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

To examine the secreted proteins in the medium after stimulation with the secretagogue, 8-Br-cAMP, 106-107 cells were treated as described (Burgess et al., 1985 (Burgess et al., , 1987 . Trypsinogen-expressing wild type AtT-20 ceils or variant HYA.15.6.T.3 cells were incubated first for 16 h in depleted DME containing 200 #Ci lzSS]cysteine, 10% FCS, glutamine, penicillin, streptomycin, and l/5th volume complete medium, and then for a 1-h interval in fresh labeling medium containing [3sS]cysteine at the same specific activity. After the labeling period, cells were washed twice with PBS and chased in normal media for three 2.5-h intervals. During the third interval one portion of ceils was incubated in the presence of 5 mM 8-Br-cAMP to stimulate release from the regulated secretory pathway. The media and cell extracts were collected at each time interval and immunoprecipitated with rabbit anti-trypsinogen, anti-proinsniin or anti-kappa antiserum as described. Immunoprecipitates were analyzed on SDS-PAGE gels and the amount of rnaterial that was secreted was quantified by cutting out the bands from the gels, fluting the labeled proteins in tissue-solubilizing solution at 60°C for 16 h and counting in a liquid scintillation counter and by scanning the autoradiograph of the gel with a soft laser densitometer (LKB Instruments, Inc., Bromma, Sweden). Proinsulin secretion were examined by essentially the same procedure except that immunoprecipitates were washed in distilled water to remove detergent and analyzed on 12-20% gradient SDS-PAGE gels. The identity of the proinsulin and mature processed insulin bands were distinguished on gels by molecular weight and by using two antiserum, a rabbit anti-human C peptide (recognizes proinsniin only) and a guinea pig anti-mature insulin (recognizes both forms). ~ chain secretion by transfected ART20 cells was determined as described for trypsinogen except that 100 t~Ci [35S]methionine was used for the labeling and cells were chased for two 3-h intervals, the latter interval in the presence or absence of 8-Br-cAMP. The amounts of the endogenous hormone, ACTH, that were released into the media during the stimulation interval was determined by a solid-phase ACTH radioimmunoassay that has been previously described (Moore et al., 1983b) . Protein determinations were assayed by an Amido Schwartz dye-binding assay (Moore et al., 1983b) .