The effects of ACTH on acid phosphatase activity in endosomes, GERL and lysosomes of cultured adrenal tumor cells

Abstract

Cultured cells derived from a mouse adrenocortical tumor transplant are unspecialized in appearance, but produce basal levels of steroids and demonstrate a near-immediate steroidogenic response to ACTH. There is biochemical evidence that ACTH induces increases in the uptake of serum lipoproteins by these cells and that this material is hydrolyzed in lysosomes to free cholesterol, a precursor for steroid end products. To investigate morphologically the role of lysosomes in the steroidogenic activity of these cells, cultures were incubated for 4 h with and without ACTH, then processed for the ultrastructural localization of acid phosphatase (ACPase), a marker enzyme for lysosomes, and for GERL, the lysosome-forming subcompartment of the Golgi, and examined by TEM and HVEM. Steroid output was determined by a fluorometric technique. Unstimulated cells secreted basal levels of steroids. By TEM, large endosomes, some containing semi-compact material and ACPase reaction product, were occasionally seen at the cell periphery and in the Golgi region. The Golgi and GERL were poorly developed. Residual bodies, a few of them ACPase+, appeared in the Golgi region and in microtubule-associated clusters near the cell membrane. ACTH-stimulated cells secreted steroids at 8-10 fold basal values. In TEM records, they displayed numerous ACPase+ endosomes between the cell periphery and the Golgi. The Golgi and GERL regions appeared to be hypertrophied and many large, inclusion-containing, strongly ACPase+ residual bodies appeared here and in elongated microtubule-containing cell processes. HVEM micrographs showed more definitively that ACTH produced distinct increases in the size of GERL and in the number of ACPase+ organelles. Our results suggest that in unstimulated cells, endosomes, presumably containing media-derived material, gain lysosomal enzymes in or near GERL, are transformed to residual bodies as their contents are hydrolyzed, and are subsequently translocated by microtubules to the cell periphery for exocytosis. ACTH appears to intensify all of these effects. The "giant" lysosomes seen in stimulated cells may result from a fusion of smaller lysosomes. Their amorphous contents may reflect an inefficient hydrolysis of LDL to free cholesterol.