A dipeptide metalloendoprotease substrate completely blocks the response of cells in culture to cholera toxin.
Prior exposure (15 min at 37 degrees C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mm N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH(2)), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH(2), an inactive analogue of Cbz-Gly-Phe-NH(2). The effect was reversible and dose- (IC(50) as low as 0.2 mm depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH(2) during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A(1) peptide of CT and NAD(+) activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH(2)-treated cells or when Cbz-Gly-Phe-NH(2) was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH(2) and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH(2) on CT cytotoxicity. Several lines of evidence indicate that a perturbation of intracellular Ca(2+) homeostasis by Cbz-Gly-Phe-NH(2) is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of (125)I-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH(2) did not completely suppress the formation of the catalytically active A(1) fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH(2) cannot be excluded.
De Wolf
M J
MJ
RUCA Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B2020 Antwerp, Belgium. mjdewolf@ruca.ua.ac.be
D000262:Adenylyl Cyclases / Q000187:drug effects; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology*; D004285:Dogs; D004336:Drug Antagonism; D004351:Drug Resistance; D004721:Endoplasmic Reticulum / Q000187:drug effects*; D006056:Golgi Apparatus / Q000187:drug effects*; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000494:pharmacology; D014709:Vero Cells
D004151:Dipeptides; D011480:Protease Inhibitors; D020126:Brefeldin A; C066375:carbobenzoxyglycylphenylalanine amide; D002772:Cholera Toxin; D000242:Cyclic AMP; D008666:Metalloendopeptidases; D000262:Adenylyl Cyclases; D002118:Calcium