Abstract
A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90 % and contained 3 % of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55 % Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80 % Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB 2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB 2, PGE 2 and 6-keto PGF 1α, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A 4 into leukotriene B 4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB 2, PGE 2 and 6-keto-PGF 1α synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A 4 hydrolase activity since they can produce leukotriene B 4 upon incubation with leukotriene A 4.
Published Version
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