Real-time imaging of renin release in vitro

Renin release from juxtaglomerular granular cells is considered the rate-limiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect, and work with significant internal errors. To directly visualize and study the dynamics of both the release and tissue activity of renin, we isolated and perfused afferent arterioles with attached glomeruli dissected from rabbit kidneys and used multiphoton fluorescence imaging. Acidotropic fluorophores, such as quinacrine and LysoTrackers, clearly and selectively labeled renin granules. Immunohistochemistry of mouse kidney with a specific renin antibody and quinacrine staining colocalized renin granules and quinacrine fluorescence. A low-salt diet for 1 wk caused an approximately fivefold increase in the number of both individual granules and renin-positive granular cells. Time-lapse imaging showed no signs of granule trafficking or any movement, only the dimming and disappearance of fluorescence from individual renin granules within 1 s in response to 100 μM isoproterenol. There appeared to be a quantal release of the granular contents; i.e., an all-or-none phenomenon. Using As4.1 cells, a granular cell line, we observed further classic signs of granule exocytosis, the emptying of granule content associated with a flash of quinacrine fluorescence. Using a fluorescence resonance energy transfer-based, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS)-conjugated renin substrate in the bath, an increase in EDANS fluorescence (renin activity) was observed around granular cells in response to isoproterenol. Fluorescence microscopy is an excellent tool for the further study of the mechanism, regulation, and dynamics of renin release.

Organic cation transport in rat choroid plexus cells studied by fluorescence microscopy

Quinacrine uptake and distribution were studied in a primary culture of rat choroid plexus epithelial cells using conventional and confocal fluorescence microscopy and image analysis. Quinacrine rapidly accumulated in cells, with steady-state levels being achieved after 10–20 min. Uptake was reduced by other organic cations, e.g., tetraethylammonium (TEA), and by KCN. Quinacrine fluorescence was distributed in two cytoplasmic compartments, one diffuse and the other punctate. TEA efflux experiments indicated that more than one-half of intracellular organic cation was in a slowly emptying compartment. The protonophore monensin both emptied that TEA compartment and abolished punctate quinacrine fluorescence, suggesting that a large fraction of total intracellular organic cation was sequestered in acidic vesicles, e.g., endosomes. Finally, quinacrine-loaded vesicles were seen to move within the cytoplasm and to abruptly release their contents at the blood side of the cell; the rate of release was greatly reduced by the microtubule disrupter nocodazole.

Quinacrine Skin Discoloration Is Associated with Fluorescence of `Y' Chromosome in Male Cells and Malarial Parasites in Malaria

Sokol et al reported yellow discoloration in a child treated with quinacrine for giardiasis. This, as the authors suggest, is a well known harmless side effect of quinacrine. During work with quinacrine fluorescence of the "Y" chromosome, we observed that malarial parasites within red cells of blood smears also fluoresced brightly. At that time we examined a smear of a Nigerian adult male with malaria, who had yellow discoloration due to quinacrine therapy. Not only did we demonstrate quinacrine fluorescence of "Y" chromosomes in white cells of his unstained blood smear but fluorescent malarial parasites were still present in some red cells.

A cytological approach to the role of guanine in determining quinacrine fluorescence response in eukaryotic chromosomes.

Photooxidation is believed to preferentially remove guanine (G) residues from chromosomal DNA. G interspersion, moreover, has been hypothesized as quenching quinacrine (Q) fluorescence in cytological preparations. Hence, we used photooxidation as a tool for inducing possible changes in the Q-banding pattern of Drosophila melanogaster, Drosophila virilis, and Mus musculus metaphase chromosomes. An enhanced Q fluorescence, which was particularly evident in certain chromosomal regions, was found. This finding would support the postulated primary role of G in determining Q bands in eukaryotic chromosomes.