Release of Chromaffin Granule Glycoproteins and Proteoglycans from Potassium‐Stimulated PC12 Pheochromocytoma Cells

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Abstract
Cultured PC12 pheochromocytoma cells were labeled with [3H]glucosamine, and the glycoproteins and proteoglycans released following K-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of K (51.5 mM in Krebs-Ringers-HEPES buffer) results in an .apprx. 6-fold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of K (6 mM). The release complex carbohydrates include chromogranins, dopamine .beta.-hydroxylase, and 2 chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(.beta.1 .fwdarw. 3)N-acetylgalactosamine, and several mono- and disialyl O-glycosidically-linked oligosaccharides, and the tetrasaccharide AcNeu(.alpha.2 .fwdarw. 3)Gal(.beta.1 .fwdarw. 3)[AcNeu(.alpha.2 .fwdarw. 6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23-68%), heparan sulfate (16-23%), and glycoprotein oligosaccharides (16-54%), which are of the tri- and tetraantennary and O-glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.