The synthesis, storage, and release of GH and PRL by a clonal strain of rat pituitary tumor cells (GH3) were studied in perifusion. GH3 cells produce GH and PRL without storing the hormones in large amounts. GH3 cells exposed to [3H]leucine during perifusion begin to release [3H]GH and [3H] PRL within 15 min, and by 120 min the rate of immunoprecipitable labeled hormone release reaches a plateau. The calculated specific activity of basally released hormone is relatively constant. Exposure to depolarizing amounts of K+ (21 mM KC1) or stimulatory concentrations of (Bu)2cAMP (1 mM) resulted in initial brief increases in the release rates of GH (204% and 243% of base) and PRL (265% and 336% of base) by RIA. With continued exposure, to excess K+, release of radioimmunoassayable hormone returned to prestimulatory rates (GH, 104% and PRL, 111% of base). With continued (Bu)2cAMP, stimulation of release was maintained (GH, 160% and PRL, 234% of base). The specific activity of GH and PRL released during the late portions of continuous K+ or (Bu)2cAMP stimulation was the same as in basal perifusion. On the other hand, the specific activity of K+− or (Bu)2cAMP-induced acute GH and PRL release fell (GH, 78% and 72% of base, respectively, and PRL, 66% and 56% of base, respectively), suggesting a dilution of released 3H-labeled hormone by a pool of unlabeled intracellular hormone. During continued perifusion in the presence of a single isotope, a second stimulatory pulse was not associated with a lowered specific activity of released hormone. However, sequential use of two isotopes permitted differential alteration of the specific activity of hormone released by serial stimuli. Finally, overnight labeling of GH3 cells that were then perifused for 4 h without the isotopic precursor resulted in a rise of the specific activity of GH and PRL released during (Bu)2cAMP stimulation (GH to 170% and PRL to 162% of base). From these observations the following conclusions were reached: GH3 rat pituitary tumor cells sequester hormone in a K+− and (Bu)2cAMP-sensitive, and slowly turning over, pool which is outside the direct path of intracellular hormone flow from synthesis to release. Once discharged, the sequestered hormone compartment is rapidly reconstituted with concurrently synthesized hormone.
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