Formation of 6-keto-PGF 1α by collecting tubule cells isolated from rabbit renal papillae

Abstract

Homogeneous populations of collecting tubule epithelial cells have been isolated from rabbit renal papillae by a sequence of procedures involving: (a) dissociation of the tissue by mincing and treatment with trypsin; (b) destruction of contaminating non-collecting tubule cells by differential lysis in hypotonic media and (c) collection and washing by repeated centrifugation. The isolated cells have been characterized as being derived from the collecting tubules on the basis of anatomical source, size and histological staining for both NADH diaphorase activity and cyclooxygenase antigenicity. The cells are judged to be viable by several criteria including their ability to exclude both trypsin and vital dyes, their capacity to metabolize glucose and leucine and their ability to retain distinctive morphology following 10–14 days in culture media. Homogenates of freshly isolated collecting tubule cells when incubated with [ 3H]-arachidonic acid yielded radioactive products identified by thin-layer chromatographic behavior in multiple solvent systems as 6-keto-PGF 1α, PGF 2α, PGE 2, PGD 2 and a monohydroxy acid, probably HHT. No lipoxygenase-like activity was detected. At arachidonate concentrations of 2 μM or less, the major product was 6-keto-PGF 1α; while at substrate concentrations of greater than 10 μM , PGE 2 was the major radioactive prostaglandin formed. Similar distributions of products were observed when homogenates of dissociated renal papillae enriched in medullary interstitial cells were incubated with arachidonic acid. Our results indicate that collecting tubule cells do contain significant prostacyclin synthetase activity and suggest that PGI 2 plays a role in the function of mammalian collecting tubules.