Abstract 108: Imaging Renin Granule Exocytosis by Total Internal Reflection (TIRF) Microscopy in Mouse Juxtaglomerular Cells: Effect of cAMP and Beta Adrenergic Stimulation

Abstract

Renin is stored in dense core granules in juxtaglomerular (JG) cells. Renin release is highly regulated. However, the kinetics of renin release and mode of granule exocytosis remains unexplored. We developed a new method for real-time visualization of renin granule exocytosis by TIRF microcopy. We hypothesize that renin exocytosis is due to a kiss-and-run mode of exocytosis, where fusion of the granule with the plasma membrane is transient and granule integrity is maintained. To study this, we generated a new adenoviral construct encoding full length mouse renin tagged with Carboxyl-terminus yellow fluorescent protein (Ad-Renin-YFP). First, we characterized its expression and activity in an endocrine pituitary cell line that does not express endogenous renin (Att20 cells). By Western Blot, we observed a band at the expected molecular mass of renin-YFP (70 kDa)(n=4). Ad-Renin-YFP retains its enzymatic activity in Att20 cells since angiotensin I conversion from angiotensinogen was only detected in Att20 cells transduced with renin-YFP (n=3; p <0.01). To monitor exocytosis of renin-YFP, we transduced primary cultures of mouse JG cells and monitored granule movement and their changes in fluorescence intensity within 250 nm of the plasma membrane by TIRF microscopy. Under baseline conditions the average number of docked granules was 16±4 granules per cell (n=12). Translational movement (X-Y planes) of docked granules was negligible. The number of events per cell during 10 minutes was low and no full fusion was detected (1.5 ±0.5 total events per cell, n=4). After stimulation with cAMP, the number and frequency of events increased to 5.3±1.0 events per cell/10 min (n=8, p<0.05). Similarly the number of events increased to 4.25±0.9 when JG cells were stimulated with isoproterenol, without a decrease in the number of granules docked (n=9). While most (73.5%) of the exocytic events occurred from docked granules; only 36.5% of events were caused by recruitment of newcomer granules to the TIRF field. We conclude that in JG cells, full fusion of granules is not the main mechanism of renin exocytosis. The rapid bursts in fluorescence intensity of docked granules suggest that kiss-and-run is the main mechanism of stimulated-renin exocytosis, in a highly regulated process.