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ATP release from freshly isolated guinea‐pig bladder urothelial cells: a quantification and study of the mechanisms involved
Author(s) -
McLatchie Linda M.,
Fry Christopher H.
Publication year - 2015
Publication title -
bju international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.773
H-Index - 148
eISSN - 1464-410X
pISSN - 1464-4096
DOI - 10.1111/bju.12954
Subject(s) - extracellular , calcium , intracellular , urothelial cell , population , calcium in biology , pannexin , purinergic receptor , guinea pig , cell , chemistry , biology , microbiology and biotechnology , biophysics , biochemistry , connexin , gap junction , urothelium , endocrinology , medicine , urinary system , environmental health , organic chemistry
Objectives To quantify the amount of ATP released from freshly isolated bladder urothelial cells, study its control by intracellular and extracellular calcium and identify the pathways responsible for its release. Materials and Methods Urothelial cells were isolated from male guinea‐pig urinary bladders and stimulated to release ATP by imposition of drag forces by repeated pipetting. ATP was measured using a luciferin‐luciferase assay and the effects of modifying internal and external calcium concentration and blockers of potential release pathways studied. Results Freshly isolated guinea‐pig urothelial cells released ATP at a mean ( sem ) rate of 1.9 (0.1) pmoles/mm 2 cell membrane, corresponding to about 700 pmoles/g of tissue, and about half [49 (6)%, n = 9) of the available cell ATP . This release was reduced to a mean ( sem ) of 0.46 (0.08) pmoles/mm 2 (160 pmoles/g) with 1.8 m m external calcium, and was increased about two‐fold by increasing intracellular calcium. The release from umbrella cells was not significantly different from a mixed intermediate and basal cell population, suggesting that all three groups of cells release a similar amount of ATP per unit area. ATP release was reduced by ≈50% by agents that block pannexin and connexin hemichannels. It is suggested that the remainder may involve vesicular release. Conclusions A significant fraction of cellular ATP is released from isolated urothelial cells by imposing drag forces that cause minimal loss of cell viability. This release involves multiple release pathways, including hemichannels and vesicular release.