Molecular organization of prolactin granules. III. Intracellular transport of sulfated glycosaminoglycans and glycoproteins of the bovine prolactin granule matrix

Abstract

The intracellular transport of sulfated glycosaminoglycans (heparan sulfate and chondroitin sulfate) and glycoproteins of the prolactin (PRL) granule matrix, as well as that of PRL, was studied using a system of double-labeled bovine anterior pituitary slices. [(35)S]sulfate was used to label sulfated macromolecules and L-[(3)H]leucine to label PRL. In membraneless granules (isolated from a PRL granule fraction after solubilization of the membrane with Lubrol PX), sulfated glycosaminoglycans and glycoproteins were considerably labeled after a 15- min pulse, while the hormone was still unlabeled. During the chase incubation, the specific radioactivity of granule PRL and the various complex carbohydrate classes first increased, reaching a peak after approximately 40 min, and then began to decline. After 4 h of chase incubation the radioactivity remaining in granule PRL and sulfated complex carbohydrates was 50-60 percent of that observed at 40 min. Thus, in pituitary mammotrophs a pool of sulfated glycoproteins and glycosaminoglycans is transported intracellularly in parallel with PRL. This finding corroborates the previous conclusion (Zanini et al., 1980 J. Cell. Biol. 86:260-272) that sulfated macromolecules are structural components of the granule matrix. The discharge of labeled PRL and complex carbohydrates from the slices to the incubation medium was also investigated. [(35)S]-glycosaminoglycans and glycoproteins were released at a rapid rate during the first 30-40 min of chase incubation, when PRL granules had not yet attained maximum specific activities. By 40 min, their release tended to level off but the radioactivity accumulating in the incubation medium was still much larger (approximately a fourfold increase) than the losses observed concomitantly in PRL granules. These discharge kinetics contrast with that of [(3)H]PRL, which was not released during the 1st h of chase incubation but then began to accumulate at a high rate in the medium, in parallel with its decrease in granules. Dopamin (5 x 10(-7) M) strongly inhibited the release of labeled PRL but had no detectable effect on the release of labeled glycosaminoglycans and glycoproteins or on the discharge of (35)S-macromolecules as revealed by SDS polyacrylamide gel electrophoresis of incubation media. Thus the releases of PRL and sulfated macromolecules have different kinetics and can be dissociated from each other. These data indicate that much of the flycosaminoglycans and glycoproteins release form pituitary slices originates from sites other than PRL granules, and that at least part of the complex carbohydrates of the PRL granule matrix might not be released with the hormone but rather remains associated with the mammotroph cells after exocytosis.