Constitutive and basal secretion from the endocrine cell line, AtT-20

To test the possibility that secretory granules were present in the variant, but lacked identifiable markers, the storage and release of free GAG chains were used as a nonspecific granule content marker. In earlier work, 3~S-sulfated GAG chains appeared to segregate as a bulk phase marker into both regulated and constitutive pathways (Burgess and Kelly, 1984) . To measure GAG storage and release from the regu- 15, 30, 45, 60, 90, 120, 150, or 210 min. Secreted (e, &) and in-traceUular (o,zx) GAG chains were recovered from SDSpolyacrylamide by dissolving the gel in tissue solubilizer and quantified by liquid scin-tiUation counting. (C and D) Wild type AtT-20 (C) and variant HYA.15.6 (D) ceils, pulsed and chased as described in A and B, were taken at 60 or 150 min of chase and either stimulated to secrete GAG chains with 5 mM 8-Br-cAMP or not. GAG chains in the secreted media were recovered at either 150 rain of chase (for the 60-min addition) or at 210 rain of chase (for the 150-min addition). (n) Without cAMP;