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Dispersed pituitary cells were labeled with [35S]sulfate (1 h), followed by a chase incubation (up to 2 h), in order to study sulfate incorporation and transport in anterior pituitary cells. The initial site of incorporation of sulfate, the kinetics of sulfate transport, and the intracellular localization of incorporated sulfate were studied by quantitative electron microscope autoradiography. Analysis of autoradiograms from estrogen-treated female rats revealed that all granulated cell types incorporate sulfate. The labeling index (relative [35S]sulfate incorporation per unit area of cytoplasm) of the various cell types was greatest for mammotrophs, slightly less for corticotrophs, gonadotrophs, and thyrotrophs (grouped together), and least for somatotrophs. Results of grain counts on mammotrophs indicated that initially (end of pulse) approximately 70% of the grains were localized in the Golgi region. After 1 and 2 h of chase, there was a decline in radioactivity in the Golgi region (to approximately 30% of the total grains), but the percentage of the grains associated with mature granules progressively increased (from 13% to approximately 40% of the total). Analysis of the relative grain density (percentage of total grains/percentage of total area) indicated that at the end of a 1-h pulse, [35S]sulfate is most concentrated in immature PRL granules and the Golgi cisternae: during 1-2 of chase it becomes progressively concentrated in mature PRL granules. Findings in somatotrophs (and other cell types) from estrogen-treated female rats or normal males were similar to those in mammotrophs. These results indicate the [35S]sulfate is initially incorporated into the Golgi complex of all anterior pituitary cell types. The majority of the sulfate-labeled macro-molecules are then packaged into immature secretion granules in the Golgi region, which become mature granules. In addition, a considerable amount (approximately 30% in mammotrophs) of the radioactivity remains associated within the Golgi region for up to 2 h post pulse. The incorporation of sulfate into the Golgi complex and its transfer to secretory granule membranes and/or contents thus appears to be a general property of anterior pituitary cells.

Dispersed pituitary cells prepared from estrogen-treated female rats were subjected to pulse labeling with [3H]leucine (5 min) followed by a chase incubation (up to 3 h) in order to study intracellular transport of PRL in mammotrophs. Sites of synthesis, rates of transport, and sites of packaging and storage of PRL were determined by quantitative electron microscopic autoradiography. Results of grain counts show that label is initially (end of pulse) distributed randomly over the rough endoplasmic reticulum (ER), but rapidly (5--15 min of chase) moves to the stacked Golgi cisternae where concentration into secretion granules takes place. The label moves successively from small (Type I) immature granules (15--55 min of chase) to large (Types II and III) polymorphic granules (55--115 min) in the Golgi region, to rounded or ovoid mature (Type IV) granules (55--185 min) usually found in the peripheral cytoplasm, indicating that these types of granules represent successive stages in granule concentration and assembly. Analysis of the relative grain density (percentage of total grains/percentage of total area) confirmed that there was progressive concentration (up to 20--150 times) along the transport route with the concentration lowest in the ER, higher in the Golgi, and highest in immature and mature secretion granules. These data indicate that synthesis of PRL occurs randomly in the ER, transport to the Golgi occurs rapidly (within 5--10 min), and is completed rapidly (90% within 15--20 min), and concentration into granules and aggregation of small granules into larger forms also occurs rapidly (by 15--20 min), but goes on over a prolonged period of time (up to 3 h). Use of dispersed cells has allowed a more precise determination of the location and kinetics of steps in the intracellular processing of PRL than has been possible previously using other systems.

Pituitary cells maintained in monolayer culture for 48 h were used for double isotope labeling to study the release of newly synthesized vs. old PRL. The intracellular pathway taken by newly synthesized PRL was studied by autoradiography. For double labeling the cells were first incubated in a 14C-labeled amino acid (4 h) and then in a 3H-labeled amino acid (1 h), each followed by a 1-h chase. Total PRL (RIA) and radiolabeled PRL (immunoprecipitation) were determined in both cells and media. Thre rate of release of PRL (RIA) was stable throughout the experimental period. In unstimulated cells the ratio of 3H- to 14C-labeled PRL in the medium at the end of a 1-h incubation following the second chase was twice that in the cells at the beginning of this incubation, indicating that newly synthesized [3H]PRL was preferentially released. Stimulation with TRH resulted in increased release of older [14C]PRL. The earliest that radiolabeled (14C or 3H) PRL could be detected in the medium was 15--30 min after exposure of the cells to isotope. For autoradiography, cells were given a 5-min pulse of [3H]leucine, followed by chase periods of 30--180 min. Grain counts indicated that mammotrophs maintained in culture for 48 h transported and packaged PRL with the same time course as those preparations studied previously. Analysis of the distribution of total grains per cell in mammotrophs revealed the presence of several functional subpopulations with different numbers of total grains per cell, indicating that some cells were manufacturing and secreting PRL at a very fast rate. It is concluded that 1) preferential release of newly synthesized PRL is due to preferential discharge of newly synthesized granules, since release occurs at a time when labeled hormone is already in the granules; 2) there is no evidence that the established secretory pathway is bypassed, since transport, concentration, and granule formation occur; and 3) preferential release of newly synthesized PRL is the result of functional heterogeneity within the mammotroph population. Functional heterogeneity is illustrated by major differences in both synthetic rates and TRH responsiveness.