Biochemical evidence for internal proteolytic damage during isolation of type II alveolar epithelial cells

Abstract

The metabolic integrity of cells isolated from tissues by proteolytic dissociation is a question of vital importance in the interpretation of studies in such cells. The integrity of cells isolated from protease-treated lung was investigated by preparing subcellular fractions from isolated cells, protease-treated lung, or alveolar macrophages. Activities and distribution of marker enzymes and enzymes of phosphatidylcholine synthesis in these fractions were compared with similar fractions from untreated whole lung and macrophages. Although the isolated cells appeared viable by virtue of trypan blue exclusion, the specific activity of the endoplasmic reticulum marker enzyme NADPH cytochromec reductase was reduced four fold in fractions from cells and tissues treated with protease. In addition, 38% of the enzyme activity was released into the 150,000 ×g supernatant. CDP choline: 1,2 diacylglycerol cholinephosphotransferase activity in similar fractions was reduced by a factor of two to seventeen, depending on the amount of trypsin used in the incubation mixture, with little apparent effect on its subcellular distribution. Phosphatidate phosphohydrolase activity in these fractions was not reduced by protease treatment and was, in fact, increased by elastase digestion. Direct protease treatment of microsomes isolated from normal lung showed that both trypsin and elastase were capable of releasing NADPH cytochromec reductase from the membrane. Under similar conditions, trypsin virtually eliminated choline phosphotransferase activity while elastase was without effect. These results suggest that during isolation of cells from lung by proteolytic dissociation, the isolated cells are damaged by intracellular uptake of protease and that the analysis of microsomal enzyme activities of cells provides a sensitive method for assessing their functional integrity after isolation. The use of this approach may aid in the development of less harsh methods of tissue dispersion and cell isolation and may yield more native cell populations in general and undamaged type II epithelial cells in particular.