Bovine Chromaffin Granule Membranes Undergo Ca"-regulated Exocytosis in Frog Oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chrornaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chrornaffin granule membranes were injected into Xenopus laevis oocytes . Exocytosis was detected by the appearance of dopamineß-hydroxylase of the chrornaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-ß-hydroxylase on the oocyte surface was strongly Cal+-dependent and was stimulated by coinjection of the chromaffin granule membranes with InSP3 or Cal+/EGTA buffer (18 p,M free Cal+) or by incubation of the injected oocytes in medium containing the Cal+ ionophore ionomycin . Similar experivariety of techniques have been developed to study the physiological and biochemical basis for regulated exocytosis from cells and neurons . The use of permeabilized cells (2, 6, 15, 24) and patch clamp techniques (19) has permitted direct control of the intracellular milieu and has greatly facilitated the analysis of factors which regulate exocytosis . However, it has not been possible to manipulate the secretory vesicle apart from the cell . In the present study we have injected bovine chromaffn granules, the secretory vesicles from adrenal chromaffin cells, into frog oocytes . We demonstrate that the injected chrornaffin granules undergo Cal+-triggered exocytosis . This approach may allow the identification of the components of the secretory vesicle necessary for regulated secretion . Frog oocytes and eggs are secretory cells . Endogenous cortical granules in frog eggs undergo exocytosis when the cytoplasmic Cal+ concentration is elevated by fertilization (3), incubation with Cal+ ionophore (4, 5), injection with Ins'-(1,4,5)P3 (4), mechanical stimulation (5, 8, 17), or expression and activation of exogenous plasma membrane receptors (14) . Manipulations to increase cytoplasmic Cal+ are less able to cause cortical granule exocytosis in frog oocytes (immature eggs) (5, 7, 12) . However, direct microinjection of Cal+ (9) or high concentrations of A23187 (12) can induce cortical granule exocytosis . Exocytosis of chromaffin granules was monitored by two © The Rockefeller University Press, 0021-9525/92/01/359/7 $2 .00 The Journal of Cell Biology, Volume 116, Number2, January 1992 359-365 ments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chrornaffin granules . Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-ß-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chrornaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis . Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals . techniques . Dopamine-ß-hydroxylase (DBH)' is both soluble within the chromaffin granule and bound as an integral membrane protein in the chrornaffin granule membrane (27) . Soluble DBH is released into the medium upon exocytosis (23) . The antigenic sites of membrane-bound DBH are intragranular and become exposed on the chromaffin cell surface upon exocytosis (21, 22) . If injected chromaffin granule membranes undergo exocytosis in the frog oocyte, then membrane-bound DBH should be exposed on the oocyte surface . Purified chromaffin granule membranes or intact granules were injected into Xenopus laevis oocytes and the oocytes stimulated with manipulations which increase cytoplasmic Cal+ . The appearance of DBH on the surface of the oocyte was measured with an immunocytochemical technique . The release of [3H]norepinephrine from oocytes injected with [3H]norepinephrine-containing intact granules was also used as a measure of exocytosis . Materials and Methods Isolation ofa PI Fraction Containing Intact Chromafn Granules and Purification ofChromafn Granule Membranes Fresh bovine adrenal medullae were homogenized in 0.3 M sucrose, 10 MM 1 . Abbreviation used in this paper: DBH, dopamine-ß-hydroxylase. 359 on S etem er 8, 2017 jcb.rress.org D ow nladed fom Hepes (pH 7.2), 1 mM EDTA, and 1 mM PMSF. The supernatant from a 800 g x 10 min centrifugation was recentrifuged at 27,000 g x 10 min to generate a large granule fraction (P2 fraction) . This was layered onto a discontinuous sucrose gradient containing 0.8, 1 .3, and 1 .7 M sucrose . All the sucrose solutions contained 1 mM EDTA and 5 mM Hepes, pH 7.2 (no PMSF) . The tubes were centrifuged at 145,000 g x 60 min . The pellet at the bottom of the tube consisted of highly purified chromaffin granules . An extensive analysis using a variety of enzymatic markers for various subcellular organelles and catecholamine for intact chrornaffin granules indicated that chrornaffin granules were purified five to eightfold with virtually no mitochondrial, lysosomal, or plasma membrane contamination . The chromaffin granules in the pellet were lysed in 10 mM Hepes, 0.2 mM EDTA, pH 7.2, frozen, and thawed . The chromaffin granule membranes were pelleted by centrifugation at 30,000 g x 20 min and resuspended in lysis buffer. They were again centrifuged at 30,000 g x 20 min and the chromaffin granule membrane pellet was resuspended (5 mg protein/ml) in 10 mM Hepes, pH 7.2 (no EDTA), aliquoted and stored at -70°C. In some experiments (e.g., see Fig . 1 E) the P2 fraction from fresh adrenal medullae was lysed in 1 mM DTT and 10 mM Hepes, pH 7.2, and the resulting membranes werewashedtwice by centrifugation and resuspension (final concentration 12 mg protein/ml) . A P 2 fraction was also prepared without osmotic lysis from primary cultures of purified, bovine chromaffin cells (25 million cells) for injection of intact granules. Manipulation ofOocytes Oocytes (1 .1-1 .3 mm diameter) were obtained from female Xenopus laevls . The follicular layer was removed after a 1-2-h incubation at room temperature with 2 mg/ml Type IV collagenase (Sigma Chemical Co ., St Louis, MO) in solution containing 82 .5 mM NaCl, 2 .5 mM KCI, 1 mM MgC12, 5 mM Hepes, pH 7.6. Oocytes were incubated overnight at 19°C in MBSH (88 mM NaCl, 1 mM KCI, 2 .4 mM NaC03, 0.82 mM MgSO4, 0.33 mM Ca(N03)2, 0.41 mM CaC12, 2.55 mM Napyruvate, and 10 mM Hepes, pH 7.4) with 1 mg/ml BSA . Oocytes were injected with suspensions of chrornaffin granule membranes or lysed or intact P2 fractions (0.05 Al) using 20-25 Am tip diameter, beveled injection pipets . As a control for the immunocytochemistry, injections were performed with buffer without chrornaffin granule membranes or a P2 fraction .