Evidence that the [3H]estradiol-binding protein in pancreas is localized in exocrine cells

Extracts of rat pancreas contain significant amounts of an [3H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [3H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [3H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [3H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [3H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [3H]estradiol-binding activity. Since amylase levels remained unchanged 2 days after streptozotocin treatment, during which time beta-islet cells were irreversibly altered, and amylase activity was restored to normal levels by insulin treatment after its depletion in chronically treated animals, it follows that neither amylase nor the [3H]estradiol-binding protein could have been associated with beta-islet cells. This was consistent with the observation that M cells (a tumor line of beta-cells only) and 14B cells (a cloned variant of this insulinoma) had neither detectable amounts of amylase nor [3H]estradiol-binding activity. To determine whether estrogen-binding activity was associated with any other type of islet cell, islets of Langerhans were isolated by the sedimentation procedure of Lacy and Kostianovsky. In this procedure, several washing steps are employed to separate the suspended acinar cells from the denser islets that sediment rapidly. During this isolation procedure, the cells from each wash were analyzed for protein, [3H]estradiol-binding protein, and amylase a