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Abstract
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.
Highlights
The subunits are arranged such that cholera toxin (CT)-A occupies the central channel of the CT-B pentamer extending well above the plane of the pentameric ring [3, 4]
Protein bands corresponding to CT-A1 and CT-A were cut from the Effect of Cbz-Gly-Phe-NH2 on the brefeldin A (BFA)-induced Inhibition of CT Cytotoxicity—As shown in Fig. 1 prior treatment of Vero cells with BFA (2 g/ml) strongly suppressed the CT-induced cyclic AMP (cAMP) accumulation which is in agreement with previous studies on other cell types (14 –16)
The dipeptide itself almost completely suppressed the CT-induced cAMP accumulation in all cell types used in this study
Summary
Materials—Highly purified CT and anticholeragenoid antibodies were obtained from List Biological Laboratories (Campbell, CA). Activation of Adenylate Cyclase—For the determination of adenylate cyclase, Vero cells (105 cells) were incubated in 1 ml of serum-free medium 199 buffered with 25 mM Hepes at 37 °C with and without 3 mM Cbz-Gly-Phe-NH2 for 15 min. Crude membranes, obtained by centrifugation of the lysate for 10 min at 16,000 ϫ g were washed and incubated for 10 min at 37 °C in 100 l containing 25 mM Tris acetate buffer, pH 7.4, 5 mM creatine phosphate, 50 IU/ml creatine phosphokinase, 5 mM magnesium acetate, 0.5 mM ATP, 1 mM dithiothreitol, 0.1 mg/ml BSA, 0.01 mM GTP, and 1 mM 3-isobutyl-1-methylxanthine. The cells were washed three times with ice-cold phosphate-buffered saline, incubated in fresh serum-free medium in the presence and absence of dipeptide for the indicated times at 37 °C, gels and analyzed for radioactivity. Values are the means Ϯ S.D. of triplicate assays from one of two similar experiments
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