41] Isolation of rabbit renomedullary cells and arachidonate metabolism

Abstract

The chapter presents a study on isolation of rabbit renomedullary cells and arachidonate metabolism. Novel arachidonic acid (AA) metabolites arising from the so-called third pathway are generated by cells of the medullary thick ascending limb of Henle's loop (mTALH). Evidence for the conversion of AA through the cytochrome P -450 pathway can be attained by the following: (1) the absolute dependency of this pathway, in broken cell preparations, on the presence of NADPH; (2) inhibition of product formation by SKF 525A and other monooxygenase inhibitors; and (3) the failure of indomethacin to affect product formation. In addition to high Na + , K + -ATPase activity, mTALH cells obtained from rabbits have distinct morphological and histochemical characteristics. These epithelial cells are morphologically recognized as being 8–10 μ m in diameter, with large nuclei, numerous mitochondria, and few apical microvilli. The chapter mentions the efficient method of isolating mTALH cells by centrifugal elutriation, a procedure that separates cells according to their sedimentation rate. This method provides a more highly purified suspension (85–95%) of mTALH cells, with sufficient yield (20–30 x 10 6 ) required for the biochemical studies, than can be achieved through microdissection techniques. By using centrifugal elutriation to separate and obtain mTALH cells, it is possible to study AA metabolism by these cells in relative homogeneity, and also to establish the conditions necessary to grow them and preserve cytochrome P -450 activity in culture.