Modulation Of Intestinal Epithelial Cell Restitution And Proliferation In Vitro By Lysophosphatidic Acid.

Gastrin, like other gut peptides, requires extensive post-translationai processing for full biological activity.Progastrin (proG) is cleaved at basic amino acids by a PC to form biologically active carboxyl-terminally amidated gastrin (G-NH2).Previously, we noted that one member of the PC family, PC2, was capable of cleaving heterologously expressed proG in endocrine cell lines.To determine if PC2 was responsible for proG cleavage within antral G-cells we sought to inhibit PC2 expression in an isolated, enriched preparation of canine antral G-ceils with antisense oligonucleotide probes (oligos).Methods: 355 bp of the canine PC2 eDNA was sequenced after amplification by PCR and 18 bp phosphoro-thioate antisense PC2 oligos were constructed.The chosen sequence was located near the translation start site of PC2 and had minimal homology to other PC family members.The same 18 bp, but in a random order, served as a control oligo.After isolation by coUagenase/EDTA digestion and counterflow elutriation, G-cells (1-2 x 106/well) were cultured overnight, incubated for 40 h with oligo (0.1 lag) and 10 lal Superfect in 2 ml standard media, and incubated in methionine-free media for 0.5 h, pulse-labeled for 2 h with -~SS-Met, and cultured for another 6 h in methionine-containing media (chase).Cell extracts and media were immunoprecipitated with antibodies 5135 (G-NH2) and GL-9 (proG).Results: PC2 expression in ceils treated with the antisense oligo was 48 ± 3% (n=6) of control as measured by Western blot.Immunoprecipitated cpms in cell extracts at each time point are depicted below: