The purpose of this investigation was to quantify free cholesterol biochemically and in radioautographs of 3H‐digitonin cholesterol complex in fasciculata cells of control and stressed rat adrenal cortex. Stress was induced by ether, laparotomy, and adrenal and intestinal handling. Control rats were anesthetized with nembutal. All animals were killed ten minutes from the beginning of anesthesia. The adrenals were excised and either fixed in glutaraldehyde containing 3H‐digitonin or homogenized for biochemical determination of free cholesterol. The plasma corticosterone level of each animal was measured. The fixed adrenals were processed, using different methods of dehydration and embedment, for light and electron microscopic radioautography. The mean number of silver grains (X̄) per unit area of zona fasciculata was counted from light microscopic radioautographs. Crystals of cholesterol‐digitonide complex were more numerous in stressed fasciculata cells, particularly over SER. Silver grains were localized over or close to the crystals. The X̄ for stressed rats was significantly higher than control values, indicating more free cholesterol in fasciculata cells of stressed rats. The results were not affected by either the method of dehydration or the type of embedding medium used. The morphologic results were substantiated by biochemical findings of increase in free cholesterol in adrenals of stressed rats. Plasma corticosterone was significantly high in stressed rats. The increase in free cholesterol in stimulated fasciculata cells is consistent with a previously reported increase in cholesterol esterase activity after ACTH stimulation.
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