FIGURE 4.

Secretion of the p.D22G activation peptide mutant from AR42J rat acinar cells and the effect of trypsin inhibitors. Dexamethasone-differentiated AR42J cells were transfected with recombinant adenovirus carrying wild-type cationic trypsinogen or the p.D22G mutant. The arrow indicates the trypsinogen band. A, trypsinogen levels in the growth medium at 24 h after infection as a function of virus concentration added. Conditioned media (15 μl per lane) were analyzed by Western blotting using an antibody against the Glu-Glu tag at the C terminus of trypsinogens. B, trypsin activity of conditioned media (10 μl) after enteropeptidase activation. Activity was expressed as percentage of the highest activity measured, which corresponded to ∼100 mOD/min reading. The average of three independent transfection experiments is shown with S.E. indicated. Statistical analysis was performed with the Tukey-Kramer multiple comparisons test. *, p < 0.05 and ***, p < 0.001. Note that the high trypsin activity measured even in the absence of transfection is due to endogenous rat trypsinogens secreted by the AR42J cells. C, trypsinogen levels in cell lysates at 24 h after infection, as a function of virus concentration added. Cell lysates (10 μg total protein per lane) were analyzed by Western blotting using an antibody against the Glu-Glu tag at the C terminus of trypsinogens. D, effect of trypsin inhibitors on the secretion of the p.D22G mutant. AR42J cells were transfected with a final virus concentration of 3 × 10⁶ pfu/ml. The inhibitors were included in the growth medium at the indicated final concentrations. Cotransfection of SPINK1 was achieved with an adenovirus construct used at 3 × 10⁶ pfu/ml final concentration. Conditioned media collected at 24 h after infection were analyzed by Western blotting. Representative immunoblots of two or three independent experiments are shown. As a control for the general secretory capacity of AR42J cells, the secretion of endogenous chymotrypsinogens was measured. Conditioned medium (20 μl) was supplemented with 0.1 m Tris-HCl (pH 8.0) and 1 mm CaCl₂ to 50 μl volume, and chymotrypsinogens were activated with 40 nm human cationic trypsin (final concentration) for 1 h at 37 °C. Chymotrypsin activity was then measured at room temperature by adding 150 μl substrate (0.2 mm N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide dissolved in 0.1 m Tris-HCl (pH 8.0) and 1 mm CaCl₂) and following 1-min time courses of p-nitroaniline release at 405 nm in a microplate reader.