Chem.-Biol Interacttons, 74 (1990) 263-274 263 Elsevier Scientific Pubhshers Ireland Ltd.

C O M P A R A T I V E E F F E C T S OF T H R E E N A T U R A L L Y OCCURRING F U R A N O C O U M A R I N S ON M I T O C H O N D R I A L BIOENERGETICS

OLUFUNSO O. OLORUNSOGO', ANTHONY O. UWAIFO b and SYLVIA O. MALOMO b*

• Laboratory for Btomembrane Research and ~Oncology Research Sectton~ Department of B~och- emistry, College of Medicine, Universtty of lbadan, lbadan (Ntgerm)

(Received May 23rd, 1989) (Revision received October 20th, 1989) (Accepted November 20th, 1989)

SUMMARY

Oxygraphic measurements of the rates of mitochondrial respiration in the presence of varying amounts of chalepin, imperatorin and marmesin, three naturally occurring furanocoumarins, revealed that the oxidation of NAD ÷- linked substrates was inhibited by chalepin and imperatorin and less significantly by marmesin. The order of potency being rotenone ) ) chalepin imperatorin > marmesin, There was no effect whatsoever on succinate oxidation by the furanocoumarins tested (up to 60 ~V[). State 3 respiration was also inhibited by these furanocoumarins; by at least 800/0 by 10 ~ cha]epin and by 48 and 290/0 with 60 ~Vl imperatorin and 60/~M marmesin, respectively. Consequently, ADP control of respiration was diminished by those concentrations of furanocoumarins that inhibited respiration. At 60 ~M, respiratory control ratio was reduced by about 88, 49 and 280/0 with chalepin, imperatorin and marmesin, respectively.

A measurement of the rate of proton and Ca2÷-movements across the mito- chondrial coupling membrane demonstrated that succinate-supported trans- port was not affected by these furanocoumarins. On the other hand, pyruvate/malate-supported proton ejection was significantly inhibited by chalepin, imperatorin and marmesin. The order of the degree of inhibition of proton flux is rotenone > ) cha]epin ~ imperatorin ~ marmesin. The pat- tern of the inhibition of pyruvate/malate-supported Ca2÷-transport was identi- cal to that seen during proton transport. A comparison of the effects of chalepin to that of rotenone suggests that cha]epin might be about 10 times less potent than rotenone.

K e y words: Furanocoumarins - Rotenone -- Mitochondrial bioenergetics

*Present address: Department of Biochemistry, University of florin, llorin, N]gerla.

0009-2797/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

264

INTRODUCTION

In tropical Africa, many plants of diverse species are used for curative purposes. These medicinal plants include species such as clausena, crotalaria and senecio [1]. For example, concoctions of the roots of Clausena anisata (wild) Rutaceae are widely used in the t rea tment of haemorrhoids in most West African countries including Nigeria [1,2]. Furthermore, in Central American countries, different parts of the plant Arura (Ruta Chalepensis) are also frequently ingested as herbal teas for the t rea tment of measles, scarlet fever, headaches, and heart conditions [3]. In order to assess the biol- ogical activity and safety of these preparations, certain furanocoumarins including chalepin (I), imperatorin (II), and oxypeucedanine (III) have been isolated and purified to homogeneity from different parts of Clausena anisata (wild) [4] (Fig. 1). Marmesin (IV) another furanocoumarin has been isolated and purified to homogeniety from Afraegle paniculata (Rutaceae) [5]. It seems likely that the frequent and uncontrolled use of these plants could have adverse effects on mitochondrial energy metabolism, because of the structural resemblance of these chemical compounds to (1) aflatoxin B 1, an uncoupler of mitochondrial respiration and a potent carcinogen produced by certain strains of the fungus, Aspergillus flavus and (2) rotenone, a naturally occurring fish poison and a potent inhibitor of mitochondrial respiration [6,7].

l I i

H'

AFLATOXI N B 1 ROTENONE '3=Cl"13

F~g. 1. Structures of four naturally occurring furanocoumarms, aflatoxm B, and rotenone: chale- pm (I), ~mperatorm (II), oxypeucedamne (III) and rnarmemn (IV)

265

Toxicological studies have shown that although only chalepin has significant necrotic and anticoagulant actions when intraperitoneally adminis- tered to rats [8], marmesin and imperatorin may be somewhat mutagenic [9]. We have investigated the possible effects of chalepin, imperatorin and mar- mesin on mitochondrial bioenergetics, in order to gain insight into the mecha- nism of interaction of these chemicals with mitochondrial metabolism. We have reported elswhere the results of a preliminary investigation on the inhibitory effect of chalepin on mitochondrial respiration [10].

In this paper, we present a detailed account of the interaction of these chemicals with mitochondrial bioenergetics. Our results show that chalepin and imperatorin inhibit the oxidation of NAD÷-linked substrates in the same manner as rotenone. In addition, Ca 2÷ accumulation and proton translocation are inhibited by these chemicals when mitochondria are respiring on NAD ÷- linked substrates. The order of potency is rotenone > > chalepin > impera- torin > marmesin.

MATERIALS AND METHODS

Materials Pure samples of chalepin, imperatorin, marmesin were obtained from

Professors D.A. Okorie and K. Adesogan, Chemistry Department, University of Ibadan, Nigeria. The purity of these compounds were determined by a comparison of their spectra (IR, UV, NMR and mass spectroscopy) and melting points with those of authentic samples [4,5]. All other chemicals were of the highest purity available and were purchased from either Sigma Chemical, London, or Fluka AG (CH-9470 Buchs), Switzerland. Mitochondria were isolated in 250 mM sucrose essentially according to the method described by Schneider and Hogeboom [11] from the livers of adult Wistar strain albino rats obtained from pathogen-free colonies of the Pre-Clinical Animal Breeding House, University of Ibadan. The respiratory control ratio of each mitochondrial preparation was not less than 4.0. Mitochondrial pro- tein was determined by use of the biuret reagent [12].

Methods Polarogrpahic measurements of oxygen uptake. The rate of oxygen con-

sumption by mitochondria was measured by use of the conventional Clark- type oxygen electrode (Yellow Springs Instruments, OH, U.S.A.) polaro- graphic technique. When a steady-recorder tracing was achieved, an aliquot of the mitochondrial fraction (final concentration 2 mg mitochondrial protein/ ml) was introduced into the reaction vessel which contained 1.1 ml reaction medium (120 mM KC1, 20 mM Tris--HC1, pH 7.4 and 5 mM KH2P04}. After the stabilization of the recorder pen, and aliquot of any of pyruvate/malate {3.98 raM/1.9 raM} or succinate {3.98 raM) was carefully introduced by use of a Hamilton syringe inserted through the opening in the glass stopper of the reaction vessel. Aliquots of ADP (final concentration 50 nmol ADP/mg mito- chondrial protein) or furanocoumarin were added at specific intervals after

266

obtaining satisfactory steady-recorder tracing. Aliquots of oligomyc~n or FCCP were also added in some experiments. Rotenone was always added together with succinate to block the transfer of electrons from 3-site substrates. Respiratory rates and control ratios were computed as described by Estabrook [13].

Measurement of proton transport. Proton ejection was measured accord- ing to the method described by Reynafarje et al. [14]. Pi-depleted mitochon- dria were prepared by pre-incubating washed mitochondria with 250 mM sucrose, 10 mM KC1 and 3 mM HEPES (pH 7.1) for 10 rain in the cold, after which the suspension was diluted with more of the incubation medium and the mitochondria re-isolated and suspended finally in a small volume of the 250 mM LiC1, 10 mM KCI, 3 mM, HEPES, 4 taM rotenone and 100 ng valino- mycin/mg protein (pH 7.2). Changes in the pH of the reaction medium were followed by means of Philips pH-glass electrode connected through a buck- ing voltage box to a Perkin-Elmer Model 56 recorder. After 2 rain of incuba- tion to ensure that endogenous substrates and ATP were depleted, 1 mM potassium succinate or 1 mM pyruvate/malate was added to induce proton translocation. In the test experiments with the furonacoumarins, an aliquot of the compound to be tested was added after the addition of the substrate. The rate of proton flux was computed as ng-ions H÷/min per mg protein. The standard proton translocator used was FCCP.

Measurement of Ca ~÷ movements across mitochondmal membrane. Changes in the extra-mitochondrial concentration of free Ca 2÷ were followed using a calibrated Ca 2÷ ion-selective electrode as modified by Reynafarje et al. [15]. The electrode potentials were amplified by a Philips pH meter linked through a bucking voltage box to a Perkin-Elmer recorder Model 56. The reaction vessel contained, in final concentrations, 150 mM KCI, 3 mM HEPES, 2 taM rotenone, 2 mM succinate and 100 taM CaC12. After obtaining a steady-recorder tracing, 7.5 mg mitochondrial protein were added to initiate the rapid uptake of Ca 2÷. This was followed by a retention of the accumu- lated Ca 2÷ by the respiring mitochondria; 1 taM FCCP or an aliquot of the test compounds was immediately added to release the accumulated Ca ~÷. The rate of flux of Ca ~÷ was computed as ng-ions CaZ÷/min per mg protein.

RESULTS

Effects of chalepin, marmes~n, and ~mperatomn on mitochondmal metabohc state $ resp~ratzon

The effects of chalepin, lmperatorin, and marmesin on pyruvate/malate- supported state 4 respiration of rat liver mitochondria are shown in Tables I, II and III, respectively. In this experiment, aliquots of the furanocoumarins being tested were added to the reaction vessel on attainment of state 4 or resting state respiration following ADP depletion in metabolic state 3 respi- ration. The effect seen consisted of a concentration-dependent inhibition of state 4 respiration by the three furanocoumarins up to 60 taM which inhib- ited respiration by 69, 40 and 20% for chalepin, imperatorin and marmesin,

267

TABLE I

MODIFICATIONS OF MITOCHONDRIAL BIOENERGETICS BY CHALEPIN °

Each value ~s a mean for five different mitochondrial preparations ± S.D. RCR or respn-atory control ratio was computed as ratio of state 3 rate to state 4 rate ~n the absence of furanocoumarin.

Chalepzn State 4 State 3 State 4 RCR Rate of H* Rate of Ca 2. (/aM) (ng A 02 m -1 mg protein) plus FCCP ejection accumulation

(ng runs H" or Ca 2÷ mm -1 mg protein -1)

0 20.1 ± 1.2 84.6 ± 2.2 90.3 ± 2.7 4.2 193.6 ± 4.6 97.3 ± 3.4 0 r 17.6 ± 0.8 73.9 ± 2.7 74.6 ± 2.1 4.2 - - 5 18.1 ± 0.9 69.9 ± 3.1 72.4 ± 1.8 3.5 186.3 ± 4.1 81.5 ± 2.8

10 15.5 ± 0.9 16.5 ± 0.7 24.0 ± 0.9 0.8 31.6 ± 1.1 19.3 ± 1.7 15 6.5 ± 0.7 12.6 ± 0.7 13.1 ± 0.8 0.6 21.7 ± 1.2 14.1 ± 1.1 20 6.7 ± 0.7 11.9 ± 0.6 11.6 ± 0.9 0.6 20.5 ± 1.0 14.6 ± 0.8 30 6.4 ± 0.6 9.3 ± 0.7 10.2 ± 0.8 0.5 17.5 ± 0.9 14.3 ± 0.7 40 6.4 ± 0.3 9.2 ± 0.6 8.4 ± 0.5 0.5 17.5 ± 0.8 14.1 ± 1.1 60 6.2 ± 0.3 9.0 ± 0.4 6.6 ± 0.4 0.4 17.1 ± 0.8 13.8 ± 0.7 60 b 10.5 ± 0.7 8.9 ± 0.6 7.3 ± 0.5 0.5 - - Rotenone 6.7 ± 0.5 8.7 ± 0.6 5.9 ± 0 5 0.4 21.0 ± 0.9 14.1 ± 0.9

(1.5 ~v~)

• Respn'atory substrate: pyruvate/malate bRespiratory substrate: 5 mM/~-hydroxybutyrate

r e s p e c t i v e l y . T h e r e s u l t s also show t h a t s t a t e 4 r e s p i r a t o r y r a t e was r e d u c e d by 68, 25, and 17°/0 a t 30 /~M of cha lep in , i m p e r a t o r i n a nd m a r m e s i n , r e s p e c t i v e l y . A t 10 /~lVl, on ly cha lep in s i gn i f i c a n t l y i n h i b i t e d s t a t e 4 r e sp i r a - t ion , by 23%. A l t h o u g h , a b o u t 10°/0 i n h i b i t i o n was s e e n w i t h 5 wM cha lep in ,

lower c o n c e n t r a t i o n s of t he t h r e e f u r a n o c o u m a r i n s (~< 5 ~M) did no t show a n y

a p p r e c i a b l e effect on s t a t e 4 r e s p i r a t i o n . As a s s e s s m e n t of t he effect of t he f u r a n o c o u m a r i n s on s u c c i n a t e - s u p p o r t e d s t a t e 4 r e s p i r a t i o n is p r e s e n t e d in

Tab l e IV. Here , i t is e v i d e n t t h a t n o n e of t he t h r e e f u r a n o c o u m a r i n s t e s t e d

had a n y effect on t he r a t e of s t a t e 4 r e s p i r a t i o n w h e n suc c i na t e was u sed as an e l e c t r o n donor . S imi l a r ly , no i n h i b i t i o n was o b s e r v e d w h e n t he fu ranocou- m a r i n s w e r e p r e - i n c u b a t e d w i th f resh m i t o c h o n d r i a l p r e p a r a t i o n s , in t he

p r e s e n c e of s u c c i n a t e a l t h o u g h t h e r e was a mi ld s t i m u l a t i o n of s t a t e 4 respi - r a t i o n a t c o n c e n t r a t i o n s g r e a t e r t h a n 60 ~M cha lep in ( resu l t s no t shown).

Effects of chalepin, marmesin and imperatorin on respiratory control by ADP

T a b l e s I, I I and I I I show the prof i les of t h e r a t e s of A D P - s t i m u l a t e d or

me tabo l i c s t a t e 3 r e s p i r a t i o n in t he p r e s e n c e of p y r u v a t e / m a l a t e a nd v a r y i n g a m o u n t s of cha lep in , i m p e r a t o r i n a n d m a r m e s i n , r e s p e c t i v e l y . The d a t a show t h a t A D P - s t i m u l a t e d r e s p i r a t i o n was i n h i b i t e d by a t l ea s t 80°/0 by 10 ~M

268

TABLE II

INFLUENCE OF IMPERATORIN ON MITOCHONDRIAL BIOENERGETICS •

Each value ~s a mean for f~ve different mitochondr~al preparations __ S.D. RCR or respiratory control raho was computed as raho of state 3 rate to state 4 rate m the absence of furanocou- marin

Imperatorin State 4 State 3 State 4 RCR Rate of H* Rate of Ca 2÷ (~I) (ng A O~ m -1 mg protein -~) plus FCCP e~echon accumulahon

(ng ~ons H ÷ or Ca 2÷ m~n -1 mg protein -l )

0 23.5 ± 1.1 96.5 ± 2.1 91.3 ± 1.8 4 1 189 7 ± 4.7 95.7 ± 3.1 0 b 19.2 ± 0.9 82.6 ± 1.9 87.5 ± 2 1 4.2 -- -- 5 22.1 ± 0.7 89.7 ± 2.1 83.4 ± 1.6 3.8 186.5 ± 4.3 94 6 ± 3.2

10 21.6 ± 0.7 84.1 ± 2.2 77 6 ± 2 1 3.6 161.2 ± 3.1 83.7 ± 2.1 15 20.0 ± 0 6 79.3 ± 1.8 70.3 ± 1.4 3.4 143.5 ± 3.7 76.6 ± 2.5 20 188 ± 0.7 744 ± 1.5 675 ± 1 3 3.2 1336 ± 2.9 69.3 ± 2.1 30 17.6 ± 0.6 63.9 ± 1.9 60.1 ± 1.5 2.7 119.6 ± 2.3 60.7 ± 1.3 40 16.5 ± 0.7 57.8 ± 2.1 52.1 ± 0.9 2.4 117.6 ± 3.1 53.6 _ 1.4 60 14.2 ± 0.6 50.1 ± 1.1 45.7 ± 0.7 2.1 114.5 ± 2 7 51.2 ± 1.2 60 b 10.9 ± 0 4 46.3 ± 1.3 80.6 ± 2.3 2.4 -- -- Rotenone 5.6 ± 0 3 9.7 ± 0.7 7.1 ± 0.6 0.4 20 6 ± 0.8 13.8 ± 0 9

(1.5 ~M)

• Respiratory substrate: pyruvate/malate. bRespiratory substrate: 5 mM/~-hydroxybutyrate.

cha lep in , i n d i c a t i n g t h a t r e s p i r a t o r y con t ro l by A D P was a l m o s t t o t a l l y los t a t th i s l eve l of cha lep in . W h e r e a s , a b o u t 13 a nd 8O/o i n h i b i t i o n o c c u r r e d wi th 10/~M i m p e r a t o r i n and 10 ~M m a r m e s i n , r e s p e c t i v e l y . A l t h o u g h t he s e v e r i t y

of i n h i b i t i o n i n c r e a s e d s t e a d i l y w i th i n c r e a s i n g a m o u n t s of i m p e r a t o r i n a nd m a r m e s i n up to 60 ~Vl, r e s p i r a t i o n was i n h i b i t e d m a x i m a l l y by approxi - m a t e l y 89o/o a t c o n c e n t r a t i o n s equa l to or h i g h e r t h a n 60 pM cha lep in . Mar-

m e s i n and i m p e r a t o r i n a t 60 /~M c o n c e n t r a t i o n i n h i b i t e d A D P - s t i m u l a t e d

r e s p i r a t i o n by 29 and 48%, r e s p e c t i v e l y . R e s p i r a t o r y con t ro l by A D P in an i m p o r t a n t c r i t e r i o n for d e t e r m i n i n g the

coup l ing of m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t to A D P p h o s p h o r y l a t i o n . I t is c o n v e n t i o n a l to e x p r e s s th i s con t ro l as a r a t i o of t he r a t e of t he A D P - s t i m u - l a t ed or s t a t e 3 r e s p i r a t i o n to t he r a t e of the ADP- l e s s or s t a t e 4 r e s p i r a t i o n .

Th i s r a t i o is t e r m e d t he a c c e p t e r or r e s p i r a t o r y con t ro l ra t io . Va lues for

m i t o c h o n d r i a l p r e p a r a t i o n s r e s p i r i n g on p y r u v a t e / m a l a t e a nd in t he p r e s e n c e of v a r y i n g c o n c e n t r a t i o n s of t h r e e f u r a n o c o u m a r i n s a r e s h o w n in

T a b l e s I - I I I . T h e r e s u l t s show t h a t t he r e s p i r a t o r y con t ro l r a t io of r a t l ive r m i t o c h o n d r i a is r e d u c e d by t h e s e f u r a n o c o u m a r i n s 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 . T h e r e s p i r a t o r y con t ro l r a t i o of t he m i t o c h o n d r i a l p r e p a r a t i o n

u sed in th i s s t u d y was no t less t h a n 4.0. Th i s r a t io was r e d u c e d by 17, 7, a nd 5O/o by a d d i n g to t he r e a c t i o n vesse l , 5 pM cha lep in , 5 pM i m p e r a t o r i n and

269

TABLE II I

INFLUENCE OF MARMESIN ON MITOCHONDRIAL BIOENERGETICS"

Each value is a mean for five different mitochondrlal preparat ions ± S.D RCR or respi ra tory control ratio was computed as ratio of s ta te 3 ra te to s ta te 4 rate in the absence of furanocou- marin.

Marmemn State 4 State 3 State 4 RCR (/~IVl) (ng A 02 m -1 mg protein -~) plus FCCP

Rate of H ÷ Rate of Ca 2~ ejectlon accumulation

(ng ions H ÷ or Ca 2÷ mln -~ mg

proteln -~)

0 23.0 ± 0 9 93.6 ± 3.1 97.3 ± 2.1 4.0 190.2 ± 2.1 96.6 ± 2 6 0 b 18.1 ± 0.6 778 ± 2.1 795 _ 2.8 4.3 -- --

5 22.8 ± 0.7 89.4 ± 3.0 91.5 ± 2.7 3.8 183.7 ± 3.4 94.6 ± 2.1 10 21.8 ± 0.8 86.2 ± 2 7 87.4 ± 2 1 3.7 180.6 ± 3.7 92,6 ± 1.8 15 21 3 ± 0.9 81.7 ± 2.1 80.6 ± 2.1 3 6 179 6 ± 3.2 92,3 ± 1.4 20 20.3 ± 0.7 79.6 ± 1.8 76.3 ± 2.0 3 4 171.2 ± 2.7 89,4 ± 1.7 30 19.7 ± 0.7 74.1 ± 2.7 71.6 ± 2.1 3 2 163.4 ± 2.1 83,6 ± 1.4 40 19.2 ± 0.6 70.1 ± 1.6 69.9 ± 1.7 3.0 160.2 ± 3 1 79.1 ± 1.6 60 18.3 ± 0.5 66.6 ± 1.5 67.3 ± 2.6 2.9 157.3 ± 2.7 73.6 ± 1.7 60 b 14.1 ± 0.6 57.6 --+ 1.1 60.1 __ 1.8 3.1 -- -- Rotenone 55.2 ± 0.3 9.9 ± 0.4 6.2 ± 0.4 0.4 21.7 ± 0.9 13.8 ± 0.7

(1.5 ~V~)

'Respira tory substra te : pyruvate/malate. bRespiratory substra te : 5 mM/~-hydroxybutyrate.

TABLE IV

MITOCHONDRIAL BIOENERGETICS IN THE PRESENCE OF CHALEPIN, IMPERATORIN AND MARMESIN DURING SUCCINATE OXIDATION

Each value is a mean for four &fferent mitochondrial preparat ions ± S.D

Additions State 4 State 3 State 4 RCR (ng A 02 m -1 mg protein-9 plus FCCP

Rate of H ÷ Rate of Ca 2÷ ejection accumulation (ng ions H ÷ or Ca 2÷ m~n -1 mg

protein -1}

None 30.6 ± 1.4 138.4 ± 4.1 141.3 ± 5.2 4.5 Chalepm 29.7 ± 1.0 140.1 ± 3.8 145.6 ± 4.1 4.6

(60 ~M) Impera tor in 31.1 ± 1.3 139.6 ± 3.3 137.0 ± 5 1 4 5

(60 ~ ) Marmesm 31.7 ± 1.7 141.7 ± 3 7 142.6 ± 4.7 4.6

(60 ~M) Rotenone 32.1 ± 0.9 140.1 ± 4 0 140 7 ± 3.7 4.6

(1.5 ~M)

249.2 + 3.7 1397 + 39 2449 ± 41 141.0 ± 36

253.1 ± 4.4 142 1 __ 4.1

250.3 ± 4.6 144.1 ± 3 7

2486 ± 3.9 139.3 _+ 42

270

5 ~M marmesin, respectively before adding ADP, the phosphate acceptor. At 10 ~M, this ratio was further reduced by 81, 13, and 8°/0, with chalepin, imperatorin and marmesin, respectively. The extent of inhibition of ADP- supported respiration with 40, 50 and especially 60 ~M chalepin was not less than 88%, suggesting that the mitochondria were no longer able to synthes- ize ATP at these levels of chalepin. Respiratory control ratio was reduced by 49 and 28°/o by 60 ~M imperatorin and 60 ~M marmesin, respectively.

Table IV summarises the results obtained when succinate was used as an electron donor. As seen from the results, state 3 respiratory rate was not affected by any of the three furanocoumarins at the highest concentrations tested. Consequently, respiratory control by ADP was not altered by these furanocoumarins during succinate oxidation.

Effect of furanocoumamns on respiration-dmven proton translocation by rat liver mitochondria

The pattern of proton transport during the oxidation of succinate in the presence of varying amounts of these furanocoumarins being investigated is shown in Table IV. Succinate-supported proton translocation was unaffected by the three furanocoumarins tested. The pattern of the effect of these fur- anocoumarins on proton translocation by mitochondria respiring on pyru- vate/malate is summarized in Tables I - I I I . As seen from these results, respiration-driven proton pumping was inhibited in a concentration-depen- dent manner by imperatorin and marmesin up to 60 ~M. Although chalepin inhibited the process more than marmesin and imperatorin, no further inhibi- tion was observed at chalepin concentrations higher than 15 wM. Thus the degree of inhibition (89o/0) seen with 20, 30, 40 and 60 ~M chalepin was not statistically different from that seen with 15 pM. The process was maximally inhibited by 30 ~M marmesin, 30 pM imperatorin and 15 ~M chalepin by 14, 37 and 89o/0, respectively (Tables I--III).

Abolition of mitochondrial Ca ~÷ uptake by furanocoumarins Ca e÷ accumulation by mitochondria is supported by the membrane poten-

tial created either by respiration or by the hydrolysis of ATP. Because of the relevance of the process in the mechanism of regulation of intracellular free Ca ~÷ ion concentration as well as its sensitivity to respiratory inhibitors, the influence of the three furanocoumarins of interest on the respiration- driven Ca~+-uptake by isolated rat liver mitochondria was studied. Changes in extramitochondrial Ca e÷ ion concentration during the oxidation of succin- ate or pyruvate/malate was followed by use of a sensitive Ca~÷-ion selective electrode as described under Materials and Methods. The results obtained with mitochondria respiring on pyruvate/malate are summarized in Tables I --III. Again chalepin inhibited Ca ~÷ accumulation by over 85% at 15 ~M even though the extent of abolition of Ca ~÷ uptake was not statistically different at concentrations ~> 15 ~M chalepin. Maximal inhibiting of about 86°/0 was seen at 60 ~M chalepin. These results show that Ca ~÷ uptake was inhibited almost totally at 15 ~M (Table I). Furthermore, the results are identical to

271

the effects of chalepin on respiratory control by ADP and respiration-driven proton translocation.

The effects seen with imperatorin and marmesin differ (Table II and III) however, from those seen with chalepin because the abolition of Ca ~÷ uptake was concentration-dependent up to about 30 ~M for these furanocoumarins and was maximal at 60 ~ I imperatorin (Table II) which reduced the rate of uptake by 460/0, and similarly at 60/~M marmesin which gave only 240/0 inhi- bition (Table III). The results show in addition, that at 15 pM, addition of imperation and marmesin to the reaction medium gave 20 and 7°/0 inhibition, respectively. A comparison of the effects of chalepin with that of rotenone revealed that the inhibition caused by the addition of 1.5 pM rotenone was statistically equal to that produced by 15 ~M chalepin, thus indicating that chalepin could be about 10 times less potent than rotenone.

Table IV shows the pattern of mitochondrial Ca 2÷ transport supported by succinate and in the presence of varying amounts of the three furanocoumar- ins. Clearly, succinate-supported Ca~÷-accumulation was not inhibited by any of the furanocoumarins studied. Furthermore, an assessment of the interac- tion of these naturally-occurring chemicals with ATP-supported Ca2÷-accu - mulation revealed a no-effect response even at 60 ~M chalepin (results not shown).

DISCUSSION

In this study, evidence is presented to show that chalepin and imperatorin have significant inhibitory effects on the generation of proton electrochemi- cal potential gradient during the NAD÷-linked oxidation of substrates by rat liver mitochondria. An assessment of the interaction of chalepin, imp~erato- rin, and marmesin with resting state respiration reveals that of the three furanocoumarins, only chalepin and imperatorin significantly inhibited the oxidation of pyruvate/malate; the degree of inhibition by chalepin (15 /~I) being equal to that produced by 1.5 ~ rotenone (Table I). Interestingly, succinate oxidation was insensitive to these chemicals even on pre-incubation of mitochondria with high levels of the furanocoumarins. These results sug- gest therefore, that chalepin and imperatorin may be blocking electron trans- fer from NADH to coenzyme Q because electron transier from succinate via FAD to coenzyme Q remains unaffected as in rotenone inhibition. The mech- anism of this interaction is, however, not clear because of the bulky nature of NADH co-enzyme Q reductase [16].

An important parameter for assessing mitochondrial bioenergetics is the respiratory control ratio, which is a ratio of the rate of resting state respira- tion to the rate of ADP-stimualted respiration. A glance at the values obtained for state 3 respiration at varying concentrations of furancoumarins reveals that state 3 respiration is almost totally (at least by 80%) inhibited by 10 ~M chalepin; indicating that ADP was not phosphorylated as such at this amount of chalepin (Table I). Furthermore, imperatorin (60 ~M) inhibited ADP phosphorylation by 480/0 while marmesin (60 ~M) gave a 29% inhibition.

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Similar results were obtained when /~-hydrobutyrate was used as substrate (Tables I--III), suggesting that the inhibition of phosphorylation of ADP by chalepin and imperatorin is due to an interaction of the furanocoumarins with the NADH-coenzyme reductase complex rather than with the NAD ÷- dependent dehydrogenase responsible for oxidizing pyruvate. Furthermore, it seems likely that because state 4 respiration is almost completely annuled by chalepin, enough protons are not translocated out of the matrix even on addition of ADP, thus preventing the formation of sufficiently high transmembrane pH difference necessary to form the proton electrochemical potential difference. Thus the respiratory control ratios obtained in the pres- ence of rotenone and/or chalepin are reduced by about 90O/o, whereas the effect of imperatorin (60 ~M) is about 48°/o that seen with chalepin. Marine- sin has a slight inhibitory effect (28O/o) on state 3 respiration (Table III).

To confirm the effect of these compounds on electron transfer-linked proton pumping, mitochondrial proton translocation was monitored by use of a sensitive pH-glass electrode during exposure of mitochondria to varying concentrations of the furanocoumarins. Our results demonstrate unequivo- cally that chalepin and imperatorin inhibit pyruvate/malate-supported proton ejection (Tables I and II). Specifically, chalepin (15 ~M) almost completely inhibited proton ejection while imperatorin (30 ~M) inhibited the process by 37O/o (Table II). Again, succinate-supported proton ejection was not affected by any of the three furanocoumarins (Table IV). Clearly, electron transfer proton pumping is obstructed by chalepin and imperatorin at the level of NADH-coenzyme Q reductase. Although these compounds are likely to pre- vent pyruvate/malate-supported proton pumping indirectly by blocking elec- tron transfer from NADH to coenzyme Q, the possibility that they could interact directly with the hydrophobic proton translocating component of the complex may not be completely ruled out.

However, because the proton electrochemical potential gradient is made up of a pH difference and an inside negative membrane potential which is normally used to drive the energy-dependent Ca 2÷ uptake by mitochondria, the effects of chalepin, imperatorin and marmesin on the process of mitochondrial Ca 2÷ uptake were investigated (Tables I--III). The results obtained by following the rate of respiration driven Ca 2÷ accumulation by mitochondria using an ion-selective Ca 2÷ electrode, reveal that chalepin abolished Ca e÷ uptake supported by pruvate/malate in the same manner as rotenone while imperatorin only slightly reduced the ability of mitochondria to accumulate Ca ~÷ (Tables I and II). Marmesin has a very mild inhibitory effect on this process (Table III). The effect of vitamin K-3 on furanocou- marin-inhibited mitochondrial respiration is shown in Table V. The data show that furanocoumarin-inhibition of pyruvate/malate oxidation was by- passed by adding catalytic amounts of vitamin K3 to mitochondria previously exposed to chalepin, imperatorin and marmesin, respectively, in the same manner as amytal [17]. This finding offers additional evidence that electron transfer from NADH to Co Q was blocked by these furanocoumarins during the oxidation of NAD-linked substrates.

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TABLE V

EFFECT OF VITAMIN K-3 ON FURANOCOUMARIN-INHIBITED RESPIRATION"

Each value is a mean for three mitochondrial preparations ± standard deviation. Reaction medium same as described under materials and methods. The additions made consisted of the following in final concentrations: antimycin (0.22 ~g), rotenone (1.5 ~I) , vitamin K~ (5 nmol), dicumarol (10 tool), and amytal (1 mM),

Additions State 4 rate State 3 rate (ng A O~ mm -1 mg protein -1)

Control 17.3 ± 0.9 79.6 ± 2.1 (no additions)

Vitamin K~ 17.4 ± 0.7 80.3 ± 1.9 Dicoumarol (a) 17.1 ± 1.0 80.1 ± 2.1 Antimycin (b) 3.2 ± 0.4 4.1 ± 0.3 Rotenone 4.7 ± 0.3 5 0 ± 0.4 Chalepin (60/~I) 4.9 ± 0.4 5.5 ± 0.3 Imperatorin (60 ~u~I) 10.4 ± 0.5 59.2 ± 1.0 Marmesin (60 ~lVl) 13.3 ± 0.7 66.4 ± 1.5 Chalep]n + Vit K-3 17.2 ± 0.8 80.1 ± 1.8 Chalepin + Vlt K-3 (a) 9.5 ± 0.6 14.1 ± 0.7 Chalep~n ÷ Vit K~ (b) 5.1 ± 0.3 6.3 ± 0.2 Imperatorin + Vit K-3 17.6 ± 0.8 81.3 ± 1.6 Marmesin + Vit K~ 17.3 ± 0.9 81.6 ± 1.7

• Pyruvate/malate was used as respiratory substrate.

Although the exact mechanism of inhibition of electron flow from NADH to Co Q by these furanocoumarins is not known, it seems possible that they could be acting like rotenone or piericidin [18] because (1) FCCP does not relieve their inhibitory effects (Tables I--III), and (2) the chemicals possess groups (Fig. 1) such as ),,),-dimethylallyl- in position 3 of chalepin, a 3-methyl- but-2-enyloxy- in position 7 of imperatorin, and isopropenyl- in position 5 of rotenone, in addition to either a furanocoumarin or coumarin ring which could carry electrons/protons probably in the same manner as the 7,8-dime- thyl-isoalloxazine moeity of FMN but unable to donate the electrons to the next carrier due to the electron donating effect of the groups mentioned above on the coumarin ring. Thus, aside from the hydrophobic characters of these chemicals, the magnitude of the electron-donating effect of these groups could determine the degree of potency of these compounds as inhibi- tors of electron transfer. Because millimolar amounts of amytal and about 1 and 0.3 nmol/mg protein of rotenone and piericidin are, respectively, required to nearly totally inhibit the NADH oxidase of electron transport particles [18], the order of potency of these inhibitors vis-a-vis chalepin may be put as piericidine Y rotenone Y chalepin ~ amytal. It seems unlikely, however, that chalepin and the other furanocoumarins studied will interact with electron transfer in the same manner as rhein (4,5-dihydroxyanthra

274

quinone-2-carboxyhi acid), a compeititive inhibitor of NADH oxidation [19] because vitamin K-3 restored respiration of furanocoumarin-inhibited mito- chondria.

It may thus be concluded from the findings reported here that an excessive and frequent ingestion of these substances could cause a serious disturbance of energy metabolism in certain tissues. The relationship between these effects and the toxicity of these substances and their metabol- ites should therefore be investigated.

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