Membranes Of The Adrenal Chromaffin Cell

The function of the adrenal medullary chromaffin cell was studied using both purified subcellular fractions and isolated intact cells. These cells are specialized to secrete catecholamines into the bloodstream in response to autonomic nervous system stimulation and have served as a model system to study secretion for many years.The polypeptide components of the chromaffin granule, the subcellular organelle of the chromaffin cell specialized to store and secrete catecholamines, were analyzed by the recently developed technique of two dimensional polyacrylamide gel electrophoresis. This method separates polypeptides on the basis of both molecular weight and isoelectric point and represents a dramatic improvement in resolution compared to previously available techniques.The secretory proteins of the chromaffin granule, referred to as the chromagranins, are highly acidic and migrate to the acidic region of the gel. Included in the secretory proteins is the soluble form of the enzyme dopamine-B-hydroxylase (DBH), which we identified by co-migration with purified DBH. This enzyme appears to be made up of multiple components, as reflected in our gels by four discrete spots with slightly different isoelectric points.The chromaffin granule membrane pattern is considerably more complex than that of the secreted proteins. The membrane form of DBH, prominent among the granule membrane proteins, appears to be identical in all respects to the soluble form. Of particular interest in the membrane pattern is a unique group of proteins at an approximate molecular weight of 35,000, but with regularly spaced isoelectric points.The topology of the membrane proteins was determined by radioiodination of intact granules followed by gel electrophoresis of the purified membranes. Most of the proteins could be labelled to some extent, suggesting that they face the outside of the granule. The membrane form of DBH does not label, and appears to face the inside of the granule. When membranes are dissolved in detergent and iodinated, all proteins are labelled.Granule membranes apear to have many proteins in common with an adrenal medullary microsome fraction which does not include the granule marker BDH. While the relative proportion of shared proteins varies, it seems clear that the granule membrane is derived biochemically from pre-existing membranes and is not synthesized de novo. Granule membranes appeared to have little homology with purified adrenal medullary mitochondria.A cell culture system was developed for collagenase digests of fresh adrenal medulla. After two days in culture, these cells acquired similar secretory characteristics to those of the fresh intact perfused gland. These cells remain stable in culture for up to two weeks.When intact cells are iodinated during secretion, labelled granule membrane could be retreived from the cells two hours later as intact granules. DBH, a protein thought to face the internal side of the granule, was labelled, satisfying the topological requirements of exocytosis. This is direct biochemical evidence for the recycling of used granule membrane after secretion, an idea inferred earlier by relative rates of membrane and secretory protein synthesis and morphological studies employing tracers