Journal of Neurochemisrry Raven Press, New York 0 1985 International Society for Neurochemistry

Fe2’-Induced Lysis and Lipid Peroxidation of Chromaffin Granules

Ronald M. Spears and Ronald W. Holz

Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan, U.S.A

Abstract: Chromaffin granules, the catecholaminergic by trace amounts of reducible polyvalent cation. Lysis storage granules from adrenal chromaffin cells, lysed in sometimes occurred when Ca2+ was added with EGTA 10-9-10-7M Fez+. Lysis was accompanied by the pro- (10 pLM free Ca2+ concentration) and was consistently duction of malondialdehyde which results from lipid per- observed together with malondialdehyde production in oxidation. Both chromaffin granule lysis and malondi- the presence of Ca2+,EGTA, and 10 pM Fez+(total con- aldehyde production were inhibited by the free radical centration). The apparent Ca2+ dependency for chro- trapping agent butylated hydroxytoluene but not by cat- maffin granule lysis and malondialdehyde production was alase and/or superoxide dismutase. The results suggest probably caused by a trace reducible polyvalent dis- that lysis resulted from a direct transfer of electrons from placed by Ca2+from EGTA and not by a Ca2+-dependent Fe2+to a component of the chromaffin granule membrane reaction involving the chromaffin granule. Key Words:

without the participation of either superoxide or hy- Chrornaffin granules- Fe2+- Malondialde hyde- Lipid drogen peroxide and may have resulted from lipid per- peroxidation-Ca2+. Spears R. M. and Holz R. W. oxidation. In some experiments, ascorbate alone induced Fe2+-induced lysis and lipid peroxidation of chromaffin chromaffin granule lysis which was inhibited by EDTA, granules. 1. Nrurochrm. 44, 1559- 1565 (1985). EGTA, or . The lysis was probably caused

Secretion of prepackaged hormones and neuro- rectly altered chromaffin granule stability by lipid transmitters generally occurs by exocytosis. peroxidation. The loss of storage granule stability Studies concerning catecholamine secretion from by low concentrations of Fe2+ has both experi- adrenal medullary chromaffin cells defined the pro- mental and clinical implications. cess biochemically and demonstrated that Ca2+in- flux (Douglas and Rubin, 1961; Viveros et a]., 1968; Kilpatrick et al., 1982; Holz et al., 1982) and a rise MATERIALS AND METHODS in cytosolic Ca2+ concentration (Knight and Kes- Cells disaggregated from bovine adrenal medulla (Fen- teven, 1983) are responsible for initiating exocy- wicket al., 1978; Kilpatrick et al., 1980; Holz et al., 1982) tosis. In the course of experiments examining the were plated at a density of 250,000-500,000 cells/cm2 in effects of Ca2+ on the stability of chromaffin gran- 60-mm diameter uncoated plastic petri dishes (Lux Sci- ules, the catecholamine-containing secretory gran- entific, Newbury Park, CA, U.S.A.) and in 75-cmz and ules from the adrenal medulla, we found that iso- 150-cm2 plastic culture flasks (Costar, Cambridge, MA, lated chromaffin granules lysed in the presence of U.S.A.) in minimum essential medium (MEM; Eagle’s) ascorbate and micromolar that was buff- (GIBCO, Grand Island, NY, U.S.A.) supplemented with ered with EGTA. The present work demonstrates 10% heat-inactivated fetal calf serum (GIBCO), 10 pM cytosine arabinoside (to inhibit fibroblast proliferation), that the lysis was caused not by Ca2+ but by trace 50 pgfml gentamycin, I00 Ulml penicillin, 100 pg/ml amounts of a reducible polyvalent ion that was dis- streptomycin, and 2.5 pglml Fungizone (Squibb, placed by Ca2+ from EGTA. The effects could be Princeton, NJ, U.S.A.). There were 1-2 million cellsiml reproduced in the absence of calcium by nanomolar culture medium. By 4 days at 34°C in 5% COz-95% air concentrations of ferrous ion, which probably di- the chromaffin cells had formed monolayers and the in-

Received June 22, 1984; accepted October 31, 1984. Abbreviations rr.trd: MEM, minimum essential medium; Address correspondence to Dr. Ronald W. Holz at Department PIPES, piperazine-N-N’-bis(2-ethanesulfonicacid). of Pharmacology, M6322 Medical Science I, The University of Michigan Medical School, Ann Arbor, MI 48109, U.S.A.

1559 1560 R.M. SPEARS AND R. W. HOLZ cubation medium was replaced with fresh medium. Ex- lipids (Tappel and Zalkin, 1959). Sample (0.45 ml) was periments were performed on days 5- 10. There were ap- mixed with 0.75 ml of 20% acetic acid (pH 3.9, 0.10 ml proximately 40 nmol catecholaminehillion cells. 8.1% sodium dodecyl sulfate, and 0.75 ml of 0.8% thio- The catecholamine stores of chromaffin cells in 60- barbituric acid. Tetramethoxypropane dissolved in 325 mM mm diameter plastic dishes were labelled with r3H]- sucrose, 10 rnM PIPES (pH 7.0) was used as a standard. norepinephrine by preincubation of the cells in 3 ml of Incubation solutions without tissue were routinely used MEM containing [3H]norepinephrine (0.3 pCi/ml, 13 Ci/ as blanks. The tubes were capped with marbles and mmol, Amersham, Arlington Heights, IL, U.S.A.) and heated in an oven at 85°C for 60 min. The samples were 0.i mM ascorbate for 1-2 h (Kilpatrick et al., 1980). The then cooled in a water bath and 0.5 ml of water added to cells were washed twice for 5 min with MEM and then each. The pink thiobarbituric acid-malondialdehyde com- once with isotonic physiological saline solution 1145 mM plex was extracted into 2.5 rnl of n-butanol-pyridine (15: 1) NaCI, 5.6 mM KC1, 15 mM N-2- hydroxyethylpiperazine- by vigorous vortex-mixing and the phases separated by N' - 2 - ethanesulfonic acid (HEPES), 0.5 mM MgC1, and centrifugation for 5 min at 3,000 r/min in a Sorvall GLC 2.2 mM CaCI,, 5.6 mM glucose, and 0.1 mM ascorbatel. desk-top centrifuge. Absorbance of the organic extracts was measured at 532 nm. Ascorbate (0.1-0.2 mM) was Preparation of chromaffin granules added to the acetic acid used in the assay to reduce the Chromaffin granules were harvested by scraping the background absorbance due to . cells from the culture dish in 260 mM sucrose, 10 mM piperazine-N-N'-bis(2-ethanesulfonicacid) (PIPES; pH Other procedures 7.0), and I mM EGTA and homogenizing the suspension Ca2+and Fe2+ion concentrations were buffered in the in a 7-ml Dounce homogenizer with a tight-fitting pestle micromolar and submicromolar ranges with 1 mM EGTA with 12 vigorous strokes. The homogenate was centri- according to Portzehl et al. (1964). The unbound ion con- fuged at 1,000 x g for 5 min. The supernatant (S,) was centrations were calculated using a value for the Ca*+- centrifuged at 40,000 x g for 10 min and the pellet (P,) EGTA dissociation constant of lo-" M (Owen, 1976) and was resuspended (using a 2-ml Dounce homogenizer) in a value for the Fe2+-EGTA dissociation constant of 260 mM sucrose, 10 mM PIPES (pH 7.0) buffer to an M (Martell and Smith, 1974). Proteins were mea- approximate protein concentration of 0.5 mg/ml. sured by the method of Bradford (1976). Highly purified chromaffin granules were prepared by Chromaffin granule lysis was monitored by measuring sedimentation of the granules through a sucrose-Ficoll- decreases in optical density at 430 nm (Johnson and D,O solution (Trifaro and Dworkind, 1970). A P, fraction Scarpa, 1976). was prepared from chromaffin cells which were main- Superoxide dismutase activity was measured according tained as a monolayer in a tissue culture flask at a density to McCord and Fridovich (1969) by the capacity of the of approximately 450,000 cellsicm' or from fresh adrenal enzyme to inhibit the reduction of cytochrome c (10 pM) medulla (Holz, 1979). The P2 pellet was resuspended in by superoxide which was generated by xanthine oxidase 1-2 ml of 260 mM sucrose, 10 mM PIPES (pH 7.0), lay- (Sigma, 1.7 mU/ml) and xanthine (50 yM). Catalase ac- ered over 7.5 rnl of 19.5% Ficoll, 260 mM sucrose, 10 mM tivity was measured by the rate of disappearance of H,O, PIPES (pD 7.0) in D20 and centrifuged at 100,000 x g according to Beers and Sizer (1952). for 60 min in a Beckman Type 40 rotor. The pellet was rinsed (without resuspension) once with 1 ml of sucrose RESULTS buffer (325 mM sucrose, 10 mM PIPES, pH 7.0) and then resuspended in 1 ml of the same buffer using a 2-ml Effects of ascorbate and Fe2+ on catecholamine Dounce homogenizer. The concentration of the suspen- release from chromaffin granules sion was adjusted to 0.25-0.5 mg proteinhl before use. Ascorbate (10-50 pM) released as much as 90% [3H]Norepinephrinerelease of the catecholamine from chromaffin granules in a Chromaffin granule suspensions were aliquoted (10 pl) P, fraction from primary cultures of adrenal med- into 2-ml plastic centrifuge tubes. Incubations were ullary chromaffin cells. The effects of ascorbate started by addition of 190 p1 of solution at 30°C. When were variable and were observed only 60% of the malondialdehyde was measured concurrently with cate- time. The release of catecholamines by ascorbate cholamine release, 30 pl of suspension was incubated was blocked by the polyvalent cation chelators under the same conditions with 470 pl of solution. Reac- EDTA and deferoxamine (Fig. l), suggesting that tions were stopped by addition of EDTA and, occasion- catecholamine release was caused by the presence ally, hydroquinone. The suspensions were centrifuged at of a contaminating polyvalent cation that was re- 40,000 x g for 10 min. Supernatants were transferred to 54plastic scintillation vials containing an equal volume duced by ascorbate. EGTA (1 mM) also inhibited of 2% Triton X-100. Pellets were solubilized with 0.4 ml ascorbate-induced lysis. Because ascorbate readily of 1% Triton X- 100 and also transferred to scintillation reduces ferric ion (Fe3+)to ferrous ion (Fe") under vials. Scintillation counting cocktail (ACS; Amersham) the conditions used in these experiments, the effect was added and radioactivity determined. Endogenous of Fe2+ on chromaffin granule stability was inves- catecholamine was measured using a fluorescent assay tigated. Fe2' could reproducibly mimic the effects (Holz et al., 1982). of ascorbate when present at very low concentra- Measurement of lipid peroxidation tions (Fig. 2). The species actually responsible for Lipid peroxidation was estimated by measuring the catecholamine release was unbound Fez+ rather reaction of thiobarbituric acid with malondialdehyde, than chelated Fe2+ since high concentrations of which is formed from the breakdown of polyunsaturated EGTA inhibited the effects of Fe2+ (Fig. 2). The Fe” -INDUCED L YSIS OF CHROMAFFIN GRANULES 1561

a? o‘io-a lo-’ 10-6 id-. id-. 10-1 l5- l5- 0 * 0 10-10. Id-. 10-8 10-7 10-6 CChelatorl (M) Lk CFe2+l (M) FIG. 1. Effects of EDTA and deferoxamine on ascorbate-in- FIG. 2. Effects of various concentrations of unbound Fez’ duced adrenal chromaffin granule lysis. Granules from a P, on chromaffin granule lysis. Purified chromaffin granules fraction were incubated at 30°C for 45 min (0.48 mg protein/ (0.52 mg proteiniml) were incubated in 325 mM sucrose, 10 ml) in 260 mM sucrose-10 mM PIPES (pH 7.0) with 50 pM mM PIPES (pH 7.0) with various concentrations of unbound ascorbate and various concentrations of EDTA (B) or defer- Fe” without added ascorbate at 30°C for 60 min. Unbound oxamine (A).Samples were also incubated with various con- Fez+ concentrations were maintained using FeEGTA (1 mM centrations of EDTA without ascorbate (0).Reactions were total EGTA). Reactions were terminated by addition of EDTA terminated by addition of EDTA to 2.5 rnM. Results are ex- and hydroquinone to 4 mM and 10 mM, respectively. Results pressed as the percentage of [3H]norepinephrine remaining are expressed as the percentage of [3H]norepinephrine re- in the granules as a function of the logarithm of the chelator maining in the granules as a function of the logarithm of the concentration. There were three samplesigroup. Fez’ concentration. Results obtained in solutions with EGTA (1 mM) without Fez* and without both EGTA and Fez* were identical and are represented as 0 Fez+ in the figure. There were three samplesigroup. effective Fe2+ concentration range was approxi- mately 10-9-10-7 M. Fe2+ concentrations of M or greater did not cause lysis. Most experiments 1954; Johnson and Scarpa, 1976). Fe2+ (21 nM) in- were performed with granules prepared from chro- duced a decrease in optical density of a suspension maffin cells maintained as monolayers in culture of purified chromaffin granules (Fig. 3). The data from 4 to 10 days. Similar results were obtained in one experiment with granules prepared from fresh indicate that the Fe2+-induced release of catechol- adrenal medulla. amines reflects a time-dependent granule lysis and not a selective permeability increase to catechol- Granule lysis and lipid peroxidation amines. Lysis was not observed in the presence of Lysis of chromaffin granules can be monitored either high concentrations of EDTA (1 mM) or Fe2+ by measuring the loss of optical density in a sus- (10 pM in the absence of added chelator). pension of purified granules (Hillarp and Nilson, Fe2+ may destabilize chromaffin granules by

FIG. 3. Time course of Fe”’-induced loss of op- tical density of a suspension of chromaffin gran- ules. Purified chromaffin granules (0.46 mg pro- tein/ml) were incubated in 325 rnM sucrose, 10 mM PIPES (pH 7.0) without added ascorbate containing either 100 pM Fez+(0); 100 pM Fez+ + 5 mM EGTA (unbound Fez+ concentration 0.65 nM) (W); 100 pM Fez” + 250 pM EGTA (unbound Fez+ concentration 21 nM) (A);or 100 p.M Fe2+ + 250 pM EGTA (unbound Fez- con- centration 21 nM) and 100 pM butylated hy- droxytoluene Absorbance at 430 nm (A43,,) + (A). C was monitored concurrently for all four suspen- 0, U sions. A430of the suspensions at the beginning L Q) a of the incubation was 0.22. The optical density of granules lysed in HzO, to which sucrose buffer was subsequently added, was 0.06 and was subtracted from the total optical densities. 04 0 5 10 15 20 25 30 Data are expressed relative to A,,, at 0 time. Time (min)

J. Nerrwchwi.. Vol, 44, No. 5, IYRS 1562 R. M. SPEARS AND R. W. HOLZ transferring electrons to acceptors on the granule cause both purified granules and granules in a P, membrane (possibly polyunsaturated lipids), fraction behaved similarly and P2 fractions were thereby generating a free radical that would initiate more readily prepared, some of the following ex- free radical reactions. Indeed, butylated hydroxy- periments were performed with a P2 fraction. toluene, which is an antioxidant and free radical trapping agent, inhibited the Fe*+-induced granule Calcium effects and Fezf-induced lysis lysis (Fig. 3 and Table 1). This entire study began because of the initial ob- Lipid peroxidation can be initiated by formation servation that chromaffin granules lysed in the pres- of free radicals in polyunsaturated fatty acid moie- ence of micromolar calcium (buffered with 1 mM ties with the subsequent rearrangement of double EGTA) (Fig. 4A). Ascorbate (10-600 pA4) was also bonds to form conjugated dimer (Buege and Aust, required for the effect. Lysis was variable, however 1978). Addition of molecular oxygen results in (occurring in 60% of the experiments), which sug- peroxy radicals and subsequent rearrangements to gested that a reducible contaminant rather than lipid hydroperoxides and lipid endoperoxides. Ca2+ was responsible for granule lysis. Some of the lipid endoperoxides break down to pro- We could consistently observe release of cate- duce malondialdehyde. Enzymatic and nonenzy- cholamine from chromaffin granules from cultured matic initiation of the chain reaction is well docu- cells in the presence of 10 pM added Fe2+ when mented (McKnight et al., 1965; Pederson and Aust, Ca2+ was buffered in the micromolar range with 1 1975; Buege and Aust, 1978; Sharma, 1979). Fe2I mM EGTA (Fig. 4B). Associated with catechol- caused the formation of malondialdehyde coinci- amine release was a decrease in optical density of dent with granule lysis in a purified granule prepa- chromaffin granule suspensions and malondialde- ration (Table 1). Superoxide dismutase (83 pg/ml) hyde production (data not shown). The apparent and/or catalase (66 pg/ml) did not inhibit Fe2+-in- Ca2+ dependency, however, was probably an arti- duced granule lysis or malondialdehyde production fact caused by the equilibria between EGTA, Cat+, (Table 2). Similar results were obtained with a P, and Fe2'. In the absence of added Ca2+,EGTA (I fraction. The results suggest that neither superoxide mM) reduced the unbound Fe2+ concentration to nor hydrogen peroxide participated in chromaffin <1 nM which was not sufficient to cause granule granule lysis induced by Fe2+ and are consistent lysis. The presence of 0.98 mM total Ca2+increased with electrons being transferred directly from Fe2+ the unbound Fe2+ concentration to 10-30 nM to the granule membrane to initiate lipid peroxida- which was sufficient to induce granule lysis. Similar tion and granule lysis. However, it should be noted that although superoxide dismutase activity was un- TABLE 2. EjJects of superoxide dismutase and altered by EGTA ? Fe2+ and catalase activity was catalase on Fez+-induced lysis and mulondialdehyde unaltered by EGTA in the incubations, the effects production in a purijied chromajjin granule preparation of EGTA and Fez+on catalase activity could not be 13H]Norepi- Malondi- determined (see footnote to Table 2). Thus, it is con- nephrine aldehyde ceivable that catalase activity was insufficient in the in pellet produced presence of Fez+ to reduce H202that may have Treatment (%) (nmol) been generated in the granule experiments. The data with purified granules suggest that the 0 Fe?+ 46 ? 2 0 10-R M Fe?+ 6 t 2" 2.0 ? 0.1" effects of Fe2+ were directly on the granules and 10 M Fe2+ 45 i I 0 not mediated by other cellular components. Be- 0 Fez+ + SOD 47 2 1 0 + Cat 4s k I 0 + SOD + Cat 40 t 1 0 TABLE 1. Chromuffh granule lysis und M Fez+ + SOD 7 ? 1" 2.1 i 0.1" malondialdehyde formation + Cat 7 t I" 2.0 -+ 0.2" + SOD + Cat 5 t 1" 1.9 t 0.2" ['HlNorcpinephrine Malondialdehyde ~~-~~ -~ Treatment in pellet (5%) produced (nmol) A purified chromaffin granule fraction (0.25 mg proteiniml) was diluted 17-fold into 325 mM sucrose, 10 mM PIPES (pH 7.0) 0 Fe2+ (with (without added ascorbate) in the presence or absence of Fe?' 1 mM EGTA) 55 f 1 n and the presence or absence of superoxide dismutase (SOD, 83 10 "' M Fez' 52 t 2 0 10 * M Fe" 5 * 2" pg/ml) and/or catalase (Cat, 66 )*.g/ml).The Fez' concentration was maintained with a FeEGTA buffer (with 1 mM total EGTA). Groups without Fe'- contained 1 mM EGTA. Incubations were + BHT (200 KM) 53 t 2 carried out for 60 min at 30°C and stopped by addition of EDTA to 4 mM. There were three samplesigroup. Superoxide dismu- Chromaftin granule: mg proteidml) purified from cultured chro- lase was not inhibited by EGTA or EGTA + Fe?' in the incu- rnaffin cells by sedirnentarwn through Ficoll/D&l were incubated for 1 h bation medium. Catalase was not inhibited by EGTA in the at 30°C in various concentrations of Fe' ' in sucrosc buffer 325 rnM su- also crose 10 mM PIPES, pH 7.0 (without added ascorbate). The free Fe'+ incubation. Catalase activity in the presence of small amounts was buffered using I mM EGTA. The reaction was stopped by adding of Fe2+ could not be assayed because of oxidation of Fe2- by EDTA to 4 mM. Therc were three smiplesigroup. H20, which was present in the catalase assay. " p < 0.001 w. 0 Fe" (with I mM EGTA). p < 0.001 vs. 0 Fez+. Fe2+-INDUCED L YSIS OF CHROMAFFIN GRANULES 1563

B r'

a'+ Fez- .- (EGTA) (EGTA) Fez+ (EGTA) FIG. 4. Apparent Caz+-dependent release of [3H]norepinephrine from chromaffin granules. A: Effects of various Ca2' concen- trations on release of catecholamine. A P, fraction was diluted 20-fold into 260 mM sucrose, 10 mM PIPES (pH 7.0),5 mgiml bovine serum albumin, 0.6 mM ascorbate, 1 mM EGTA, and various amounts of Caz+ at 30°C. After 5 min the reaction was stopped by placing the tubes on ice and centrifuging the remaining intact granules at 40,000 x g for 10 min. The percent of [3H]norepinephrine in the pellet is plotted as a function of the logarithm of the unbound Ca2' concentration. There were three samples/group. B: Requirement of Fe2' for apparent calcium-induced lysis of chromaffin granules. A P, fraction was diluted 20-fold into 260 mM sucrose, 10 mM PIPES (pH 7.0) with various additions as follows: Fez-, 10 pM Fez+;EGTA, 1 mM EGTA; Ca,', 10 pM unbound Ca2- buffered with 1 mM EGTA; Fe", 10 pM added Fe2' + 1 mM EGTA, unbound [Fe"] = 0.3 nM; Ca", Fez+,10 pM unbound Caz- buffered with 1 mM EGTA + 10 pM added Fez+,unbound [Fez+] = 10-30 nM. Incubations were carried out for 60 min at 30°C in the absence of ascorbate. Reactions were stopped by addition of EDTA to 2.5 mM and centrifugation of the remaining intact granules at 40,000 x g for 10 min. Data are expressed as percent [3H]norepinephrine remaining in the pellet. There were three samplesigroup. In this preparation 50 pM ascorbate caused release of approximately half of the granular catecholamine within 60 min (data not shown). The ascorbate-induced release was totally inhibited by 1 mM EGTA. but variable results were obtained using chromaffin molar (or greater) concentrations of Fe' ' did not granules prepared from fresh adrenal medulla. cause lysis. Lysis was associated with the produc- Various factors altered granule lysis induced ei- tion of malondialdehyde which is a product of lipid ther with ascorbate alone, or with ascorbate and peroxidation. Both granule lysis and malondialde- M calcium (buffered with 1 mM EGTA). High hyde production were inhibited by the free radical medium concentrations of ascorbate (e.g., 5 mM) trapping agent butylated hydroxytoluene but were or epinephrine (>lo0 FM), both of which are not altered by superoxide dismutase and/or cata- present in chromaffin granules, prevented granule lase. It is possible that lysis resulted from a direct lysis. Bovine serum albumin also occasionally in- transfer of electrons from Fe2+ to a component in hibited ascorbate-induced granule lysis. the chromaffin granule membrane without the par- ticipation of either superoxide or hydrogen per- oxide. However, we cannot rule out the possibility DISCUSSION that membranes or substances contaminating the purified granule preparations were also involved. We have demonstrated that Fe2+ (10p9-10-7 M) The evidence is consistent with lysis being caused in vitro caused lysis of chrornaffin granules, the se- by lipid peroxidation. The inability of micromolar cretory granules from the adrenal medulla. Micro- Fe2+to cause granule lysis or malondialdehyde pro- duction may have resulted from free radical quenching effects of high concentrations of Fez+ ' In two of three preparations of chronmffin granules purified (Could, 1959). from fresh adrenal medulla, there was a Ca2+-dependent (0.98 mhf total Ca2+, approximately 10 p~ free ca2+concentration) Recently it was demonstrated that liver lyso- catecholamine release or a decrease in optical density in the somes are also susceptible to Fez+-inducedlipid presence of Fez' (10 IJ,Mtotal), EGTA (I mM total), and ascor- peroxidation and lysis. The effects of Fez+ did not bate (50 pM). The lack of effect in one experiment may have involve reduced oxygen intermediates (Mak et al., been caused by high Concentrations of inhibitory substances such as ascorbate, which is found in higher concentrations in 1983) and the mechanism may be similar to that fresh adrenal medulla than in cultured chromaffin cells (Diliberto which caused chromaffin granule lysis in the eta]., 1983). present experiments.

.I. Nutiroclrrnt.. V01, 44. No. 5, 1985 1564 R. M. SPEARS AND R. W. HOLZ

An apparent Calf dependence for granule lysis REFERENCES was sometimes observed in the presence of EGTA Beers R. F, and Sizer 1. W. (1952) A spectrophotometric method and ascorbate and was consistently observed when for measuring the breakdown of hydrogen peroxide by cat-

Fez+ (10-600 FM total concentration) was present. alase. J. Bid. Chem. 195, 133-140. Lysis in the presence of Ca2+ probably resulted Bradford M. M. (1976) A rapid and sensitive assay for the quan- titation of microgram quantities of protein utilizing the prin- from displacement of contaminating reduced poly- ciples of protein dye binding. Anal. Biochem. 72, 248-254. valent ion from EGTA to yield a concentration of Buege J. A. and Aust S. D. (1978) Microsomal lipid peroxida- reduced ion in the concentration range adequate for tion, in Methods in Enzymology, Vol. 52, Part C (Fleisher granule lysis and lipid peroxidation. We have not S. and Packer L., eds), pp. 302-310. Academic Press, New identified the ion. There are numerous reports de- York. Diliberto E. J., Heckman G. D., and Daniels A. (1983) Charac- scribing a similar calcium-induced lysis of secretory terization of ascorbic acid transport by adrenomedullary vesicles (Pinchasi et al., 1979; Kuo et al., 1979; chromaffin cells. J. Bid. Chem. 258, 12886-12894. Konings and DePotter, 1982). In these studies, ves- Douglas W. W. and Rubin R. P. (1961) The role of calcium in the icle lysis was observed in the presence of M secretory response of the adrenal medulla to acetylcholine. J. Physiol. (Lond.) 159, 40-57. free calcium (buffered with EGTA). Low levels of Fenwick E. M., Fajdiga P. B., Howe N. S. B., and Livett B. G. ascorbate (1 - 10 pM) were also sometimes present. (1978) Functional and morphological characterization of iso- Our results suggest that calcium-induced storage lated bovine adrenal medullary cells. J. Cell Biol. 76, vesicle lysis in solutions in which Ca2+is buffered 12-30. Gould E. S. (1959) Mechrtnism and Structrrre in Organic Chem- by polyvalent ion chelators may be caused by con- istry, p. 690. Holt Rinehart and Winston, New York. taminating Fe2+ or other polyvalent that can Hillarp N. and Nilson B. (1954) The structure of the adrenaline initiate free radical reactions. and noradrenaline containing granules in the adrenal med- We have no evidence that the Fe2+-induced lysis ullary cells with reference to the storage and release of the of chromaffin granules is related to exocytosis. No sympathomimetric amines. Acra Physiol. Scand. 31 (Suppl. I13), 79-107. detectable malondialdehyde is released upon cate- Holz R. W. (1979) Measurement of membrane potential of chro- cholamine secretion from chromaffin cells and the maffin granules by the accumulation of triphenylmethyl- free radical scavenger butylated hydroxytoluene phosphonium cation. J. Bid. Chem. 254, 6703-6709. does not alter secretion (R. M. Spears and R. W. Holz R. W., Senter R. A,, and Frye R. A. (1982) Relationship unpublished observations). However, chro- between Ca‘+ uptake and catecholamine secretion in pri- Holz, mary dissociated cultures of adrenal medulla. J. Neuro- maffin granules contain large amounts of reducing chem. 39, 635-646. equivalents in the form of ascorbate (10 mM), and Johnson R. G. and Scarpa A. (1976) Ion permeability of isolated the granule membrane contains cytochrome bS6, chromaffin granules. J. Cm. Physiol. 68, 601 -63 I. which can conduct electrons across the membrane Kilpatrick D. L., Ledbetter F. H., Carson K. A,, Kirshner A. G., Slepetis R., and Kirshner N. (1980) Stability of bo- (Njus et al., 1983). It is possible that the effects of vine adrenal medulla cells in culture. J. Neiirochern. 35, Fe2+ on chromaffin granule stability are related to 679-692. the redox system of chromaffin granules. Kilpdtrick D. L., Slepetis R. J., Corcoran J. J., and Kirshner N. Lipid peroxidation is one of the primary causes (1982) Calcium uptake and catecholamine secretion by cul- tured bovine adrenal medulla cells. J. Neurochem. 38, 427- of tissue damage in ischemia. In the heart, ischemia 435. and lipid peroxidation are associated with the re- Knight D. E. and Kesteven N. T. (1983) Evoked transient intra- lease of norepinephrine from sympathetic nerves cellular free Ca” changes and secretion in isolated bovine (Rao and Mueller, 1983). The present study raises adrenal medullary cells. Proc. R. Soc. Lond. [Biol.] 238, the possibility that the release of norepinephrine re- 177- 199. Konings F. and DePotter W. (I 982) The chromaffin granule- sults, in part, from a lipid peroxidation-induced loss plasma membrane interaction as a model for exocytosis: of storage vesicle stability. Fe2+ also induces lipid quantitative release of the soluble granular content. peroxidation in vivo in the CNS (Triggs and Will- Biochem. Biophys. Res. Commun. 104, 254-258. more, 1984). Thus, it is possible that CNS hemor- Kuo C. H., Hata F., Yoshida H., Yamatodani A,, and Wada H. (1979) Effects of ascorbic acid on release of acetylcholine rhage could release neurotransmitters from storage from synaptic vesicles prepared from different species of granules within CNS neurons because of the release animals and release of noradrenaline from synaptic vesicles. of Fez+ from extravasated red blood cells and sub- Life Sci. 24, 91 1-916. sequent lipid peroxidation. Mak I. T., Misra H. P., and Weglicki W. B. (1983) Temporal re- lationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lyso- somes. J. Bid. Chem. 258, 13733-13737. Acknowledgment: We are grateful to Dr. Marshal Martell A. E. and Smith R. M. (1974) Criticul Stability Con- Shlafer (Department of Pharmacology, University of stants, Vol. I: Amino Acids, pp. 269-271. Plenum Press, Michigan Medical School) for help and advice in mea- New York. McCord J. M. and Fridovich I. (1969) Superoxide disrnutase: an suring malondialdehyde and superoxide dismutase ac- enzyme function for erthrocuprein (hemocuprein). J. Biol. tivity. R.M.S. is a recipient of a Michigan Heart Asso- Chem. 244, 6049-6055. ciation postdoctoral fellowship. R.W.H. is a recipient of McKnight R. C., Hunter F. E. Jr., and Oehlert W. H. (1965) Mi- NSF Grant BNS-8211493 and PHS Grant RO1-AM27959 tochondrial membrane ghosts produced by lipid peroxida- in support of this work. tion induced by ferrous ion. J. Biol. Chem. 240, 3439-3446.

J. Neuroclrrm.. Vd.44, No. 5, 198.5 Fe2+-INDUCED LYSIS OF CHROMAFFIN GRANULES 1565

Njus D., Knoth J., Cook C., and Kelley P. M. (1983) Electron Rao P. S. and Mueller H. S. (1983) Lipid peroxidation and acute transfer across the chromaffin granule membrane. J. Biol. myocardia ischemia. Adv. Exp. Med. Bid. 161, 347-363. Chem. 258, 27-30. Sharma 0. P. (1979) Ascorbic acid, iron and nonenzymatic lipid Owen J. D. (1976) The determination of the Stdbihty constant of peroxidation in rat brain mitochondria. Ind. J. Biochem. calcium-EGTA. Binchim. Biophys. Actu 451, 321-325. Biophys. 16, 139-142. Pederson T. C. and Aust S. D. (1975) The mechanism of liver Tappel A. L. and Zalkin H. (1959) Lipid peroxidation in isolated microsomal lipid peroxidation. Biochirn. Biophys. Actu 385, mitochondria. Arch. Biochem. Biophys. 80, 326. 232-241. Trifaro J. M. and Dworkind J. (1970) A new and simple method Pinchasi I., Michaelson D. M., and Sokolovsky M. (1979) Cho- for isolation of adrenal chromaffin granules by means of an linergic nerve terminals contain ascorbic acid which induces isotonic density gradient. Anal. Bioc,hrm. 34, 403-412. Ca2+-dependentrelease of acetylcholine and ATP from iso- Triggs W. J. and Willmore L. J. (1984) In vivo lipid peroxidation lated Torpedo synaptic vesicles. FEES Lrfr. 108, 189- 192. in rat brain following intracortical Fe?' injection. J. Neu- Portzehl H., Caldwell P. C., and Ruegg J. C. (1964) The depen- rochem. 42, 976-980. dence of contraction and relaxation of muscle fibres from Viveros 0. H., Arqueros L., and Kirshner N. (1968) Release of crab Muju Squinado on the internal concentration of free catecholamines and dopamine-P-oxidase from the adrenal calcium ions. Biochim. Biophys. Actu 79, 581-591. medulla. Life Sci. Part I, 7, 609-618.