Recent findings in cell biology have demonstrated there are several kinds of active sorting from the trans-Golgi network in all cells. The presence of several sorting pathways, using more than one sorting signal, in neuroendocrine cells means that mutations that direct a hormone to a constitutive pathway instead of a regulated one may not simply be interpreted as a signal for sorting to a regulated pathway. The use of three-dimensional electron microscopy of lactotrophs and the possibility that the trans-Golgi network is consumed during sorting has suggested a major role for hormone aggregation, not only as a sorting mechanism, but also as a mechanism for granule formation, in that other transport vesicles may bud from the trans-Golgi network, leaving the aggregated protein as the dense core granule. If aggregation plays such a role, it is unclear how it works in cases where the prohormone must be processed one or more times; does a porous aggregate or colloid form? Obtaining information about the kinds of aggregates that occur in cells is difficult, because at this time there is not a definitive way of knowing whether an aggregate that occurs in solution also occurs in cells. Although secretory granule proteins tend to aggregate relatively easily in solution, the separate treatment of different secretory granule proteins in the same cell make it unlikely that aggregation is a purely passive process, but suggests that the process of aggregation of each hormone is actively controlled in cells. Even if the ability to aggregate accounts for most of the sorting of cargo-secretory granule proteins into granules, other sorting must still occur to get correct membrane proteins necessary for transport and exocytosis into secretory granule membranes. Possible recognition sites for these secretory granule membrane proteins include the cargo itself in an aggregated form, membrane lipids in some unrecognized way, or the proteins and factors that specifically control aggregation of the cargo.

GH4C1 cells are a rat pituitary tumor cell strain that secretes PRL and GH but contains almost no secretory granules. Treatment of GH4C1 cells with a combination of estradiol (1 nM), insulin (300 nM), and epidermal growth factor (10 nM) increased the cellular content of PRL by more than 30-fold above control levels but only increased PRL accumulation in the medium 6-fold. To determine whether the increase in intracellular PRL was accompanied by an increase in secretory granules, we compared the numbers of granules in ultrathin sections from untreated GH4C1 cells and from cells treated with the combined hormone regimen and found a nearly 50-fold increase in granule number. Only 75% of the granules stained for PRL by the protein-A gold technique; the other 25% stained for neither PRL nor GH. The occasional granules found in untreated GH4C1 cells stained for PRL. The data demonstrate that the number of granules in GH4C1 cells can be regulated by hormone treatment and that the increase in intracellular PRL is found in storage granules.

A novel PTH-like peptide has recently been purified and cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. We surveyed the expression of mRNAs encoding this peptide in normal tissues by Northern analysis. One or more low abundance hybridizing transcripts was identified in poly(A)+ RNA prepared from human keratinocytes, thyroid, bone marrow, and fibroblasts, from bovine hypothalamus, pituitary, parathyroid, adrenal cortex, and adrenal medulla, and from rat brain, stomach mucosa, and fetal but not adult liver. One or more hybridizing transcripts was also identified in poly(A)+ RNA prepared from a number of established lines, including rat pituitary (GH4), rat pheochromocytoma (PC 12), human osteosarcoma (TE-85), and human medullary carcinoma (TT) cells. Northern analysis of mRNAs from abnormal human parathyroid tissue revealed an overexpression of transcripts for the PTH-like peptide which appeared to be specific for adenomatous or autonomous glands. These findings suggest that the PTH-like peptide is expressed in a number of endocrine and nonendocrine tissues, that it is developmentally expressed in at least one tissue (fetal liver), and that the regulation of its expression is abnormal in human parathyroid adenomas.