Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels

Abstract

The water permeability of ADH target epithelial cells is believed to be regulated by a cycle of exo-endocytosis of vesicles containing functional water channels. These vesicles were selectively labeled in intact frog urinary bladders with an impermeant fluorescent marker, 6-carboxyfluorescein. Vesicle suspensions containing the labeled endosomes were obtained by homogenization and differential centrifugation of bladder epithelial cells. The osmotic permeability of the endocytic vesicles was measured, using a stopped-flow fluorescence technique, in the absence or in the presence of HgCl2. This permeability was found very high (500 microns/sec) and inhibited by 1 mM HgCl2 (90%), thus confirming the presence of water channels. The labeled endosomes were then separated from the other membrane vesicles by flow cytometry and sorting. Their protein content was analyzed by electrophoresis on ultrathin polyacrylamide gels. Two double bands were found at 71 and 55 kDa as well as a small band at 43 kDa. They respectively correspond to 31, 38 and 10% of the total amount of silver-stained proteins present in the sorted endosomes, while they only represent 2, 4, and less than 1% of the proteins contained in the vesicle suspension, before sorting. These highly enriched proteins (or at least one of them) are likely to be involved in the mechanism of water transport. Associated to their partial purification by differential centrifugation, the sorting of the endosomes by flow cytometry seems a good way to further characterize the water channel.

Distribution and morphology of sacral spinal cord neurons innervating pelvic structures in Xenopus laevis

“…Urine can flow into the bladder from the cloaca but does not necessarily do so (Deucher, 1975). Anurans lack a renal loop of Henle; however, the bladder functions in water and electrolyte regulation (Bagrov et al, 1991;Chester-Jones et al, 1972;Erlij et al, 1992;Frings et al, 1990;Mia et al, 1991;Van der Goot et al, 1992;Whittembury et al, 1987;Yorio et al, 1991). Acetylcholine (ACh) decreases water uptake from the bladder (Bagrov et al, 1991) and causes contraction of bladder smooth muscle, which empties the bladder contents into the cloaca (Burnstock, 1963).…”

Complementary methods to assist subcellular fractionation in organellar proteomics

“…However, due to technical constraints, most of these studies were analytical in nature, though some preparative ones still achieved a significant level of enrichment. Among the latter, we can mention the preparative sorting of phagosomes [73], mitochondria [74] and endosomes [75]. Interestingly, endosomes can be accessed from outside the cell through loading with fluorescent dyes or fluorescently labeled ligands under various conditions [57].…”