Cisplatin, an antineoplastic drug, inhibits catecholamine secretion from bovine adrenal chromaffin cells

European Journal of Pharmacology, 236 (1993) 355-361

355

© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

EJP 53095

Cisplatin, an antineoplastic drug, inhibits catecholamine secretion from bovine adrenal chromaffin cells Eiichi T a c h i k a w a , H i r o k o Y o s h i n a r i 1, S a b u r o T a k a h a s h i , T a k e s h i K a s h i m o t o , K e n z o M i z u m a a n d Eiji T a k a h a s h i " Department of Pharmacology, School of Medicine and a Department of Medicine, School of Dentistry, lwate Medical Unit;ersity, Morioka 020, Japan Received 29 October 1992, revised MS received 22 February 1993, accepted 9 March 1993

Long-term pretreatment (12-120 h) of cultured bovine adrenal chromaffin cells with cis-diamminedichloroplatinum (cisplatin, Pt(NH3)2C12) (33 ~M), an antineoplastic drug, resulted in a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine. Acetylcholine-induced 45Ca2+ influx into the cells was also reduced in the cells pretreated with cisplatin for 48 h. The concentration-response curves (3-66 txM) for cisplatin inhibition of the secretion and 45Ca2+ influx were quite similar. Pretreatment of ceils with 33 #M Pt 4+ or carboplatin, an analog of cisplatin, for 48 h also led to a decrease in acetylcholine-evoked secretion, but not with 33 p.M Pt 2+ or other metals (Au +, Au 3+, Ni 2+, Os 3+, Pd 2+, Ir 3+, and Ir 4+) that have properties similar to Pt 4+. These results strongly suggest that in bovine adrenal chromaffin cells, cisplatin (3-66 /xM) inhibits catecholamine secretion by the suppression of the Ca 2+ influx into the cells evoked by acetylcholine and that the inhibitory effect of cisplatin is attributable to the tetravalent platinum ion in its molecule. Cisplatin; Catecholamine secretion; Adrenal chromaffin cells; Acetylcholine; Ca 2+ influx

I. Introduction

cis-Diamminedichloroplatinum (cisplatin, Pt(NH3) 2 CI 2) is an inorganic water-soluble, pl0tinum-containing complex, and recently, has been commonly used in the chemotherapy of neoplastic diseases. Rosenberg et al. (1965, 1967) have observed an inhibition of cell division in Escherichia Coli when they studied the influence of an electrical field on bacteria growth using platinum electrodes. The inhibition of Escherich& coli growth was due to the formation of inorganic platinum-containing compounds. Accordingly, with inhibition of c a n c e r cell division expected, m a n y inorganic platinum-containing compounds have been tested in experimental tumor systems, and cisplatin was found to be the most active (Rosenberg et al., 1969). The action mechanism of cisplatin is based on the theory that, entering cells by diffusion, the drug is activated by hydrolysis of the chlorides and binds D N A molecules,

Correspondence to: E. Tachikawa, Department of Pharmacology, School of Medicine, Iwate Medical University, Uchimaru 19-1, Morioka 020, Japan. Tel. 81-196-51-511 ext. 3366, fax 81-196-51-8055. i Present address: Department of Surgery I, School of Medicine, Iwate Medical University, Morioka 020, Japan.

and thus inhibits D N A functions (Eastman, 1985; Reed et al., 1986). In addition to its reactivity with DNA, cisplatin can react with nucleophiles, such as sulfhydryl groups. The sulfhydryl group, which is found in most proteins and enzymes, is highly reactive and participates in many functions in vivo (Rothstein, 1970). The effect of cisplatin on proteins may also be one of its mechanisms of antineoplastic action or may be responsible for the side-effects of the drug. Nausea, vomiting, nephrotoxicity, and ototoxicity are known as the main side-effects of cisplatin (Calabresi and Chabner, 1990). Cisplatin at higher doses also causes peripheral neuropathy (Calabresi and Chabner, 1990). However, there is no report directly showing the effect of cisplatin on the autonomic nervous system. The adrenal medulla is used in studies of catecholamine secretion and catecholamine biosynthesis as a useful model of sympathetic nervous system, though it is an endocrine organ. Bovine adrenal chromaffin cells can secrete catecholamines by exocytosis via stimulation of the nicotinic acetylcholine receptor. The process of catecholamine secretion is as follows: (1) acetylcholine, a physiological secretagogue, binds to the receptor; (2) influx of Na + occurs through receptor-operated Na + channels; (3) the cell m e m b r a n e depolarizes and the influx of Ca 2+ occurs through

356 voltage-dependent Ca 2+ channels; and (4) the intracellular free Ca 2+ concentration increases and results in exocytotic catecholamine secretion (Douglas and Poisner, 1961; Wilson and Kirshner, 1977; Holz et al., 1982; Wada et al., 1985). In this study, therefore, we examined the influence of cisplatin on catecholamine secretion from bovine adrenal chromaffin cells. The results show that longterm pretreatment of the cells with cisplatin causes the inhibition of the Ca 2+ influx into the cells evoked by acetylcholine and that catecholamine secretion is diminished. The mechanism by which cisplatin inhibits Ca 2+ influx was also investigated.

2. Materials and methods

2.1. Materials Cisplatin and carboplatin were supplied by Nippon Kayaku Ltd. (Tokyo, Japan), and Bristol-Myers Squibb Co. (Tokyo, Japan), respectively. Oxygenated KrebsRinger-4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES) buffer ( K R H buffer) (pH 7.4) was used as incubation medium and was composed of (mM) 125 NaC1, 4.8 KCI, 2.6 CaC12, 1.2 MgSO 4, 25 HEPES, 5.6 glucose, and 0.5% bovine serum albumin. In 56 mM KC1-KRH buffer, NaCI was reduced to maintain the isotonicity of the medium. Tissue culture instruments were obtained from the Falcon Plastics Co. (Cockeysville, MD, U.S.A.). Eagle's minimum essential medium was from Nissui Seiyaku (Tokyo, Japan). Calf serum, acetylcholine, and various inorganic metals were obtained from Nacarai Tesque Inc. (Kyoto, Japan). 45CAC12 (0.5-2.0 C i / m m o l ) was from Amersham International Ltd. (Arlington Heights, IL, U.S.A.). All other chemicals were of the highest grade available for commercial sources.

2.2. Isolation and primary culture of bovine adrenal chromaffin cells Bovine adrenal glands were kindly provided by the Center of Iwate Livestock Industry. Adrenal chromaffin cells were prepared by collagenase digestion as described elsewhere (Tachikawa et al., 1989). The isolated cells were suspended in Eagle's minimum essential medium containing 10% calf serum and antibiotics (100 u n i t s / m l penicillin, 100 p~g/ml streptomycin, and 0 . 3 / z g / m l amphotericin B) and were plated on 35-mm dishes at a density of 2 × 10 6 cells. The cells were cultured at 37°C in a CO 2 incubator (95% air and 5% CO 2) for 2 days. A total of 2 × 106 cells contained 37.1 + 1.2 /xg of catecholamines as epinephrine and norepinephrine.

2.3. Measurements 45Ca2 + influx

of catecholamine

secretion and

After 2 days of culturing, the chromaffin cells were further cultured at 37°C for 2 days with culture medium, plain, or containing cisplatin- or inorganic metal except if described otherwise below. The cells were washed twice with KRH buffer and then preincubated with K R H buffer for 10 min at 37°C. The cells were washed once more with prewarmed K R H buffer and incubated with or without acetylcholine or 56 mM K + for 10 min. The reaction was terminated by transferring the incubation medium to tubes in an ice-cold bath. The catecholamines secreted into the medium were extracted with 0.4 M perchloric acid and adsorbed to aluminum hydroxide. Their amounts were estimated by the ethylenediamine condensation method (Weil-Malherbe and Bone, 1952), using a fluorescence spectrophotometer (650-10S; Hitachi, Tokyo, Japan) at an excitation wavelength of 420 nm and an emission wavelength of 540 nm. At these wavelengths, epinephrine and norepinephrine showed the same fluorescence intensity. 'The amount of catecholamines secreted from the cells was expressed as a percentage of total cellular catecholamines. After preincubation of the cells with KRH buffer for 10 min, the cells were incubated with 45Ca2+ (1 /xCi) in 1.0 ml of the medium, in the presence or absence of acetylcholine for 10 rain. The medium was removed, and the cells were immediately cooled on ice and washed three times with 2.0 ml of ice-cold Ca 2+free K R H buffer. The cells were scraped and solubilized in 1.0 ml of 10% Triton X-100. Radioactivity was determined using a liquid scintillation counter (LSC900; Aloka, Tokyo, Japan) (Tachikawa et al., 1991). The Ca 2+ influx was expressed as nanomoles of Ca 2+ per 2 × 106 cells.

2.4. Preparation of digitonin-permeabilized cells and catecholamine secretion from the cells The cells pretreated with or without cisplatin for 2 days were washed twice with K R H buffer and then preincubated with K R H buffer for 10 min at 37°C. They were washed once more with prewarmed K R H buffer and incubated for 5 min at 37°C with 2 0 / z g / m l digitonin in potassium glutamate H E P E S buffer (pH 7.4), consisting of (mM) 150 potassium glutamate, 15 H E P E S - K O H , 5 EGTA, and 5.6 glucose (Dunn and Holz, 1983; Wilson and Kirshner, 1983). The digitonin-permeabilized cells were washed with potassium glutamate H E P E S buffer and incubated with or without 600 nM free Ca 2+ in potassium glutamate H E P E S buffer containing 2 mM ATP and MgSO 4. The concentrations of free Ca 2+ were adjusted using an

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Fig. 1. Effects of cisplatin pretreatment time on catecholamine secretion from bovine adrenal chromaffin cells. The isolated bovine adrenal chromaffin cells were cultured for 2 days, and then were further cultured without ( • , o ) or with 33 ~ M (o, • ) cisplatin for the times indicated. The cultured cells were washed twice with K R H buffer and then preincubated with K R H buffer for 10 min at 37°C. They were washed once more with prewarmed K R H buffer and incubated with ( • , e ) or without ( o , • ) 5 0 / z M acetylcholine for 10 min at 37°C. Catecholamines secreted into the m e d i u m were determined as described in Materials and methods. Data are m e a n s + S.D. from four experiments. * P < 0.05, significantly different from acetylcholine-evoked secretion.

EGTA-Ca 2+ buffer and were calculated according to Portzehl et al. (1964).

3. Results

3.1. Effect of cisplatin on catecholamine secretion from bovine adrenal chromaffin cells When the cultured chromaffin cells were previously exposed to 3 3 / x M cisplatin for 6-120 h, the secretion of catecholamines from the cells stimulated by 5 0 / x M acetylcholine was reduced (fig. 1). The inhibitory effect of cisplatin on the secretion was dependent on pretreatment time; inhibition was detectable after 12 h of cell pretreatment with cisplatin and after 48 and 120 h, it was 52 and 87%, respectively. Cisplatin at 33/xM did not affect the secretion from non-stimulated cells (Basal secretion) even 120 h after pretreatment. On the other hand, direct incubation of the cells with 17-264 /xM cisplatin for 10 min in the presence of 5 0 / z M acetylcholine did not result in inhibition of the secretion (data not shown). Figure 2 shows the effects of different concentrations of cisplatin on the secretion from the cells. Cisplatin (3-264 /zM) inhibited acetylcholine-evoked secretion in a concentration-dependent manner without affecting the basal secretion, when the cells were pretreated with cisplatin for 48 h (fig. 2). The inhibitory effect was detectable with 3 /xM cisplatin. The inhibi-

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Fig. 2. Effects of different concentrations of cisplatin on acetylcholine-evoked catecholamine secretion. After the cultured cells were pretreated with different concentrations of cisplatin for 2 days, they were washed twice with K R H buffer and preincubated with K R H buffer for 10 min at 37°C. The cells were washed once more with prewarmed K R H buffer and incubated with (e) or without ( o ) 50 ~ M acetylcholine for 10 min at 37°C. Catecholamines secreted into the m e d i u m were determined as described in Materials and methods. Data are means_+ S.D. from four experiments. * P < 0.05, significantly different from acetylcholine-evoked secretion.

tion by cisplatin at 17/xM was 28% and at 33 and 66 /xM it was 52 and 65%, respectively, and at 2 6 4 / z M it was almost complete.

3.2. Effects of cisplatin on catecholamine content in the cells and on catecholamine secretion from digitonin-permeabilized chromaffin cells After pretreatment of the cells with different concentrations of cisplatin for 48 h, the catecholamine content in the cells was estimated. Cisplatin (17-264 /~M) had no effect on catecholamine content (table 1). As shown in fig. 3, addition of 600 nM free Ca 2 + to digitonin-permeabilized cells resulted in an increase in

TABLE 1 Effects of different concentrations of cisplatin on catecholamine content in adrenal chromaffin cells. T h e cells were pretreated with different concentrations of cisplatin for 2 days, and then washed twice with K R H buffer. The cells were scraped and homogenized with 0.4 M perchloric acid. The homogenate was centrifuged at 2 5 0 0 0 x g for 10 rain. Catecholamines in the supernatant were determined as described in Materials and methods. Data are m e a n s + S.D. from four experiments. Cisplatin (/xM)

Catecholamine content ( / z g / 2 × 10 6 cells)

0 17 33 66 132 264

37.4 4- 1.4 37.3 _+ 1.1 36.5 _+ 1.8 37.7 _+ 1.0 39.5 _+ 1.0 37.1 _+ 1.7

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Fig. 3. Effects of different concentrations of cisplatin on catecholamine secretion from digitonin-permeabilized chromaffin cells. The cells were pretreated with different concentrations of cisplatin for 2 days, and then washed twice with KRH buffer. They were preincubated with KRH buffer for 10 rain at 37°C and incubated with 20/zg/ml digitonin in potassium glutamate HEPES buffer for 5 min at 37°C. The digitonin-permeabilized cells were washed with potassium glutamate HEPES buffer and incubated for 5 min with (e) or without (©) 600 nM free Ca 2÷ in potassium glutamate HEPES buffer containing 2 mM ATP and 2 mM MgSO4. Catecholamines secreted into the medium were determined as described in Materials and methods. Data are means+ S.D. from four experiments. *P < 0.01, significantly different from CaZ+-evoked secretion. the s e c r e t i o n o f c a t e c h o l a m i n e s f r o m the cells (10.2% s e c r e t i o n of total cellular c a t e c h o l a m i n e s ) . T h i s C a 2+i n d u c e d s e c r e t i o n was not significantly a f f e c t e d by cisplatin at c o n c e n t r a t i o n s up to 132 /~M, b u t was greatly d i m i n i s h e d by 2 6 4 / z M cisplatin (63% inhibition), w h e n the intact cells w e r e p r e t r e a t e d with cisplatin for 48 h pr i or to t h e d i g i t o n i n t r e a t m e n t .

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Fig. 4. Recovery of cisplatin inhibition of catecholamine secretion. The cells were pretreated with (A,e) or without (©,ll) 33 /xM cisplatin for 2 days and then treated with (A) or without (©,e, ll) 33 /zM cisplatin for additional times as indicated in the figure. The cells were washed twice with KRH buffer, preincubated with KRH buffer for 10 min at 37°C, and washed once more with prewarmed KRH buffer. They were incubated with ( l l , e , - ) or without (©) 50 /xM acetylcholine for 10 rain at 37°C. Catecholamines secreted from the cells into the medium were determined as described in Materials and methods. Data are means-%S.D. from four experiments. * P < 0.02, significantly different from the secretion from cisplatin-treated cells evoked by acetylcholine.

n m o l / 2 × 106 cells (fig. 5). Cisplatin up to a c o n c e n t r a tion of 132 /~M d i m i n i s h e d a c e t y l c h o l i n e - i n d u c e d 45Ca 2+ influx in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r w i t h o u t affecting the basal 45Ca 2+ influx, w h e r e a s at 264 /~M, the cisplatin inhibition was w e a k e r t h a n that at 33, 66, or 1 3 2 / ~ M . T h e inhibitory effect of cisplatin was significant with 1 7 / ~ M (25% inhibition). Cisplatin

5q

3.3. Recocery of cisplatin inhibition of acetylcholineecoked catecholamine secretion

4 T h e c u l t u r e d cells w e r e p r e t r e a t e d with or w i t h o u t 3 3 / z M cisplatin in the m e d i u m for 48 h, r e p l a c i n g e a c h m e d i u m with e i t h e r n o r m a l (plain) or 3 3 / z M cisplatinc o n t a i n i n g m e d i u m , a n d t h e n c u l t u r e d for an additional 2 4 - 7 2 h. A s shown in fig. 4, cisplatin time d e p e n d e n t l y i n h i b i t e d the s e c r e t i o n e v o k e d by acetylc h o l i n e ( 5 0 / z M ) d u r i n g 120 h of t r e a t m e n t . In contrast, the cells p r e t r e a t e d with cisplatin for 48 h and t h e n i n c u b a t e d in plain m e d i u m , s h o w e d significantly r e s t o r a t i o n of a c e t y l c h o l i n e - e v o k e d secretion. R e c o v e r y was significant aft er i n c u b a t i o n with plain m e d i u m for 24 h, a n d was 30 an d 5 2 % a f te r 48 a n d 72 h, respectively.

3.4. Effect of cisplatin on acetylcholine-induced 45Ca2+ influx into the cells A c e t y l c h o l i n e (50 /xM) i n c r e a s e d 45Ca 2+ influx into the cells; the influx o f C a 2+ a m o u n t e d to 4.8_+ 0.5

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Fig. 5. Effects of cisplatin on 4SCa2+ influx into the chromaffin cells. The cells were pretreated with different concentrations of cisplatin for 2 days, and washed twice with KRH buffer. They were then preincubated with KRH buffer for 10 min at 37°C and washed once more with prewarmed KRH buffer. The cells were incubated for 10 min at 37°C with (e) or without (©) 50 /xM acetylcholine in KRH buffer containing 1 tzCi of 45CAC12.The radioactivity in the cells was determined as described in Materials and methods. Data are means + S.D. from four experiments. * P < 0.05, significantly different from acetylcholine-induced ~SCa2+ influx,

359

at 33 and 66 /.~M inhibited the acetylcholine-induced 45Ca2+ influx by 55 and 70%, respectively, and at 132 tzM, the inhibition was complete. Thus, the concentration-response curves (up to 66 IzM) for cisplatin inhibition of catecholamine secretion and 45Ca2+ influx were quite similar. 3.5. Effect of cisplatin on cholamine secretion

high-K+-induced

cate-

T h e e x c h a n g e o f n o r m a l i n c u b a t i o n m e d i u m for 56 m M K + ( h i g h - K +) m e d i u m c a u s e d t h e s e c r e t i o n o f c a t e c h o l a m i n e s f r o m t h e cells (fig. 6). P r e t r e a t m e n t o f t h e cells w i t h d i f f e r e n t c o n c e n t r a t i o n s o f c i s p l a t i n for 48 h d e c r e a s e d t h e h i g h - K + - i n d u c e d s e c r e t i o n in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . C i s p l a t i n at 17 a n d 66 / z M i n h i b i t e d t h e s e c r e t i o n by 9 a n d 4 2 % , r e s p e c tively. T h u s , t h e c i s p l a t i n i n h i b i t i o n o f h i g h - K + - i n d u c e d s e c r e t i o n w a s m u c h less t h a n t h a t o f a c e t y l c h o l i n e evoked secretion.

3.6. Effects of external Ca 2+ and acetylcholine concentrations on cisplatin inhibition of catecholamine secretion The inhibitory effect of cisplatin on acetylcholineevoked catecholamine secretion from the chromaffin cells was e x a m i n e d w i t h d i f f e r e n t c o n c e n t r a t i o n o f C a 2+ or acetylcholine. Cisplatin (33/xM) inhibited the secret i o n by 5 8 % in t h e p r e s e n c e o f 2.6 m M C a 2+ (fig. 7, left). In t h e p r e s e n c e o f 5.2, 7.8, a n d 10.4 m M C a 2+, c i s p l a t i n i n h i b i t i o n w a s 55, 54, a n d 5 3 % , r e s p e c t i v e l y . A s s h o w n in fig. 7 (right), c i s p l a t i n (33 t z M ) i n h i b i t i o n o f t h e s e c r e t i o n w a s m a i n t a i n e d e v e n at h i g h e r c o n c e n t r a t i o n s o f a c e t y l c h o l i n e (100 a n d 200 /~M). T h u s , t h e i n h i b i t i o n by c i s p l a t i n was n o t o v e r c o m e by i n c r e a s i n g t h e e x t e r n a l C a 2+ o r a c e t y l c h o l i n e c o n c e n trations.

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Fig. 6. Effects of cisplatin on acetylcholine- and high-K+-induced

3. 7. Effects of various metals on catecholamine secretion evoked by acetylcholine To further understand the mechanism of cisplatin inhibition of acetylcholine-evoked catecholamine secretion, we examined the effects of Pt 4+, carboplatin, an analog of cisplatin that also contains Pt 4+ in the

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catecholamine secretion. The cells were pretreated with different concentrations of cisplatin for 2 days, and then washed twice with KRH buffer. They were preincubated with KRH buffer for 10 min at 37°C and washed once more with prewarmed KRH buffer. The cells were incubated for 10 rain at 37°C without or with 50 /xM acetylcholine (e) or 56 mM K + (©). Catecholamines secreted from the cells into the medium were determined as described in Materials and methods. Basal values were subtracted from the data and secretagogue-induced responses were assigned a value of 100%. Percentages of catecholamine secretion induced by acetylcholine and high K + were 23.3+ 1.4 and 19.6_+ 1.2%, respectively. Basal secretion was 0.9 +-0.2%. Data are means + S.D. from four experiments. * P < 0.05, significantly different from acetylcholine- or high-K+-induced secretion.

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Fig. 7. Effects of various concentrations of C a 2+ and acetylcholine on cisplatin inhibition of catecholamine secretion. The cells were pretreated with or without 33 /~M cisplatin for 2 days, and then washed twice with KRH buffer. They were preincubated with KRH buffer for 10 rain at 37°C and washed once more with prewarmed KRH buffer. (Left) The cells were incubated for 10 min at 37°C with or without 50 /xM acetylcholine under various concentrations of Ca 2+ (2.6, 5.2, 7.8, and 10.4 mM). (Right) The cells were incubated for 10 min at 37°C without or with 50, 100, and 200/zM acetylcholine. Catecholamines secreted from the cells into the medium were determined as described in Materials and methods. Data are means_+ S.D. from four experiments. *P < 0.01, significantly different from acetylcholine-evoked secretion.

360 TABLE 2 Effects of cisplatin, carboplatin and various metals on catecholamine secretion from bovine adrenal chromaffin cells evoked by acetylcholine. The cells were treated with or without 33 /zM cisplatin, carboplatin or various metals for 2 days, and then washed twice with KRH buffer. They were preincubated with KRH buffer for 10 min at 37°C and washed once more with KRH buffer. The cells were incubated with or without 50/xM acetylcholine. Catecholamines secreted from the cells into the medium were determined as described in Materials and methods. Data are m e a n s + S.D. from four experiments. Metals

Catecholamine secretion (% of total)

Basal Acetylcholine + AuCN + NaAuC14 + OsCI 3 + NiC12 + PdC12 + PtCI 2 +PtCI 4 + cisplatin + carboplatin + IrCl 3 + IrCl 4

0.4 + 0.1 21.9 + 0.9 21.9 _+1.2 21.8 + 1.0 21.7 + 0.4 23.6 +_ 1.4 22.8 +_ 1.3 22.2_+ 1.1 16.6+ 1.4 11.8 _+0.4 15.2 + 1.4 23.4 _+0.5 21.3 _+1.1

a p < 0.05, significantly different from acetylcholine-evoked secretion.

molecule, and various other metals having properties similar to Pt 4+ on the secretion. Au +, Au 3+, Os 3+, Ni 2+, Pd 2+, Ir 3+, Ir 4+, and even Pt 2+ (33/xM) did not inhibit either acetylcholine-evoked (table 2) or basal secretion (data not shown), while Pt 4+ and carboplatin (33 /zM) only inhibited acetylcholine-evoked secretion by 25 and 31%, respectively (table 2) without affecting the basal secretion (data not shown).

4. Discussion

We found that long-term pretreatment (12-120 h) of bovine adrenal chromaffin cells with cisplatin (3-264 /zM) resulted in a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine. The cisplatin inhibition appeared to be time-dependent (fig. 1), concentration-dependent (fig. 2), reversible (fig. 4), and attributable to the presence of Pt 4+ that is contained in the cisplatin molecule (table 2). The inhibitory effect of cisplatin may be considered to be due to the killing of chromaffin cells or suppression of cell growth, since cisplatin is an antineoplastic agent. However, the possibility that this concept applies, except for the case of cisplatin at higher concentrations, is negated by the following reasons: (1) cisplatin (17-132 /3,M) treatment did not affect either catecholamine content in the intact ceils (table 1) or

catecholamine secretion from digitonin-permeabilized cells evoked by Ca 2+ (fig. 3), and (2) the cisplatin inhibition of the secretion was reversible (fig. 4). In bovine adrenal chromaffin cells, acetylcholine causes a Ca 2+ influx into the ceils, increases the intracellular free C a 2+ concentration, and consequently induces exocytotic catecholamine secretion from the cells (Douglas and Poisner, 1961; Wilson and Kirshner, 1977; Holz et al., 1982; Wada et al., 1985). Cisplatin, up to 132 /zM, did not inhibit the secretion evoked by Ca 2+ from digitonin-permeabilized cells (fig. 3), indicating that cisplatin does not affect the intracellular secretory process following Ca 2+ influx. Cisplatin (17-264 /.~M) reduced 45Ca2+ influx into the cells evoked by acetylcholine, and the concentration-response curve (17-66 p~M) for cisplatin inhibition was quite similar to that for cisplatin inhibition of catecholamine secretion (figs. 2 and 5). Further, cisplatin also inhibited high K+-in duced secretion, but the inhibition was less than that of the acetylcholine-evoked secretion (fig. 6). These results suggest strongly that cisplatin at low concentrations (17-66 /~M) diminishes not only Ca 2+ influx through voltage-dependent Ca 2+ channels but also Ca 2+ influx associated with the acetylcholine receptor, and inhibits catecholamine secretion from the cells stimulated by acetylcholine. On the other hand, cisplatin at high concentrations (132-264 /~M) probably damages the cell. The mechanism of action of cisplatin as an antincoplastic drug is based on the theory that cisplatin enters cells by diffusion, reacts with purine or pyrimidine bases in DNA molecules and inhibits protein synthesis and cell division (Eastman, 1985; Reed et al., 1986). In the present experiments, the cisplatin inhibition of secretion was not reversed by an increase in acetylcholine or Ca 2+ concentration (fig. 7A and B). Furthermore, inhibition required long-term pretreatment of the cells with cisplatin (fig. 1) and was considerably diminished by removing cisplatin from the culture medium (fig. 4). Therefore, cisplatin inhibition of Ca 2+ influx into the cells may be the result of the suppression of protein synthesis by its specific binding to D N A molecules for C a 2+ channels or acetylcholine receptor rather than the result of the direct inhibition of voltage-dependent Ca 2+ channels or the nicotinic acetylcholine receptor. Cisplatin can also react with nucleophiles like sulfhydryl groups of proteins. It has been reported that N-ethylmaleimide (Ferris et al., 1970) and p-chloromercuribenzoate (Tachikawa et al., 1989), sulfhydrylreacting agents, affect catecholamine secretion from bovine adrenal chromaffin cells. The former agent inhibits the secretion evoked by acetylcholine, and the latter itself stimulates secretion. Hence, another mechanism of the cisplatin inhibition of Ca 2+ influx into the cells may include the possibility that cisplatin acts on

361

the sulfhydryl groups of proteins responsible for Ca 2+ influx. It is well known that aminoglycoside antibiotics such as streptomycin, kanamycin, and gentamicin show serious nephrotoxicity and ototoxicity (Sande and Mandell, 1990). Aminoglycoside antagonism to the effect of Ca 2+, as observed in blocking of transmission in the neuromuscular junction (Fiekers, 1983) and inhibition of catecholamine secretion in adrenal medullary cells (Kashimoto et al., 1982), has been considered one of the mechanisms of their toxicity (Humes et al., 1984). Cisplatin also induces nephrotoxicity and ototoxicity (Calabresi and Chabner, 1990). Therefore, the cisplatin-induced side-effects may occur through suppression of Ca 2+ influx into the tissues. At high concentrations, cisplatin has been reported to cause peripheral neuropathy, which may worsen after discontinuation of the drug (Calabresi and Chabner, 1990). When the usual dose (about 30 mg/60 kg per day) of cisplatin is administered continuously into a human vein for 5 days, the blood level of cisplatin is maintained at about 5 IzM for 5 days after 3 days of this administration (Sawada et al., 1982). In this experiment, cisplatin at 3 tzM significantly inhibited catecholamine secretion and at 5/xM, it showed about 20% inhibition after 48 h of treatment (fig. 2). Therefore, the inhibitory effect of cisplatin on adrenal chromaffin cells may be related not only to suppression of sympathetic neuron activity but also to the peripheral neuropathy. References Calabresi, P. and B.A. Chabner, 1990, Chemotherapy of neoplastic diseases, in: The Pharmacological Basis of Therapeutics, eds. A.G. Gilman, T.W. Rail, A.S. Nies and P. Taylor (Pergamon press, New York) p. 1202. Douglas, W.W. and A.M. Poisner, 1961, Stimulation of uptake of 45calcium in the adrenal gland by acetylcholine, Nature (London) 192, 1229. Dunn, L.A. and R.W. Holz, 1983, Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells, J. Biol. Chem. 258, 4989. Eastman, A., 1985, Intrastrand cross-links and sequence specificity in the reaction of cis-dichloro (ethylene-diamine) platinum (II) with DNA, Biochemistry 24, 5027. Ferris, R.M., O.H. Viveros and N. Kirshner, 1970, Effect of various agents on the Mg2+-ATP stimulated incorporation and release of catecholamines by isolated bovine adrenal medullary storage vesicles and on secretion from the adrenal medulla, Biochem. Pharmacol. 19, 505. Fiekers, F.T., 1983, Effects of aminoglycoside antibiotics, streptomycin and neomycin, on neuromuscular transmission. I. Presynaptic considerations, J. Pharmacol. Exp. Ther. 225, 487.

Holz, R.W., R.A. Senter and R.A. Fry, 1982, Relationship between Ca 2+ uptake and catecholamine secretion in primary dissociation cultures of adrenal medulla, J. Neurochem. 39, 635. Humes, H.D., M. Sastrasinh and J.M. Weinberg, 1984, Calcium is a competitive inhibitory of gentamicin-renal membrane binding interactions, and dietary calcium supplementation protects against gentamicin nephrotoxicity, J. Clin. Invest. 73, 134. Kashimoto, T., C. Shimizu, S. Takahashi, E. Tachikawa, N. Ohtsubo and E. Takahashi, 1982, Inhibitory effect of kanamycin on catecholamine secretion from adrenal medullary cells, Res. Commun. Chem. Pathol. 37, 151. Portzehl, H., P.C. Caqldwell and J.C. Ruegg, 1964, The dependence of contraction and relaxation of muscle fibers from the crab Maisa squinado on the internal concentration of free calcium ions, Biochim. Biophys. Acta 79, 581. Reed, E., S.H. Yopsa, L.A. Zwelling, R.F. Ozols and M.C. Poirier, 1986, Quantitation of cis-diamminedichloroplatinum II (cisplatin)-DNA-intrastrand adducts intesticular and ovarin cancer patients receiving cisplatin chemotherapy, J. Clin. Invest. 77, 545. Rosenberg, B., L. VanCamp and T. Krigus, 1965, Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode, Nature 205 698. Rosenberg, B., L. VanCamp, E.B. Grimley and A.J. Thomson, 1967, The inhibition of growth or cell division in Escherichia coli by different ionic species of platinum (IV) complexes, J. Biol. Chem. 242, 1347. Rosenberg, B., L. VanCamp, J.E. Trosko and V.H. Mansour, 1969, Platinum compounds: A new class of potent antitumor agents, Nature 222, 385. Rothstein, A., 1970, Sulfhydryl groups in membrane structure and function, in: Current Topics in Membranes and Transport, eds. F. Bronner and A. Kleinzeller (Academic Press, New York) p. 135. Sande, M.A. and G.L. Mandell, 1990, The aminoglycosides, antimicrobial agents (continued), in: the Pharmacological Basis of Therapeutics, eds. A.G. Gilman, T.W. Rail, A.S. Nies and P. Taylor (Pergamon press, New York) p. 1098. Sawada, M., Y. Okudaira, Y. Matsui, H. Nishiura and T. Yanagida, 1982, Pharmacological studies of cis-platinum diammine dichloride in repeated administration, Cancer Chemother. 9, 55. Tachikawa, E., S. Takahashi and T. Kashimoto, 1989, p-Chloromercuribenzoate causes Ca2+-dependent exocytotic catecholamine secretion from cultured bovine adrenal medullary cells, J. Neurochem. 53, 19. Tachikawa, E., S. Takahashi, K. Furumachi, T. Kashimoto, A. Iida, Y. Nagaoka, T. Fujita and Y. Takaishi, 1991, Trichosporin-B-III, an ct-aminoisobutyric acid-containing peptide, causes Ca2+-de pendent catecholamine secretion from adrenal medullary chromaffin cells, Mol. Pharmacol. 40, 790. Wada, A., H. Takara, F. Izumi, H. Kobayashi and N. Yanagihara, 1985, Influx of 22Na through acetylcholine receptor-associated Na channels: relationship between 22Na influx, 45Ca influx and secretion of catecholamines in cultured bovine adrenal medulla cells, Neuroscience 15, 283. WeiI-Malherbe, H. and A.D. Bone, 1952, The chemical estimation of adrenaline-like substances in blood, Biochem. J. 51,311. Wilson, S.P. and N. Kirshner, 1977, The acetylcholine receptor of the adrenal medulla, J. Neurochem. 28, 687. Wilson, S.P. and N. Kirshner, 1983, Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells, J. Biol. Chem. 258, 4994. i