TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

49,209-217

(1979)

Effect of Ifenprodil on Mitochondrial Respiration of Guinea Pig Brain YOSHIAKI YAMASHITA,’

MASAO

NISHIKAWA, SHIGEO MITSUHIRO, AND TATSUO FURUKAWA~

EIICHI SAKAKIBARA,

General Research Center, Funai Pharmaceutical Industries, Ltd., Hirakata, and Department of Pharmacology, School of Medicine, Fukuoka Fukuoka 814, Japan

Osaka 573, Japan; University,

Received August 1, 1978; accepted December 28, 1978 Effect of Ifenprodil on Mitochondrial Respiration of Guinea Pig Brain. YAMASHITA, NISHIKAWA, M., MITSUHIRO, S., SAKAKIEWU, E., AND FURUKAWA, T. (1979). Toxicol. Appl. Pharmacol. 49, 209-217. The effects of ifenprodil on respiratory activity in both the presence and absence of cyanide were examined using guinea pig brain mitochondria and mouse brain cytochrome oxidase preparations. Ifenprodil stimulated respiratory activity, exhibiting maximal effect at a concentration of 0.8 mM in the presence of succinate and potassium phosphate (P,) among the various substrates, anions, and phosphonate compounds tested. The stimulatory action of ifenprodil on respiration could not be observed with frozen and thawed brain mitochondria. The stimulatory action was not affected by oligomycin but was reduced markedly by mersalyl. Dinitrophenol and flufenamic acid did not require the presence of Pi for their stimulatory action on respiration. Ifenprodil abolished completely the cyanide-induced inhibition of respiration with succinate as a substrate, but elicited no significant effects against the cyanide-induced inhibition of cytochrome oxidase activity in both in vivo and in vitro experiments. The results suggest that ifenprodil seems to act on the mitochondrial membrane by accelerating P, and succinic acid transport through the’membrane as well as by stimulating succinate oxidation, and that these stimulatory actions of ifenprodil may be involved in its protective effects against cyanide toxicity. Y.,

Ifenprodil [4-benzyl-a-(p-hydroxyphenyl)+ methyl-I-piperidineethanol], a 2-piperidinoalkanol derivative, has a-adrenergic blocking, weak p-mimetic and moderate antihistaminic activities (Carron et al., 1971; Mizusawa and Fujiwara, 1974; Maeda and Furukawa, 1975), as well as peripheral and cerebral vasodilator effects (Carron et al., 1971). It was also observed that ifenprodil showed a therapeutic effect on human cerebrovascular diseases (Otomo and Kodama, 1976). 1 Address for reprints: Y. Yamashita, General Research Center, Funai Pharmaceutical Industries, Ltd.,

Hirakata,

Osaka

573,

Japan.

2 Department of Pharmacology,‘ School of Medicine, Fukuoka University, Fukuoka, Japan. 209

The present authors (Furukawa et al., 1976) have found that ifenprodil exhibits a protective effect against cyanide toxicity determined by lethality, decline in respiration, blood pressure, and electroencephalogram in rats, guinea pigs, rabbits, and mice. The protective effects of this drug are not due to chemical trapping of cyanide (Furukawa et al., 1976). In addition ifenprodil protected against the decrease of respiratory control index in brain mitochondrial respiration elicited by the experimental injury of cerebral blood flow in rabbits (Furumi et al., 1975). Therefore, the present investigation was performed in an attempt to understand the mechanism involved in the antagonism of 0041-008x/79/080209-09s02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

210

YAMASHITA

ifenprodil against cyqnide poisoning, by determining the effect of this drug on KCN inhibition of brain mitochondrial respiration of guinea pig in vitro and brain cytochrome oxidase activity of mouse in vivo and in vitro.

METHODS The animals used were healthy male guinea pigs weighing about 300 g and ddK male mice weighing about 25 g. Ifenprodil was prepared by the method of Carron et al. (1971) and used as its L-tartaric acid salt. The ifenprodil sample used in this study is believed to be pure as judged from chemical and physical analysis using ultraviolet, infrared, and proton nuclear magnetic resonance techniques, as well as polarimetric and elementary analysis, and also from chromatographic data in our laboratory. Adenosine+-diphosphate, Na salt (ADP), oligomycin, tris(hydroxymethyl)aminomethane (Tris), and o - (3 - hydroxymercuri - 2 - methoxypropyl)car bamoyl-phenoxyacetate, Na salt (mersalyl) were purchased from Sigma Chemical Company. Pyrophosphate, Na salt was from Wako Chemicals. Cytochrome c was from Sankyo Company, sodium dodecyl sulfate from Difco Company, and antimycin A from P-L Biochemicals. N-(3-Trifluoromethylphenyl)anthranilic acid (flufenamic acid) was prepared by purifying Arlef 100 which was obtained from Taisho Pharmaceutical Company. All the other reagents were purchased from Nakarai Chemicals, Kyoto. The reagents were dissolved in redistilled water unless otherwise specified. If necessary all solutions were adjusted to pH 7.4. Preparation of bruin mitochondria. Mitochondria were prepared from guinea pig brain according to the method of Ozawa et al. (1966). After the animals were sacrificed by decapitation, the brain was removed and placed immediately into an ice-cold suspension medium containing 0.3 M o-mannitol and 0.1 mM ethylenediaminetetraacetic acid Na, salt (EDTA). The suspension was mildly homogenized in a hand homogenizer of the Potter type, followed by centrifugation at 6OOg for 8 min. The supernatant was centrifuged at 10,000g for 10 min, and the resulting precipitate was resuspended in the suspension medium described above. The suspension was centrifuged at 5OOOgfor 20 min, and the resulting precipitate consisted of three layers. The lower brownish layer, the mitochondrial preparation, was resuspended by homogenization in a suspension medium to give a protein concentration of approximately 32.5 mg/ml. The protein content of the mitochondrial suspension was determined by the biuret method (Gornall et al., 1948). Determination of mitochondrial respiratory activity.

ET AL.

Respiratory activity was measured polarographically by the method described by Hagihara (1961) using an automatic recording apparatus (Yanagimoto PO-100 A). Incubations were carried out at 25°C in an assay medium (pH 7.4) containing 0.22 M Dmannitol, 7.25 mM KCI, 7.25 mM Tris-HCI, 29.0 mu KH,PO,, and 0.145 mM EDTA in a total volume of 2.0ml. The standard assay system was as follows: mitochondrial suspension (0.4 ml) was added after the assay medium indicated above was preincubated for 2 min. One minute later, the reaction was started with the addition of substrate. Sequentially, after 2 min, ifenprodil or the other reagent was added. In the experiment on antagonism against cyanide inhibition, KCN was present at a concentration of 20~~ as reported previously with guinea pig liver mitochondria (Furukawa et al., 1976). The effect without the reagent was observed by adding water instead of the reagent unless otherwise specified. The respiratory control index was calculated according to the method of Hagihara (1961). The respiratory control index with mitochondria for assay was 3.54 0.4 (n = 75) in the presence of succinate as a substrate. Rates of respiratory activities are expressed as nanomoles of O2 consumed per minute per milligram of protein, unless otherwise specified. The preparation and assay of brain cytochrome oxiduse. The mice, which had been fasted overnight, received 6 mg/kg ifenprodil by ip administration. The administration’of KCN and the sacrifice of animals were carried out according to the method of Schubert and Brill (1968); KCN (3 mg/kg) was injected ip 55 min after the administration of ifenprodil. Five minutes later, the animals were sacrificed by decapitation. The brain was removed and placed immediately into an ice-cold suspension medium for mitochondria to give a 10% solution, followed by homogenization. The protein content per 0.4 ml of this homogenate was 9.4 + 0.15 mg of protein, the mean f SE of 36 animals. Cytochrome oxidase (cytochrome c : oxygen oxidoreductase, EC 1.9.3.1) activity was assayed as follows: the brain homogenate (0.4 ml) prepared was preincubated for 2 min in an assay medium with an added 1.25 mg of sodium dodecyl sulfate. After the antimycin A and the reducing solution containing L-ascorbate, p-phenylenediamine, and EDTA were added successively at intervals of 30 set, the reaction mixture was incubated for 2 min. Antimycin A, cytochrome c, L-ascorbate,p-phenylenediamine, and EDTA were respectively present at the final concentrations of 0.5 PM, 1.5 ,uh4, 40 mM, 1.88 mM, and 40 mM in the total volume (2 ml) of the reaction mixture. Statistical analysis. The values were expressed as means + SE. The statistical analysis was calculated using Student’s t test, and the level of significance chosen was P-C 0.05.

IFENPRODIL ON MITOCHONDRIAL

211

RESPIRATION

RESULTS Eficts of Ifenprodil on Brain Mitochondrial Respiration

As shown in Fig. 1, ifenprodil, which was added to the incubation medium containing guinea pig brain mitochondria, after the addition of succinate, stimulated the respiratory activity in a dose-dependent manner. This ifenprodil-induced stimulation in I 8 the respiration was maximal at a concentra01’ 0 0.5 1.0 tion of 0.8 mM ifenprodil. IFENPRODIL (mM) None of these actions were observed by an FIG. 1. Effects of ifenprodil on brain mitochondrial addition of L-tartaric acid or KC1 instead of respiration. Succinate used as substrate was present ifenprodil or KH,P04 (Pi). at a concentration of 12.5 mM. Ifenprodil was added Ifenprodil did not elicit stimulative effects as indicated. Each point and vertical bar represent when frozen and thawed mitochondria were the mean value + SE of five experiments. An asterisk indicates a significant difference from respiratory used. activity without ifenprodil.

Eflects of Ifenprodil on Respiratory Activity with Various Substrates

strates in the presence and absence of P,. As summarized in Table 1, the stimulatory effect was obtained in the presence of Pi and with succinate as substrate, but not in the

The stimulatory effect of ifenprodil on respiration was examined using various subTABLE

1

EFFECT OF IFENPRODIL ON RESPIRATORY ACTIVITY OF GUINEA PIG BRAIN MITOCHONDRIA WITH VARIOUS SUBSTRATES, IN THE PRESENCE AND ABSENCE OF P,”

Respiratory activity (nmol/min/mg

protein)

-pi

+p,

Ifenprodil Substrate Succinate or.-a-Glycerophosphate a-Ketoglutarate L-Malate DL-Isocitrate DL-B-Hydroxybutyrate L-Glutamate L-Ascorbate + TMPD”

Ifenprodil

-

+

2.17kO.15 1.49kO.13 0.93 k 0.08 0.94kO.15 1.13f0.13 0.72kO.12 1.04*0.10 21.53+0.20

2.04kO.13 0.93+0.10 0.80 + 0.05 0.71+ 0.08 0.94kO.12 0.39kO.18 0.75rto.10 11.98kO.18

+ 2.7OkO.10 1.28 f 0.07 1.00+0.08 1.08+0.10 1.15rtO.18 0.45kO.13 1.02rtO.15 18.55kO.20

4.09+_0.15* 1.27kO.10 0.88kO.13 0.96kO.12 0.95kO.15 0.44 + 0.11 0.80 rf:0.08 14.58 + 0.22

a Results were expressed as the mean value f SE of five experiments. An asterisk indicates a signficant stimulative effect from respiratory activity without ifenprodil. Mitochondria were incubated in an assay medium in the presence and absence of 21.8 mu KH2P0,. Reactions were carried out as described in the text except as follows: each substrate indicated above was added 1 min after the addition of mitochondria, and then 2 min later ifenprodil was added. Ifenprodil was present at a concentration of 0.8 mM. L-Ascorbate was 8.6 mM and the other substrates 12.5 mM. (-), Absence; ( + 1, presence. * N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrcchloride.

212

YAMASHITA ETAL.

absence of Pi and with the other even in the presence of Pi.

substrates

Influences of Various Anions on the Stimulatory E#ect of Ifenprodil

As shown in Fig. 2, the stimulative effect of ifenprodil with succinate was markedly increased when POd3- was added to the medium, and was increased to a certain degree by addition of CH,CH,COO-, SOd2-, CH,COO-, and AsOd3-. Influences of Varying Concentrations of Pi on the Stimulatory E#ect of Ifenprodil

As shown in Fig. 3, mitochondrial respiration without jfenprodil was increased by the addition of P, (5 mM) but hardly changed by further increasing the concentration of Pi. The respiration with ifenprodil was stimulated by increasing in concentration of P,, the maximal effect being observed at a concentration of 30 mM Pi.

I

CONTROL P043-

CH3CH2' coo-

SO4'-

0 I, 0

10 20 30 POTASSIUM PHOSPHATE (mMi

8 40

FIG. 3. Effects of increasing concentrations of Pi on respiratory activity with or without ifenprodil. Reactions were carried out as described in the legend to Fig. 2, except that KHjPO, instead of anions was added. Respiratory activity was measured with various concentrations of KH,PO, in the absence (0) and presence (m) of 0.8 mM ifenprodil. Succinate used as substrate was present at a concentration of 12.5 mM. Each point and vertical bar represent the mean value +SE of five experiments. An asterisk indicates a significant difference from the respiratory activity without KH,PO,.

* I

CH3COO‘

AsO3'

FIG. 2. Effects of anions on respiratory activity stimulated by ifenprodil. Reactions were carried out as described in the text except as follows: anions were added to the assay medium from which KH*PO., was omitted and preincubated for 1 min, after which mitochondria and succinate were added successively at intervals of 1 min. Two minutes later, ifenprodil was added. KHZPOd, CH,CH,COONa, K,SO,, CH,COONa, and Na,HAsO, were used at a concentration of 40 mM. Ifenprodil was 0.6 mM and succinate 12.5 mM. Each column and vertical bar represent the mean value + SE of five experiments. An asterisk indicates a significant difference from control.

Effects of Pi on respiration stimulated by representative uncouplers were studied. The mitochondria were preincubated for 1 min in the assay medium in the absence and presence of 23.0 mM KH,PO,, and then succinate was added. Two minutes later, 2,4dinitrophenol (DNP) or flufenamic acid dissolved in ethyl alcohol was added to a concentration of 15 PM. The effect of the solvent was tested by the addition of ethyl alcohol alone. DNP stimulated the respiration to 260% of control without Pi and to 220% with 23.0 mM P,, and flufenamic acid to 220% without Pi and to 170% with 23.0 mM Pi, respectively. Furthermore, varying the concentrations of Pi from 0.5 to 40 mM did not affect the stimulation effects of these agents. Eflects of Phosphonate Compounds on the Action of Ifenprodil

Figure 4 shows the influences of various phosphonate compounds on mitochondrial respiration in the absence and presence of ifenprodil. Mitochondrial respiration without

IFENPRODIL

ON MITOCHONDRIAL

RESPIRATION

213

ifenprodil was stimulated by Pi and phenylphosphonate but reduced by pyrosphonate and p-nitrophenylphosphate. In the presence of ifenprodil, the respiration was accelerated by P, but inhibited by the other phosphonate compounds.

CONTROL KH2P04 C6H6.

Na4P207 C6H4(N02)

OPO(OH+

OPO(OH)2

FIG. 4. Effects of phosphonate compounds on respiratory activity with or without ifenprodil. Reactions were carried out as described in the legend to Fig. 2, except for the use of phosphonate compounds instead of anions. Respiratory activity was measured with various phosphonate compounds (25.0 mM) in the absence (0) and presence (m) of 0.8 mM ifenprodil. Succinate used as substrate was present at a concentration of 12.5 mM. Further explanations as in Fig. 2.

KHzPO,,

-

-

++++++

HERSALYLtuM) IFENPROOIL

-

t

- 100 --

IIII

+

60 t

100 160 f-+

FIG. 5. Effects of Pi and mersalyl on respiratory activity with or without ifenprodil. Mitochondria were preincubated for 1 min in an assay medium in the absence or presence of 21.5 mM Pi, and then succinate was added. Then 2 min later, mersalyl was added at the indicated concentration. Succiqate and ifenprodil were present at concentrations of 12.5 and 0.8 mM respectively. Each column and vertical bar represent the mean value + SE of five experiments.

Eflects of Mersalyl on the Action of Ifenprodil The effect of mersalyl on ifenprodilstimulated respiration with Pi was tested. As demonstrated in Fig. 5, mersalyl not only significantly reduced the significant stimulation of respiration induced by Pi but also elicited a concentration-dependent inhibition in stimulatory effects of ifenprodil obtained in the presence of Pi. Effects of Oligomycin on the Action of Ifenprodil The effects of oligomycin on the stimulatory actions of ifenprodil and ADP were examined in the presence of succinate and Pi. Mitochondria were preincubated for 1 min in the assay medium, and then succinate was added. Two minutes later, oligomycin (70 pg) dissolved in ethyl alcohol was added. After a I-min incubation, ifenprodil or ADP was added. The effect without oligomycin was tested with the addition of ethyl alcohol alone. Ifenprodil, succinate, and ADP were present at the concentrations of 0.8, 12.5, and 0.42 mM, respectively. Oligomycin did not influence the stimulatory action of ifenprodil but eliminated that of ADP. Effects of Ifenprodil on Inhibitory Action of KCN KCN caused a concentration-dependent inhibition of mitochondrial respiration at the range of 10 to 100~~. As shown in Fig. 6, ifenprodil (0.1-I .O mM) exhibited a concentration-dependent stimulatory effect even in the presence of 20 PM KCN which depressed 25 % of the respiratory activity. Thus, the cyanide-induced reduction in respiration was almost eliminated by 0.5 mM of ifenprodil, and was restored to a higher level of

214

YAMASHITA

ET Al..

piration.

stimulation in respiration was

P,

markedly potentiated by the addition of ifenprodil (0.8 mM). Thus, the cyanideinduced reduction in respiration was almost

01 0’

, 0.5

1.0

IFENPRODIL(mM)

FIG. 6. Effects of various concentrations of ifenprodil on respiratory activity in the presence of KCN. Succinate was added after mitochondria were preincubated for 1 min in the assay medium. Two minutes later then, KCN (20p(M) wasadded.After incubation for 1 min, ifenprodil was added. (m) Respirationwithout KCN and ifenprodil;(0) effect of ifenprodil in the presence of KCN. Each point

and vertical bar represent the mean value+ SE of 10 experiments. An asterisk indicates a significant difference from respiratory activity without ifenprodil in the presence of KCN.

respiration at a concentration of 0.08 mM

than those obtained before KCN. None of this action was observed by addition. of L-tartaric acid instead of ifenprodil. The influences of various substrates in place of succinate on the antagonistic actions of ifenprodil against the inhibitory effect of cyanide were examined. KCN (20 p(M) caused a 22.7, 5.6, and 13% inhibition, when succinate, malate, and a-ketoglutarate were used as substrate, respectively. As shown in Fig. 7, ifenprodil restored the cyanide-induced inhibition of respiration with succinate but not with malate and cr-ketoglutarate as sub-

strate. As shown in Fig. 8, ifenprodil induced no effect against cyanide-inhibited respiration

when P, was absent. Pi brought about a stimulation of respiration inhibited by cyanide even in the absence of ifenprodil, Pi eliminating the inhibition in respiration produced by cyanide (20 ,UM) at 10 mM but further increase in Pi concentration from 10 to 20 mM eliciting no additional stimulation in the res-

SUCCINATE

u MALATE

&KETOGLUTARATE

FIG. 7. Effects of substrates on restorative action of ifenprodil against KCN-inhibited respiration. Reactions were carried out as described in the legend to Fig. 6 in the absence (0) and presence (m) of 0.8 mM ifenprodil. The concentration of substrate was 12.5 mM, and that of KCN 20 PM. Each column and vertical bar represent the mean value+ SE of five experiments. An asterisk indicates a significant increase with ifenprodil.

OL,

0

4 10

20 KHzP04 (mM)

30

40

FIG. 8. Effects of various Pi concentrations on cyanide action in the absence and presence of ifenprodil. Mitochondria were incubated in the presence of various concentrations of KHIPO, as indicated. After a I-min incubation, succinate was added at a concentration of 12.5 mM. Two minutes later, KCN and ifenprodil were added successively at intervals of 1 min. (A) Absence of both KCN and ifenprodil; (0) 20 PM KCN and 0 mM ifenprodil; (m) 20 pM KCN and 0.8 mM ifenprodil. Further explansltions as in Fig. 3.

IFENPRODIL

ON MITOCHONDRIAL

eliminated in the presence of ifenprodil at 10 mM of Pi, followed by even higher levels of respiration at over 20 mM P, than that obtained without cyanide. Eflects of Ifenprodil Activity

on Cytochrome

Oxidase

The effect of ifenprodil on mouse brain cytochrome oxidase activity inhibited by cyanide was determined in both in vivo and in vitro experiments. The assay for cytochrome oxidase activity in in vivo experiments was carried out as described under Methods. In vitro experiments on the enzyme assay were carried out as follows: the mouse brain mitochondria were preincubated for 1 min in an assay medium. Sequentially succinate, antimycin A, and the reducing solution were added successively at intervals of 30 set, followed by the addition of KCN. The enzyme activity was measured in the absence and presence of 12.5 mM succinate. KCN and ifenprodil were present at concentrations on 20 PM and 0.8 mM, respectively. Other reagents were at the same concentrations as those in in vivo experiment. Neither the oxidase activities without KCN, nor those reduced by in vivo treatment with KCN (3 mg/kg) were affected by ifenprodil (6 mg/kg) administered in vivo. In in vitro experiments on the oxidase activities, ifenprodil (0.8 mM) did not influence either the activities without KCN or those reduced previously by KCN (20 ,UM) in both the absence and presence of succinate (12.5 mM) as substrate. DISCUSSION Ifenprodil displays a stimulative effect on guinea pig brain mitochondrial respiration. This stimulative effect was obtained with succinate as substrate but not with other substrates, whereas, in liver mitochondria (Furukawa et al., 1976), the stimulative effect was observed not only with succinate but also with a-ketoglutarate, DL-isocitrate, and Lglutamate as substrate, though the actions

RESPIRATTON

215

were weak. P,, however, is similarly important in the stimulative action of ifenprodil in respiration of both brain and liver mitochondria. ’ As reported previously (Lehninger, 1964), oligomycin inhibits the ‘stimulatory effect of ADP which elicites an appearance of a state 3 respiration (Chance and Williams, 1955). However, oligomycin did not affect the stimulative effect of ifenprodil on mitochondrial respiration. The insensitivity of oligomycin with respect to the stimulative effect of ifenprodil had been similarly observed in effects of DNP and flufenamic acid (Cross and Wang, 1970; Saeki et al., 1972), typical uncouplers of respiration. However, the presence of Pi in the medium is not necessary for the stimulatory effect of DNP and flufenamic acid on respiration, as reported previously by Sugano et al. (1972). Thus, the DNP- and flufenamic acid-uncoupled respiration is not affected by P,, whereas Pi is essential for the action of ifenprodil. The stimulatory effect of DNP appeared with L-glutamate (Sugano et al., 1972) and p-hydroxybutyrate (unpublished observations), as well as with succinate as substrates, while that of ifenprodil appeared only with succinate. Accordingly, ifenprodil is different from ADP, DNP, and flufenamic acid in the mode of stimulatory action on mitochondrial respiration. The protective effect of ifenprodil against cyanide toxicity observed in vivo (Furukawa et al., 1976) was shown to be also observed in brain mitochondrial preparations. Absorbed cyanide is distributed in the brain (Ballantyne et al., 1972) and produces a rapidly toxic effect involving inhibition of cytochrome oxidase activity (Albaum et al., 1946). Brain cytochrome oxidase activity was not affected by either in vivo treatment with ifenprodil at a dose sufficient to protect against cyanide intoxication (Furukawa et al., 1976) or by its in vitro treatment. If the drug were to stimulate the cytochrome oxidase activity, the stimulatory effect on mitochondrial respiration should be elicited with all substrates, since

216

YAMASHITA ET At.

the electrons released from each substrate are transferred through a common transport system (Lehninger, 1964). However, ifenprodil stimulates mitochondrial respiration only with succinate as electron donor. On the other hand, it is well documented that methemoglobin completely abolishes the inhibitory effect of cyanide on cytochrome oxidase activity by combining with cyanide (Schubert and Brill, 1968). Our previous experiments using ultraviolet absorption and proton nuclear magnelic resonance spectra did not show that ifenprodil reacts directly with free cyanide ion to trap it (Furukawa et al., 1976). Therefore, it is presumed that ifenprodil does not react directly with the cytochrome oxidase and does not chemically react with cyanide, either. DNP stimulated oxygen consumption to an even larger extent than did ifenprodii, but did not protect against cyanide toxicity (unpublished observations), whereas ifenprodil stimulated oxygen consumption and apparently protected against toxicity of KCN. This seems to imply that oxygen consumption is not necessarily a part of mechanism for protection against KCN toxicity, unless the increase in oxygen consumption is correlated with ATP production. However, ATP production has not yet been measured with ifenprodil. Pi, which has been proposed to function as a Pi, dicarboxylate and tricarboxylate carrier at mitochondrial membranes (Johnson and Chappell, 1973, 1974), enhanced the stimulatory activity of ifenprodil, whereas other phosphonate compounds which do not function as Pi carriers (Johnson and Chappell, 1974) did not. On the contrary, Pi did not affect the stimulation effect of DNP and flufenamic acid in mitochondrial respiration. The stimulative effect of Pi on mitochondrial respiration was markedly potentiated by ifenprodil. The respiratory stimulation by ifenprodil was accelerated with increasing concentrations of Pi. Pi was reported to restore the pyruvate efflux inhibited by cyanide in adrenal cortex mitochondria (Sauer and

Park, 1973). In the present studies on brain mitochondrial preparations, Pi itself protected against the inhibitory effect of cyanide on respiration when the concentration of Pi was increased. Ifenprodil induced no effects against cyanide-inhibited respiration when Pi was absent. In addition, the ifenprodil stimulation of respiration was inhibited by mersalyl, an inhibitor of Pi efflux through the mitochondrial membrane (Tyler, 1969), while DNP stimulation was reported not to be inhibited by mersalyl (Tyler, 1969). The stimulatory effect of ifenprodil could not be observed with frozen and thawed mitochondria, probably because the mitochondrial membrane was broken. Accordingly, although Pi efflux has not yet been determined after treatment with ifenprodil, ifenprodil may well act on the mitochondrial membrane to increase Pi efflux, and increased Pi in the mitochondria may also function as part of the antagonistic effect of ifenprodil against cyanide toxicity. From these results, it is likely that ifenprodil acts directly on the mitochondrial membrane, accelerates Pi and succinate transport through the membrane, and stimulates succinate oxidation, and that these stimulative effects of ifenprodil are involved in its protective effect against cyanide toxicity.

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CROSS,R. L., AND WANG, J. H. (1970). Evidence of a phosphorylated intermediate in mitochondrial oxidative phosphorylation. Eiochem. Biophys. Res. Commun. 38, 848-854. FURUKAWA, T., MAEDA, Y., YAMASHITA, Y., UEDA, H., MIZUSAWA, H., AND SAKAKIBARA, E. (1976). lfenprodil : Protective effect in experimental cyanide poisonmg. Toxicol. Appt. Pharmacol. 37, 289300. FURUMI, K., MORI, K., MAEDA, K., AND YAMADA, R. (1975). Effect of ifenprodil on experimental cerebrovascular disease. Geria Med. 13, 13541358. [In Japanese]. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1948). Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751-766. HAGIHARA, B. (1961). Techniques for the application of polarography to mitochondrial respiration. Biochem. Biophys Acta 46, 134-142. JOHNSON, R. N., AND CHAPPELL, J. B. (1973). The transport of inorganic phosphate by the mitochondrial dicarboxylate carrier. Biochem. J. 134, 769-774. JOHNSON,R. N., AND CHAPPELL, J. B. (1974). The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphate compounds. Biochem. 1. 138, 171-175. LEHNINGER,A. L. (1964). In The Mitochondrion. Moiecular basis of structure and function, pp. 121-123. Benjamin, New York. MAEDA, Y., AND FURUKAWA, T. (1975). Pharmacolo-

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gical studies on ifenprodil. Folia Pharmacol. Japon. 71, 585-595. [In Japanese]. MIZUSAWA, H., AND FUJIWARA, H. (1974). Effect of ifenprodil on isolated arteries, Folia Pharmacol. Ja@m. 70, 785-799. [In Japanese]. OTOMO, E., AND KODAMA, R. (1976). Double blind study of FX-505 (ifenprodil) on cerebrovascular diseases. Phase III study. Clin. Evalu. 4, 419-458. [In Japanese]. OZAWA, K., SETA, K., TAKEDA, H., AMDO, K., HANDA, H., AND ARAKI, C. (1966). On the isolation of mitochondria with high respiratory control from rat brain. J. Biochem. Tokyo 59, 501-510. SAEKI, K., MURAOKA, S., AND YAMASAKI, H. (1972). Anti-inflammatory properties of N-phenylanthranilic acid derivatives in relation to uncoupling of oxidative phosphorylation. Japan J. Pharmacof. 22, 187-199. SAUER, L. A., AND PARK, R. (1973). A stimulation by phosphate of malate transport and oxidation in rat adrenal mitochondria. Biochemistry 12, 643-649. SCHUBERT, J., AND BRILL, W. A. (1968). Antagonism of experimental cyanide toxicity in relation to the in vivo activity of cytochrome oxidase. J. Pharmacol. Exp.

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Effect of ifenprodil on mitochondrial respiration of guinea pig brain.

TOXICOLOGY AND APPLIED PHARMACOLOGY 49,209-217 (1979) Effect of Ifenprodil on Mitochondrial Respiration of Guinea Pig Brain YOSHIAKI YAMASHITA,’...
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