Abstract. The biochemical events and components responsible for ATP-dependent Ca2+-activated secretion remain to be identified. To simplify the molecular dissection of regulated secretion, we have resolved norepinephrine (NE) secretion from semi-intact PC12 cells into two kinetically distinct stages, each of which was studied separately to discern its molecular requirements. The first stage consisted of MgATPdependent priming of the secretory apparatus in the absence of Ca 2+. MgATP-dependent priming was readily reversible and inhibited by a broad range of protein kinase inhibitors. The second stage consisted of Ca2+-triggered exocytosis which, in contrast to priming, occurred in the absence of MgATP. Both priming and triggering were found to be dependent upon or stimulated by cytosolic proteins. The priming and triggering activities of cytosol were functionally distinct as indicated by differing thermolability. Furthermore, active components in cytosol resolved by gel filtration were found to support either priming or triggering, but not both. For both priming and triggering reactions, several peaks of activity were detected; one of each type of factor was partially purified from rat brain cytosol, and found to be enriched for stage-specific activity. Two partially purified factors exhibiting stagespecific activity, a ~,,20-kD priming factor and "o300-kD triggering factor, were able to support regulated secretion as effectively as crude cytosol when used sequentially in the partial reactions. Further characterization of stage-specific cytosolic factors should clarify the nature of MgATP-and Ca2+-dependent events in the regulated secretory pathway.

Renal tubular fluid is supersaturated with calcium and oxalate ions, which can nucleate to form crystals of calcium oxalate monohydrate (COM), the most abundant constituent of kidney stones. However, the mechanisms by which nascent crystals are retained in the nephron and then grow into kidney stones are unclear. An interaction of COM crystals with the surface of renal epithelial cells could be a critical initiating event in nephrolithiasis. To investigate this possibility we used cultures of monkey kidney epithelial cells (BSC-1 line) as a model system and found that [14CJCOM crystals bound to the cell surface within seconds. Scanning electron microscopy revealed that crystals bind first to apical microvilli, which subsequently migrate over the crystalline surface. When visualized by transmission electron microscopy, intracellular crystals were located within vesicles. Cytoskeletal responses to crystal uptake were sought by immunofuorescence microscopy, which revealed concentration of (3). Before use, crystals were sterilized by heating to 180TC overnight and then suspended in distilled water to form a slurry from which they were added to the culture medium (3). X-ray crystallography, performed by S. Deganello (University of Chicago), demonstrated that heating did not alter the structure of COM crystals.[14C]COM crystals (10-300 Mig/ml; 2.4-70.8 pg/cm2 cell surface) were added to high-density, quiescent cultures in 60-mm dishes (Nunc) to define the kinetics of association between a cell and crystal. After a specified period, the medium was aspirated, and the monolayer was washed three times with phosphate-buffered saline (PBS; 5 ml). Each culture was inspected under a microscope and the number of cells with adherent crystals was counted in five separate fields. Subsequently, the cell monolayer with adherent crystals was scraped directly into a scintillation vial containing 6 M HC (0.5 ml), 4.5 ml of Ecoscint (National Diagnostics) was added, and the amount of radioactivity was measured. To investigate the effect of crystal exposure for up to 15 days on renal epithelial cells, COM crystals (50 pg/ml) were added to near-confluent cultures of BSC-1 cells (106 cells per 60-mm dish) containing 0.5% calf serum. Every 4 days thereafter, the medium was aspirated and replaced with fresh medium containing 0.5% calf serum with no additional crystals. At 1, 8, and 15 days after addition of crystals, the medium was aspirated, and a solution of crystalline trypsin was used to detach the cells, which were then inspected under a microscope as described (4). The total number of cells in each culture was counted with a hemocytometer, and 100 cells from each culture were scored for the presence of internalized crystals.Abbreviations: COM, calcium oxalate monohydrate; TEM, transmission electron microscopy; SEM, scanning electron microscopy. tTo whom reprint requests should be addressed. 6987The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...