Rat atrial secretory granules and pro-ANF processing enzyme

We examined the possibility that rat atrial granules may contain a pro-ANF processing protease. Isolated atrial granules were lysed either by detergent, osmotic shock or sonication and incubated at 37° C. Pro-ANF processing and/or degradation were followed by radioimmunoassays and Western blotting using three antibodies which are specific either to the N-terminus, the C-terminus or the processing site (98–99) of pro-ANF. Whatever the method used for the lysis of the granules, we failed to detect any production of ANF (99–I26) and ANF (1–98). However, slight degradation of pro-ANF was recorded probably due to contamination by lysosomal proteases. The in vitro system was validated by addition of thrombin to lysed granules which resulted in a rapid disappearance of the immunoreactivity related to the processing site. These results suggest that the rat atrial granules do not contain any active processing enzyme unless adequate incubation conditions were not met to express its enzymatic activity. The atrial granules may not be directly involved in the maturation of pro-ANF.