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Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29)
Abstract:
The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.
Insights
Vasoactive-intestinal-peptide (VIP) binding sites on colon cancer cells rapidly disappear after VIP exposure, indicating receptor internalization. This process is reversible, suggesting VIP receptor regulation in HT 29 cells.
Area of Science:
- Cell Biology
- Molecular Endocrinology
- Gastroenterology
Background:
- Vasoactive-intestinal-peptide (VIP) plays a role in various physiological processes, including cell proliferation and differentiation.
- Understanding VIP receptor dynamics is crucial for investigating its signaling pathways in conditions like colorectal cancer.
Purpose of the Study:
- To investigate the disappearance of VIP binding sites on human colonic adenocarcinoma (HT 29) cells after VIP exposure.
- To characterize the kinetics, specificity, and reversibility of this phenomenon.
- To determine if VIP internalization is receptor-mediated.
Main Methods:
- Studied the loss of 125I-VIP binding sites on HT 29 cells after pre-incubation with VIP.
- Varied time, VIP concentration, and temperature to determine optimal conditions for binding site disappearance.
- Assessed the specificity by measuring epidermal growth factor binding.
- Investigated reversibility by incubating cells in VIP-free medium.
- Quantified 125I-VIP internalization using independent experimental methods.
Main Results:
- Maximum loss of VIP binding capacity (60-80%) occurred after 5-10 min at 37°C with 100 nM VIP.
- The half-life of binding site disappearance was less than 2 min.
- VIP binding site loss was specific and did not affect epidermal growth factor binding.
- Binding capacity was restored by 90% within 60 min in VIP-free medium.
- Maximal internalization of bound 125I-VIP occurred at 10 min at 37°C.
Conclusions:
- VIP internalization is a receptor-mediated process in HT 29 cells.
- Exposure to native VIP leads to VIP receptor downregulation or sequestration.
- These findings provide insights into VIP receptor regulation in colorectal cancer cells.