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Brefeldin a Affects Early Events but Does Not Affect Late Events Along The Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7242-7246, August 1992 Cell Biology Brefeldin A affects early events but does not affect late events along the exocytic pathway in pancreatic acinar cells (Golg complex/membrane trafflcking/exocytosis) LINDA C. HENDRICKS*t, SUSAN L. MCCLANAHAN*, GEORGE E. PALADEO, AND MARILYN GIST FARQUHARt *Division of Cellular and Molecular Medicine and tCenter for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093 Contributed by George E. Palade, April 30, 1992 ABSTRACT Brefeldin A (BFA) blocks protein export from In rat exocrine pancreatic cells we have seen yet another the endoplasmic reticulum (ER) to Golgi complex and causes variation of the effects of BFA on the compartments of the dismanting of the Golgi complex with relocation of resident exocytic pathway: Golgi cisternae disassemble to produce Golgi proteins to the ER in some cultured cells. It is not known persistent clusters ofuncoated Golgi tubules and vesicles and whether later steps in the secretory process are affected. We nonclathrin coated vesicles. Furthermore, no relocation of previously have shown that in BFA-treated rat pancreatic Golgi enzymes and antigens into the ER is detected even after lobules, there is no detectable relocation ofGolgi proteins to the prolonged BFA incubation (19). The response of the pancre- ER and, although Golgi clsternae are rapidly ntled, atic acinar cell to BFA treatment warrants further study to clusters of small smooth vesicles consisting of both bona fide determine whether this cell type represents another variant of Golgi remnants and associated vesicular carriers persist even BFA resistance and, more importantly, whether the drug with prolonged BFA exposure. We now report the effects of affects basal and regulated release of secretory proteins, BFA on transport of proteins through the secretory pathway in topics not addressed by previous investigations. In this study exocrine pancreatic cells; we pulse-labeled pancreatic lobules we used pulse-chase analysis ofpancreatic secretory protein with 35Slmethionine and then chased for various times before release to (i) answer the questions posed above, (ii) pinpoint adding BFA. When BFA was added at pulse, treated lobules the site(s) of drug action, and (iii) determine whether single released <10% of radlactive protein in comparison with or multiple steps in the exocytic pathway are affected by controls, regardless of whether or not the lobule cultures were BFA. These inquiries can be ideally addressed in the exo- simulated with carbamoylcholine. However, when lobules crine pancreatic cell because the kinetics ofsecretory protein were pulsed and then chased for 30, 45, or 60 min before BFA transport are well established (20-22) in this cell type which addition, the amount of labeled protein released was compa- provided the original model for transport along the exocytic rable in both BFA-treated and untreated cultures. Further- pathway (23). more, the kinetics and amounts ofbasal and carbamoylcholine- stimulated release of unlbeed a-amylase from storage in MATERIALS AND METHODS zymogen granules were smilar in both control and BFA- treated lobules. Therefore, in the rat pancreas, BFA blocks ER Materials. Animals, reagents, and supplies were obtained to Golgi transport but does not affect later stages along the from the following sources: Male rats (50-125 g) from Har- secretary pathway, includin intra-Golgi transport, exit from lan-Sprague-Dawley, BFA from Epicentre Technologies the Golgi complex, formaton and concentration of secretory (Madison, WI), Ham's F-12 medium from the University of granules, and exocytosis. California, San Diego Core Facility, fetal calf serum from GIBCO, HL-1 from Ventrex Laboratories (Portland, ME), Nutridoma from Boehringer Mannheim, Nu-Serum IV from Brefeldin A (BFA) is a macrocyclic fungal metabolite that Collaborative Research, L-[35S]methionine, cell-labeling rapidly and reversibly blocks the intracellular transport of grade (>28 TBq/mmol) from NEN, Nitex nylon monofila- secretory (1-4), lysosomal (5), and membrane (6-10) proteins ment (100-gnm mesh) from Tetko (BriarcliffManor, NY); and beyond the endoplasmic reticulum (ER) and causes the the Phadebas a-amylase test from Pharmacia. All other concomitant rapid vesiculation of Golgi cisternae and loss of reagents and chemicals were purchased from Sigma. Golgi membranes (2, 8, 11) in most cell types. Recently BFA Pancreas Lobule Preparation. Pancreatic lobules were pre- also has been shown to affect both endocytosis and transcy- pared as described (19). Briefly, the gland was distended by tosis, depending on cell type (12). Thus, in MDCK and PtKj injecting Ham's F-12 medium containing 1% HL-1, Nutri- kidney cell lines, which are "resistant" to the effects of BFA doma, Nu-Serum IV, aprotinin at 0.1 trypsin inhibitor unit/ on Golgi membranes (12-14), the drug induces membrane ml, and soybean trypsin inhibitor at 10 mg/ml into its fusion within the endocytic system (13, 15). In contrast, BFA interstitia to separate lobules. The latter were minced be- affects both endocytic and exocytic pathways in NRK cells tween scalpel blades into individual =2-mm3 lobules (24), (8, 15, 16). Thus, the drug has more pleiotropic effects on which were placed in groups of -20 on Nitex rafts into vials intracellular membranes than originally reported (8). containing 10 ml of medium with or without BFA (7.2 ,uM, 2 Although BFA clearly inhibits vesicular traffic from ER to iug/ml) and allowed to equilibrate in a 95% 02/5% CO2 Golgi complex in many systems, whether it also affects later atmosphere at 370C for 5-10 min with agitation before the steps in the secretory process is unknown. Because BFA beginning of each experiment. apparently generates cation-selective channels in planar lipid Release of Secretory Proteins. To determine the effect of bilayers (17), it may have more nonselective effects on proteins, membranes than reported to date. In fact, BFA has been BFA on release of newly synthesized secretory suggested to be a membrane fusogen (18). Abbreviations: BFA, brefeldin A; LDH, lactate dehydrogenase; ER, endoplasmic reticulum. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Division of payment. This article must therefore be hereby marked "advertisement" Cellular and Molecular Medicine, 0651, 9500 Gilman Drive, Uni- in accordance with 18 U.S.C. §1734 solely to indicate this fact. versity of California, San Diego, La Jolla, CA 92093-0651. 7242 Downloaded by guest on September 24, 2021 Cell Biology: Hendricks et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7243 lobules were pulse-labeled (5 min) with [35S]methionine (100 700- puCi/ml; 1 Ci = 37 GBq), rinsed quickly (five times) with medium containing lOOx excess unlabeled methionine, and 600- then chased for up to 7 hr in the same medium. When BFA was added at 0 min, it was included in both the pulse and 500 chase media. Where indicated, the secretagogue carbamoyl- choline (20 AM) was added 60 min into the chase. In prelim- we400 inary experiments, we established that our enriched medium ~300- allowed retention of acinar cell function for up to 8 hr (data E not shown), which is longer than the experiments reported o here. To define the site of the block in exocytic traffic, BFA 200 was added at 10, 30, 45, or 60 min after the pulse. To examine the time course of recovery from a BFA block, pulse-labeled 100 - | W - lobules were chased for 60 min in the presence ofBFA before I carbamoylcholine addition and further chased for an addi- 0 120 240 360 480 tional 60 min before BFA was washed out by five rapid minutes medium exchanges, and the chase was continued in BFA-free medium containing carbamoylcholine. In these experiments, FIG. 1. BFA blocks basal and stimulated release of newly syn- 0.1-ml samples were removed from the medium at 20-min thesized proteins in rat pancreas. Lobules were pulsed for 5 min with intervals throughout the chase, and release of newly synthe- [35S]methionine and then chased with or without 7.2 AM BFA. In sized proteins was quantitated by trichloroacetic acid pre- some experiments, the secretagogue, carbamoylcholine (20 AuM) was cipitation and scintillation counting of each precipitate. This added 60 min into the chase (arrowhead). Trichloroacetic acid- assay is valid because =95% of newly synthesized proteins in precipitable counts were assayed in medium removed from the lobules at intervals throughout a o7-hr chase. Negligible amounts of the adult pancreas are secretory (25). Data are expressed as radioactive secretory proteins were released from unstimulated cpm per aliquot and normalized to Ag ofdry weight oflobules BFA-treated lobules (0) as compared with controls (o). Radioactive (recovered and air-dried for 48 hr). We found this normal- secretory proteins released by carbamoylcholine-stimulated, BFA- ization more reproducible than normalizing to recovered treated lobules (o) were <10%o ofcontrols (a). Data are expressed as protein (because of the difficulty in obtaining complete sol- cpm//Ag of dry weight of tissue shown as mean ± SD of triplicate ubilization of lobules) or DNA (unreliable due to endogenous samples. The graph is representative of -10 experiments. Cell DNase activity). In all experiments, lactate dehydrogenase integrity as assessed by LDH release was maintained at -98% (LDH) release was also monitored throughout the chase to throughout the experiments. ensure that the radioactive proteins in the medium were from secretion rather than from cell lysis. LDH activity was oylcholine, a secretagogue that stimulates release of pancre- determined by a modification (sodium pyruvate was not atic secretory proteins from zymogen granules (27), resulted added due to adequate levels in the medium) of Sigma in <10% release of radioactive proteins from BFA-treated procedure no. 500. Cell integrity (expressed as percent of lobules past 240 min in comparison with nontreated controls maximal release from detergent-lysed lobules) was typically (Fig.
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