Novel Gastrin Receptors Mediate Mitogenic Effects of Gastrin and Processing Intermediates of Gastrin on Swiss 3T3 Fibroblasts ABSENCE OF DETECTABLE CHOLECYSTOKININ (CCK)-A AND CCK-B RECEPTORS

Abstract We have reported previously mitogenic effects of gastrin on several immortalized and neoplastic cell lines, including Swiss 3T3 fibroblasts. Receptor subtypes, cholecystokinin (CCK)-A and CCK-B, for a closely related peptide, cholecystokinin, were recently cloned. These studies were undertaken to investigate if CCK-A- and CCK-B receptors were perhaps mediating the mitogenic effects of gastrin on Swiss 3T3 cells. Receptor antagonists that inhibit the biological effects and binding of peptides to the CCK-A (L-364,718 (L18)) and CCK-B (L-365,260 (L60)) receptors were ineffective toward inhibiting the binding and proliferative effects of gastrin on Swiss 3T3 cells. Radiolabeled L18 and L60 demonstrated no binding to the cells, indicating that CCK-A and CCK-B receptors may be absent on Swiss 3T3 cells. Radiolabeled CCK-8, gastrin, L18, and L60, on the other hand, demonstrated specific binding to a pancreatic cancer cell line (AR42J cells) (used as a positive control). In cross-linking studies the molecular mass of the major band of gastrin receptors (GR) on Swiss 3T3 cells was determined to be 45 kDa. The mitogenic potency of 0.1-1.0 n M gastrin-like peptides on Swiss 3T3 cells was in the order of G1-17 ≥ G1-17-Gly > G5-17 ≥ G5-17-Gly > G2-17 > CCK-8-Gly ≥ G1-17-Lys ≥ CCK-8. The relative binding affinity of the peptides (based on the dose-dependent inhibition of binding of I-G1-17 to Swiss 3T3 cells) was similar to the relative mitogenic potency of the peptides as given above. Furthermore, G1-17-Gly was equally effective as G1-17 in displacing the binding of I-G1-17 to the 45-kDa GR from the Swiss 3T3 cells. Based on these studies it became evident that the novel gastrin preferring GR, expressed by Swiss 3T3 cells, binds and mediates the mitogenic effects of not only the mature (amidated) forms of gastrin-like peptides but also binds and meditates the mitogenic effects of glycine-extended forms of gastrin-like peptides. Possible mRNA expression of CCK-A and CCK-B receptor subtypes by gastrin-responsive rodent intestinal and fibroblast cell lines (Swiss 3T3, IEC-6, CA) was measured by the methods of Northern blot analysis and reverse transcriptase-polymerase chain reaction. mRNA from rat pancreas, AR42J cells, and rat antrum served as positive controls. We were unable to detect CCK-A and CCK-B receptor mRNA in the three cell lines by both the methods. The positive control samples, on the other hand, gave the expected results. Thus the three gastrin-responsive rodent cell lines examined in this study lacked CCK-A and CCK-B receptors, but were positive for the gastrin preferring GR that demonstrated physicochemical and pharmacological attributes distinct from CCK-A and CCK-B receptors. Since colon cancers are known to express high concentrations of non-amidated gastrin-like peptides, a possible autocrine role of glycine extended forms of gastrin becomes a highly relevant clinical question in the light of the presence of novel GR on the fibroblast and intestinal cell lines.