scattering of epithelial cells. Results were analyzed in relation to clinicopathological parameters (cpTNE) including tumor size (T), lymph node status (N), periesophageal tissue invasion (E), disease recurrence, and overall survival. The ST3, BM-40/SPARC, and MET genes were found to be overexpressed in tumor samples compared to control mucosa. BM-40/SPARC and MET mRNA levels were not linked to any one of the cpTNE, indicating that this overexpression occurs at an early stage of neoplastic progression. In contrast, ST3 expression, identified by immunohistochemistry in fibroblastic cells surrounding neoplastic islets, correlated with tumor size and periesophageal tissue invasion. Of the 36 patients studied, those with high ST3 levels had shorter disease-free survival than those with low levels, but there was no relationship between the cpTNE and disease recurrence or survival. Our study demonstrates that ST3, BM-40/SPARC, and MET are involved in different steps of esophageal carcinogenesis and that ST3 overexpression is a marker of aggressive clinical behavior. We conclude that in esophageal cancer, ST3 might help to assess survival and the risk of recurrence after surgical resection.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.