Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29)

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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°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°C. All the data strongly suggest that: (1) internalization of VIP is receptor-mediated; (2) upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.