Fig. 3. Potentiation of isoproterenol-stimulated amylase secretion by CCh. Parotid lobules were pulse-labeled and chased as in Fig. 1. (A) Secretion of amylase enzyme from samples stimulated with 40 nM CCh alone (CCh), 1 µM Iso (Iso), 40 nM CCh and 1 µM Iso in combination (CCh + Iso). A parallel sample was treated with 40 nM CCh and 1 µM Iso for 10 minutes and subsequently with 1 µM Iso alone (CCh take-away). At each timepoint the medium was removed in entirely and replaced with medium to which additives had been freshly added. (B) Specific radioactivity of amylase in samples stimulated with 1 µM Iso, 1 µM Iso and 40 nM CCh, or 1 µM Iso and 200 nM CCh during the first 10 minutes of stimulation. At each time point the medium was completely removed and replaced. (C) Effect of pre-stimulation with 40 nM CCh on initial secretion and specific radioactivity of amylase. Lobules were pulsed and chased as above. Amylase enzyme (left panel) and specific radioactivity of amylase (right panel) were determined in secretion from the first 1 minute of stimulation in samples treated with 1 µM Iso (open bar), 1 µM Iso and 40 nM CCh added simultaneously (hashed bar), and a sample pre-treated with 40 nM CCh 5 minutes prior to addition of 1 µM Iso plus 40 nM CCh (filled bar). Medium was completely replaced at the end of the pretreatment. (D) Time course of secretion of radiolabeled PSP as a marker of newly formed granules. The gels shown were those from the experiment shown in B.