Combined ultrastructural and biochemical study of cellular processing of vasoactive intestinal peptide and its receptors in human colonic carcinoma cells in culture.
Desensitization of human carcinoma colonic cells in culture (HT-29) to vasoactive intestinal peptide (VIP) has been reported previously (C. Boissard, J. C. Marie, G. Hejblum, C. Gespach, and G. Rosselin, Cancer Res., 46: 4406-4413, 1986). In the present study, we have determined the ultrastructural localization of VIP and its receptor after exposure of HT-29 cells to VIP monoiodinated on tyrosyl residue 10 together with the molecular forms and the activity of the internalized VIP receptor. Quantitative electron microscope autoradiography showed that after binding at the cell surface, VIP is internalized in heterogeneous endosomes. Cross-linking experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis were performed in different experimental conditions allowing us to selectively obtain cell surface-associated, internalized, or recycled receptors. No detectable alteration of the labeled VIP-receptor complex occurred during the internalization and recycling processes. Furthermore, a loss of the forskolin potentiation of the VIP-induced stimulation of adenylate cyclase was observed after VIP exposure. This feature was time and temperature dependent as was the VIP-induced loss of cell surface receptors, indicating that the internalized VIP receptor is dissociated from the adenylate cyclase.
Hejblum
G
G
Unité de Recherches sur les Peptides Neurodigestifs et le Diabète, INSERM U.55, Paris, France.
Gali
P
P
Boissard
C
C
Astesano
A
A
Marie
J C
JC
Anteunis
A
A
Hui Bon Hoa
D
D
Rosselin
G
G
D000262:Adenylyl Cyclases / Q000032:analysis; D000643:Ammonium Chloride / Q000494:pharmacology; D002277:Carcinoma / Q000378:metabolism* / Q000648:ultrastructure; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D000242:Cyclic AMP / Q000096:biosynthesis; D004705:Endocytosis; D006801:Humans; D008854:Microscopy, Electron; D008970:Molecular Weight; D011964:Receptors, Gastrointestinal Hormone / Q000032:analysis / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D000643:Ammonium Chloride; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D000262:Adenylyl Cyclases