Calcium-sensing receptor-mediated L-tryptophan-induced secretion of cholecystokinin and glucose-dependent insulinotropic peptide in swine duodenum

Abstract

This study aimed to elucidate the effect of tryptophan (Trp) on gut hormone secretion as well as the roles of the calcium-sensing receptor (CaSR) and its downstream signaling pathway in gut hormone secretion by assessing swine duodenal perfusion in vitro. Swine duodenum was perfused with Krebs-Henseleit buffer as a basal solution. Various concentrations (0, 10, and 20 mM) of Trp were applied to investigate its effect on gut hormone secretion. A CaSR antagonist was used to detect the involvement of CaSR and its signal molecules. The 20 mM Trp concentration promoted the secretion of cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP), elevated the mRNA level of CaSR, and upregulated the protein levels of CaSR, protein kinase C (PKC), and inositol trisphosphate receptor (IP3R). However, NPS 2143, an inhibitor of CaSR, attenuated the CCK and GIP release, reduced the mRNA level of CaSR, and decreased the protein levels of CaSR, PKC, and IP3R with 20 mM Trp perfusion. The results indicate that CCK and GIP secretion can be induced by Trp in swine duodenum in vitro, and the effect is mediated by CaSR and its downstream signal molecules PKC and IP3R.

Keywords: calcium-sensing receptor; gut hormone; signaling pathway; swine; tryptophan.

Fig. 1. Schematic of the perfusion system (A) and the perfusion chamber (B). In (A): 1, oxygen bomb (mixture of 95% O₂ and 5% CO₂); 2, media reservoir; 3, peristaltic pump; 4, silicone tube; 5, perfusion chamber; 6, water bath (37℃); 7, collector. In (B): 1, nylon filter platform; 2, needle; 3, rubber stopper; 4, syringe; 5, rubber plunger.

Fig. 2. The mRNA expression of CaSR (A) and the secretion of gut hormones (B and C) regulated by different concentrations of tryptophan (Trp). The duodenal tissues were perfused with 0 (control), 10, and 20 mM Trp for 120 min. Tissues were collected at the end of perfusion. CaSR (A) mRNA expression was analyzed by quantitative polymerase chain reaction (qPCR) and glyceraldehyde phosphate dehydrogenase was used as the internal control. The concentrations of cholecystokinin (CCK, B) and glucose-dependent insulinotropic peptide (GIP, C) in the perfusate samples were detected every 20 min by using enzyme-linked immunosorbent assay kits. Values are expressed as mean ± SEM (n = 4). A statistical difference was determined by one-way ANOVA followed by Tukey's multiple comparison tests. Bars with the different letters show significant differences compared with the control group (A) (p < 0.05); ^* and δ (B and C); 20 mM Trp and 10 mM Trp treatments are significantly different from the control, respectively (p < 0.05). The influence of Ca²⁺ on the ability of Trp to induce the expression of CaSR (D) and the secretion of gut hormones (E and F). Tissue samples of duodenum were perfused with Krebs-Henseleit buffer containing 0 mM Trp without [Ca²⁺]_e (control) and 20 mM Trp with or without 10 mM [Ca²⁺]_e for 120 min. After perfusion, the expression of CaSR (D) was determined by qPCR. Perfusate samples were collected every 20 min to measure concentrations of CCK (E) and GIP (F). Values are expressed as mean ± SEM (n = 4). Statistical differences were determined by one-way ANOVA followed by Tukey's multiple comparison tests. Bars with the different letters showed the significant differences compared with the control group (D) (p < 0.05); γ (E and F); the combination of 20 mM Trp and 10 mM Ca²⁺ was significantly different from the control (p < 0.05). Effects of CaSR antagonist NPS 2143 on the expression of Trp-induced CaSR (G) and the secretion of gut hormones (H and I). Duodenal tissues were treated with 0 (control), 20, or 20 mM Trp and NPS 2143 (25 µM). At the end of perfusion (120 min), the duodenum tissues were collected to determine the mRNA expression of CaSR (G) by qPCR. The perfusate samples were obtained every 20 min to evaluate the concentration of CCK (H) and GIP (I). Values are expressed as mean ± SEM (n = 6). Statistical differences were determined by one-way ANOVA followed by Tukey's multiple comparison tests. Bars with the different letters showed the significant differences compared with the control group (G) (p < 0.05); χ (H and I); 20 mM Trp and the combination of 20 mM Trp and NPS 2143 treatments are significantly different from the control, respectively (p < 0.05).

Fig. 3. Protein levels of calcium-sensing receptor (CaSR) and its signal molecules regulated by tryptophan (Trp). (A) The protein images of CaSR, protein kinase C (PKC), and inositol trisphosphate receptor (IP3R) at 0 and 20 mM Trp treatments. The optical densities of CaSR, PKC, and IP3R are shown in (B–D), respectively. Values are shown as mean ± SEM. ^**p < 0.01.

Fig. 4. The protein levels of calcium-sensing receptor (CaSR) and its signal molecules regulated by CaSR antagonist NPS 2143. (A) The protein images of CaSR, protein kinase C (PKC), and inositol trisphosphate receptor (IP3R) in the presence of 20 mM tryptophan and NPS 2143. The optical densities of CaSR, PKC, and IP3R are shown in (B–D), respectively. Values are shown as mean ± SEM. ^**p < 0.01.