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

Sulfated, free GAG chains were generated as described (Burgess and Kelly, 1984) by preincubating cells for 2.5-3 h in DME H21 growth medium containing 0.5 mM 4-methylumbelliferyl ~-v-xyloside and then incubating the cells in sulfate-depleted DME H21 containing xyloside and -35 5 rnCi [ S]sulfate. Cells were chased by incubating for 2.5 h in serum-free medium containing xyloside. The medium was collected, and then analyzed directly on 12-20% polyacrylamide gels that were processed for autoradiography as described above. Pulse-chase experiments to follow free GAG chains were performed by preparing confluent six-well dishes (12 wells), preincobating for 1 h in DME containing 0.5 mM xyloside, shortening the labeling time in 5 mCi [35S]sulfate to 1 h, washing the cells with PBS, and incubating for 15, 30, 45, 60, 90, 120, 150, or 210 rain in complete growth medium. At 60 min of chase, 5 mM 8-Br-cAMP was added to one well of ceils and the media collected at 150 rain. At 150 rain of cbase, cAMP was added to a second and the media collected at 210 rain. At the end of the chase intervals, media were removed from the cells, dialyzed for 24 h at 4°C against three changes of 50 mM NI-LtHCO3, dried in a Speed Vac (Savant Instruments, Hicksviile, NY) for 6 h, dissolved in SDS-PAGE sample buffer, and then analyzed on 12-20% polyacrylamide gels as described above. Cell extracts were prepared by direct lysis in NDET buffer (1% NP-40, 0.4% deoxycholate, 66 mM EDTA, 10 mM Tris, pH 7.4; Burgess et al., 1985) , removing the nuclei, adding SDS-PAGE sample buffer, and loading portions directly on polyacrylamide gels. GAG chains were quantiffed by cutting out the parts of the gels containing the GAG chains, dissolving in NCS tissue-homngenizing solution (Amersbam Corp.) or TS-1 tissue solubilizer (Research Products International Corp., Mt. Prospect, IL), and counting in a liquid scintillation counter.