Trifluoperazine inhibition of electron transport and adenosine triphosphatase in plant mitochondria

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 229, No. 1, February 15, pp. 287-294, 1984

Trifluoperazine inhibition of Electron Transport and Adenosine Triphosphatase in Plant Mitochondria PAUL P. J. DUNN,*

ANTON1 R. SLABAS,t IAN R. COTTINGHAM,* ANTHONY L. MOORE*,’

AND

*Department of Biochemistry, School of Biological Sciences, University of Sussex, Falmer, Brighton, and TApplied Biosciences, Unilever Research, Bedford MK4.4 ILQ, United Kingdom Received August 4, 1983, and in revised form October 23, 1983

Trifluoperazine inhibits ADP-stimulated respiration in mung bean (Phaseohs aweus) mitochondria when either NADH, malate, or succinate serve as substrates (I& values of 56, 59, and 55 PM, respectively). Succinate:ferricyanide oxidoreductase activity of these mitochondria was inhibited to a similar extent. The oxidation of ascorbate/TMPD was also sensitive to the phenothiazine (I& = 65 PM). Oxidation of exogenous NADH was inhibited by trifluoperazine even in the presence of excess EGTA [ethylene glycol bis(P-aminoethyl ether)-N,N’-tetraacetic acid] (I&,, = 60 PM), indicating an interaction with the electron transport chain rather than with the dehydrogenase itself. In contrast, substrate oxidation in Voodoo lily (Sauromatum guttatum) mitochondria was relatively insensitive to the phenothiazine. The results suggest the bci complex to be a major site of inhibition. The membrane potential of energized mung bean mitochondria was depressed by micromolar concentrations of trifluoperazine, suggesting an effect on the proton-pumping capability of these mitochondria. Membrane-bound and soluble ATPases were equally sensitive to trifluoperazine (IC& of 28 PM for both), implying the site of inhibition to be on the Fi. Inhibition of the soluble ATPase was not affected by EGTA, CaCl,, or exogenous calmodulin. Trifluoperazine inhibition of electron transport and phosphorylation in plant mitochondria appears to be due to an interaction with a protein of the organelle that is not calmodulin. Trifluoperazine is one of a series of antipsychotic phenothiazine drugs that have been extensively used to demonstrate CaM’-mediated events. Levin and Weiss (1) showed that phenothiazines are competitive antagonists of CaM-activated, Ca’+dependent CAMP phosphodiesterase of brain. CaM is a ubiquitous, low-molecularweight (iVfr N 17,000) Ca2+-binding protein

(for a review see (2)) that has been isolated from a range of metazoa and protozoa (3) and fungi (4). CaM has been shown to have a central role in many metabolic events. CaM has been isolated from some plants (5,6) and has been implicated in a number of enzyme reactions including NAD kinase (7,8), microsomal ATPase (9), the external NADH dehydrogenase (10) and Ca”+ATPase-mediated Ca2+ uptake into apple fruit mitochondria (11). The interaction between CaM and phenothiazine is so great that immobilized phenothiazines have been used to purify CaM from various sources (5,12). However, it has been reported that phenothiazines also have membrane anesthetic effects (13) and, therefore, careful interpretation is

r To whom correspondence should be addressed. ‘Abbreviations used: CaM, calmodulin; FCCP, ptrifluoromethoxycarbonylcyanidephenylhydrazone; TMPD, N,N,N’,N’-tetramethylphenylenediamine; Mops, 4-morpholinepropanesulphonic acid; PEP, phosphoenolpyruvate; EGTA, ethylene glycol his@aminoethyl ether)-N,N’-tetraaeetic acid. 287

0003-9861/84 $3.00 Copyright All rights

G 1984 by Academic Press. Inc of reproduction in any form reserved

288

DUNN

required if these drugs are to be used as probes for CaM-mediated events. Observations that local anesthetics and CaM antagonists inhibit mitochondrial electron transport (14-16), the F1 ATPase (17,18), and the report of CaM association with animal mitochondria (19) prompted us to investigate the effects of trifluoperazine on plant mitochondria. There is controversy as to whether or not CaM is associated with mitochondria (20). In this paper we report the effects of trifluoperazine on mitochondria isolated from mung bean hypocotyls and the spadices of Sauromatum guttatum. Trifluoperazine was found to inhibit succinate:ferricyanide oxidoreductase and the oxidation of succinate, malate, and exogenous NADH in mung bean mitochondria but not the oxidation of these substrates in 5’. guttatum mitochondria, suggesting inhibition to be at the level of the be1 complex. Furthermore, it was found that micromolar concentrations of the phenothiazine inhibited ATPase activity in plant mitochondria. MATERIALS

AND

METHODS

Preparation of mitochvndria. Mitochondria were isolated from etiolated hypocotyls of mung bean (Phaseolus aureus), tubers of Jerusalem artichoke (Helianthus tu&-osus), and from the spadices of Arum maculntum and S. guttatum as described previously (21). Mitochwbial respiratory assays. Mitochondrial respiratory activity was assessed using a Rank O2 electrode (Rank Bras., Cambridge, U. K.) thermostatted at 25°C. Recordings of the electrode response were made with a Servoscribe chart recorder. The reaction medium (1 ml) contained 0.3 M mannitol, 5 mM MgC&, 10 mM KCI, 10 mM KzHPO,, 10 mM Mops (pH 7.2) (medium A). Succinute:~~rricyonide oxidnreductase was assayed by following the decrease in absorbance at 420 nm with a Cary 210 spectrophotometer (21). The assay medium (2.5 ml) contained medium A, mitochondria (0.6 mg), 0.8 mM KCN, 11 mM succinate, 0.6 mM ATP, and 1 m&l potassium ferricyanide. Meusurement of merniw-a?le potential. Membrane potentials of plant mitochondria were determined using a tetraphenylphosphonium (TPP+)-specific electrode which was prepared as described in (22). Preparaticm of oli~omycin-seriaitiuE ATPase. Submitochondrial particles were prepared from mitochondria of Jerusalem artichoke and A. maculatum using a French pressure cell as previously described

ET AL. (21). The particles were washed once in 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-SO1 (pH 7.7) and pelleted by centrifugation at 110,OOOgfor 45 min. Particles prepared in this manner were either used immediately or stored at -80°C. The ATPase activity of such preparations was inhibited 90% by 1 pg oligomycin. Preparatimz of FlATPose. The oligomycin-insensitive ATPases of Jerusalem artichoke and A. madatum were prepared by a chloroform release method modified from Beechey et al (23). Submitochondrial particles were diluted to 10 mg protein/ml with 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-SOI (pH 7.7) at room temperature. The diluted particles were then added to glass tubes and, with continuous vortex mixing (20 s), 200 mM chloroform (Jerusalem artichoke particles) or 150 mM chloroform (A. maculatum particles) was added. The chloroform concentration chosen was determined by titration of oligomycin-insensitive ATPase from submitochondrial particles as described by Fisher et al. (24). Centrifugation of the chloroform-treated particles at 110,OOOg for 60 min (room temperature) yielded the released FiATPase in the supernatant. The enzyme was stored as a 60% ammonium sulfate precipitate at 4°C. Before use the ammonium sulfate suspension was centrifuged (Eppendorf bench centrifuge) and the pellet was dissolved in 0.25 M sucrose, 1 mM EDTA, 10 mM TrisSO, (pH 7.7). Such preparations were oligomycin insensitive. ATPase activity. ATPase was assayed by coupling the production of ADP to the oxidation of NADH via the pyruvate kinase and lactic dehydrogenase reactions. The decrease in absorbance at 340 nm (room temperature) was measured using a Cary 210 spectrophotometer (25). The assay medium (1 ml) contained 0.3 M mannitol, 65 mM Mops (pH 8.0), 500 PM PEP, 500 PM ATP, 600 PM MgCIz, 250 FM NADH, 8 units pyruvate kinase,3 and 13 units lactic dehydrogenase.’ The reaction was started by the addition of ATPase; sufficient ATPase was added to ensure it was rate limiting. A unit of ATPase activity is defined as the amount resulting in the oxidation of 1 pmol NADH/min; specific activity being units per milligram protein. Each determination was performed at least in duplicate. Phenothiazine did not affect the ATPregenerating system since ATPase assayed by the calorimetric determination of Pi (as described by Le Bel (26)) yielded identical results. In this assay, a unit of ATPase is defined as the amount resulting in the hydrolysis of 1 Frnol ATP/min; specific activity being units per milligram protein.

’ One unit of pyruvate kinase will convert 1.0 rmol phosphoenolpyruvate to pyruvate per minute. 4 One unit of lactic dehydrogenase will reduce 1.0 pm01 of pyruvate to lactate per minute.

INHIBITION

OF OXIDATIVE

PHOSPHORYLATION

Protein determination and reagents. Protein was determined by the method of Peterson (26) using bovine serum albumin, fraction V, as standard. ATP and phosphoenolpyruvate were purchased from Boehringer, Mannheim, F. R. G. Bovine brain CaM, pyruvate kinase, and lactic dehydrogenase were obtained from Sigma Chemical Company, Poole, Dorset, U. K. All other biochemicals were purchased from either Sigma or BDH, Poole, Dorset, U. K.

RESULTS

Inhibition of Mitochmdrial by Tri,fuoperaxine

Respiration

Figure 1A illustrates the effect of micromolar concentrations of trifluoperazine on ADP-stimulated respiration of mung bean mitochondria oxidizing either malate, succinate, or exogenous NADH. As indicated in Fig. lA, trifluoperazine inhibited state 3 respiration with either substrate; 50% inhibition achieved at phenothiazine concentrations (herewith termed I(&,, values) of 59,55, and 56 pM for NADH, malate, and succinate, respectively. These IC& values are similar to those reported for inhibition of ADP-stimulated succinate oxidation in rat liver (66 PM (15)). Considerably higher concentrations of trifluoperazine were required for inhibition A

130_

BY TRIFLUOPERAZINE

of ADP-stimulated succinate oxidation in porcine liver mitochondria (>200 PM (16)). The reason for the discrepancy in the I(& values is not apparent. The I&, values for trifluoperazine inhibition of plant mitochondria are considerably higher than the 10 @M reported for inhibition of CaM-sensitive, Ca2+-dependent CAMP phosphodiesterase of brain (1); therefore, the inhibition observed does not appear to be due to an effect on a CaM-sensitive reaction. In contrast to the data of Ruben and Rassmussen (15), but in agreement with that of Cheah and Waring (16), inhibition of oxidation supported by any of the substrates could not be relieved by 1 PM FCCP, suggesting that trilfluoperazine could be affecting electron transport and perhaps phosphorylation. Succinate: ferricyanide oxidoreductase activity of mung bean mitochondria was also inhibited by trifluoperazine (Fig. 2), the level of inhibition being identical to that observed in Fig. 1A (i.e., IC,, = 55 PM). In addition, ascorbate/ TMPD oxidation was inhibited by the drug with an I& value of 65 PM (data not shown). It would therefore seem that trifluoperazine is quite a potent inhibitor of electron transport in plant mitochondria and, since malate, NADH, and succinate B

130_

i

2 .-2 E

0

2

z

q

65

;;:,.

50

289

100 0 TRIFLUOPERAZINE

50

100

PM

FIG. 1. Effects of triiluoperazine on ADP-stimulated respiration. Mung bean mitochondria (0.4 mg) were incubated in the presence (B) or absence (A) of 4 mM EGTA in 1 ml of medium A. Oxygen consumption was monitored as described under Materials and Methods. Malate (20 mM) (0), NADH (0.89 mM) (A), or succinate (10 mM) (0) were added followed by ADP (0.95 mM) for malate or 0.48 mM for succinate or NADH). Trifluoperazine was added to achieve the final concentrations shown.

290

DUNN

35 TRIFLUOPERAZINE

70 p’M

FIG. 2. Inhibition of succinate:ferricyanide oxidoreductase activity in mung bean mitochondria by trifluoperazine. The assay was performed as described under Materials and Methods in 2.5 ml reaction medium (plus 0.6 mg protein). Potassium ferricyanide (1 mM) initiated the reaction and trifluoperazine was added as indicated.

gave similar I&, values, it appears that one site of inhibition is located in the bci complex and another possible site is cytochrome oxidase as suggested recently (16). It is well documented that divalent cations (particularly Ca2+) enhance the oxidation of exogenous NADH by plant mitochondria (28, 29). In view of the finding that the dehydrogenase is stimulated by micromolar free [Ca2+] (30), it has been suggested that this cation may be a modulator of the external NADH dehydrogenase in vivo (10). Whether this modulation is mediated by CaM is at present uncertain. In an attempt to resolve this question, the effect of trifluoperazine on exogenous NADH oxidation was investigated (Fig. 1B). The addition of 4 mM EGTA to chelate endogenous Ca2+ considerably reduces NADH oxidation rates (=SO%), whereas it has little effect on the coupled oxidation rates in the presence of succinate or malate. Even under these conditions, however, it is apparent from Fig. 1B that trifluoperazine still inhibits NADH oxidation since the I& of 60 PM is similar to that observed in Fig. lA, suggesting that the

ET AL.

effect of this drug is on electron transport rather than on the dehydrogenase itself. In order to more clearly define its site of action, the effect of this drug on S guttat-urn mitochondria was investigated. S. guttatum mitochondria possess a cyanideand antimycin A-insensitive alternative oxidase, the branchpoint of which is considered to be at the level of ubiquinone. In the presence of antimycin A, 75 ELM trifluoperazine only elicited inhibitions of 10, 14, and-25% for the oxidation of succinate, malate, and NADH, respectively (Table I). Under these conditions, reducing equivalents from the dehydrogenases pass via the nonphosphorylating alternative pathway to molecular oxygen, bypassing the bci complex and cytochrome oxidase. In conjunction with the data expressed in Figs. 1 and 2, the results show that the site or sites of action of this drug in intact plant mitochondria lie on the phosphorylating cytochrome pathway, possibly in the region of the bci complex. It is also conceivable that the inhibition of ADP- and uncoupler-stimulated respiration may also be due to membrane anesthesia as opposed to an interaction with a specific site in the electron transport chain. The membrane potential measurements illustrated in Table II reveal, however, that TABLE

I

EFFECTSOFTRIFLUOPERAZINE ON SUBSTRATE OXIDATIONBY S guttatum MITOCHONDRIA

Substrate Malate Succinate

Trifluoperazine + +

NADH

f

Rate of 02 uptake (nmol/min/mg)

Percentage inhibition

325 278

0 14

440 394

10

834 626

0 25

0

Note. Effects of trifluoperazine on substrate oxidation by S. guttatum mitochondria.ADP (0.48mM) and antimycin A (100nM)were addedto mitochondria (0.12mg) and medium A (1 ml) in the oxygenelectrode.Other conditionsasdescribed under Materials and Methodsand in Fig. 1. Trifluoperazine (75PM) was addedOncea linear rate of substrate oxidation had been achieved.

INHIBITION

OF OXIDATIVE

TABLE

PHOSPHORYLATION

II

DEPRESSION OF THE MITOCHONDRIAL MEMBRANE POTENTIAL BY TRIFLUOPERAZINE

Substrate

Trifluoperazine (PM)

(2)

NADH

0 25 50 75 100

183 180 168 148 128

Ascorbate/TMPD

0 25 50 75 100

183 179 164 146 127

Note. Depression of the mitochondrial membrane potential by trifluoperazine. A* was measured as described under Materials and Methods. The incubation contained 2 ml medium A, 1 PM TPP+, mung bean mitochondria (1.25 mg), and either 1 mM NADH or 5 mM ascorbate plus 0.25 mM TMPD. Membrane potentials were calculated according to the Nernst equation, A$ = -60 log [TPP+ in]/[TPP+ out] on the basis of a mitochondrial matrix volume of 1 rl/mg protein.

increasing concentrations of trifluoperazine depress the membrane potential, which is more consistent with decreasing the rate of respiratory-linked proton pumping rather than an effect on membrane fluidity. Such results are more consistent with the idea that the effect of the drug is due to a direct interaction with a protein rather than a general perturbation of the lipid bilayer. Trifluoperazine, up to 100 pM, does not affect swelling of intact mung bean mitochondria in potassium acetate. However, in the presence of 300 pM trifluoperazine these mitochondria swell spontaneously in potassium acetate. Valinomycin and uncoupler had no effect on this spontaneous swelling, suggesting that the drug had made the mitochondrial membrane freely permeable to K+ and H+. It would therefore appear that trifluoperazine only affects mitochondrial membrane fluidity at concentrations in excess of those used in this study (unpublished observations).

291

BY TRIFLUOPERAZINE

Interaction of Tri$uoperaxine the ATPase

with

Since trifluoperazine inhibits both ADPand uncoupler-stimulated respiration in intact mitochondria, it is difficult to assess whether it also affects phosphorylation. Consequently, both membrane-bound and soluble ATPase were prepared by subjecting mitochondria to French pressing followed by solubilization using chloroform. As seen in Fig. 3 both the membrane-bound and soluble ATPases of Jerusalem artichoke mitochondria are equally sensitive to trifluoperazine; 28 PM phenothiazine required for 50% inhibition of both enzymes. This is somewhat higher than the K, of ‘7 PM reported for trifluoperazine inhibition of Lubrol-treated mitoplasts of rat liver (15). The ATPase of A. maculatum was also solubilized by chloroform release. In terms of specific activity, oligomycin insensitivity, and cation requirement, this enzyme was identical to that isolated from Jerusalem artichoke. Figure 4 shows the effects of trifluoperazine on this enzyme in the presence of 1 mM Ca2+ or 1 mM EGTA.

00 0

50 TRIFLUOPERAZINE

100 pbl

FIG. 3. Comparison of the effects of trifluoperazine on the membrane-bound and soluble ATPases of Jerusalem artichoke mitochondria. The enzymes were prepared as described under Materials and Methods and assayed by the coupled assay system. Soluble ATPase (2.1 pg) (0); membrane-bound ATPase (50 f.4 (0).

292

DUNN

ET AL.

ml, was added to the soluble ATPase there was no effect on ATPase activity, or ATPase activity in the presence of 1 InM EGTA, in the presence of 100 PM Ca2+, or in the presence of 30 PM trifluoperazine (Table III). The results suggest that either CaM is already present in the enzyme preparation or it is not a modulator of the plant mitochondrial F1 ATPase. The same seems to apply to rat liver mitoplasts (15).

TABLE EFFECT OF CALMODULIN OF

A.

maculatum

Control 50

25 TRIFLUOPERAZINE

IN

FIG. 4. Trifluoperazine inhibition of the soluble ATPase of A. maculatum mitochondria. The ATPase was isolated as described under Materials and Methods and assayed by the calorimetric determination of Pi. Control ATPase (2.8 pg) (0); 1 mM CaCl* (A) or 1 mM EGTA (0) preceded the addition of ATPase (2.8 pg) and phenothiazine.

Under the three conditions shown in Fig. 4 the concentrations of phenothiazine required for 50% inhibition of the ATPase were 29 FM (control), 33 PM (plus EGTA), and 28 pM (plus Ca’+). Phenothiazines inhibit CaM-mediated reactions by binding to the Ca2+-CaM complex. In the absence of calcium, phenothiazines are unable to bind to CaM and hence cannot cause inhibition of the CaM-mediated reaction. Since the potent inhibition of F1 ATPase by trifluoperazine illustrated in Fig. 4 was not Ca2+ dependent, it does not appear to be due to a trifluoperazine-CaM interaction. In preparation of the membrane-bound and soluble ATPases it is conceivable that any CaM associated with the inside of the mitochondria may have been lost. Table III shows that when CaM, up to 0.75 pg/

ON THE SOLUBLE MITOCHONDRIA

ATPase

0 0.10 0.50 0.75 ATPase plus 1

1.21 1.21 1.15 1.19 EGTA

mM

1.07 1.07 1.07 1.09

0 0.10 0.50 0.75 ATPase plus 100

pM

CaClz

0 0.10 0.50 0.75 ATPase plus 30 0 0.10 0.50 0.75

ATPase

ATPase activity (wmol ATP hydrolyzed/min/mg)

Calmodulin (be)

01 0

III

1.31 1.31 1.30 1.31 pM

trifluoperazine 0.59 0.54 0.58 0.59

Note. Effect of calmodulin on the soluble ATPase of A. maculatum mitochondria. ATPase activity was assayed by the calorimetric determination of Pi. Bovine brain calmodulin was made up in 300 mM mannitol, 1 mM EGTA, 65 mM Mops (pH 7.4) and added to the ATPase assay to give the final concentrations shown. EGTA (1 mM), CaCl* (0.1 mM), and trifluoperazine (75 pM) were added prior to the addition of calmodulin and ATPase (2.8 pg) as indicated.

INHIBITION

OF OXIDATIVE

PHOSPHORYLATION

DISCUSSION

Inhibition by phenothiazine is generally considered to be indicative of CaM involvement in the regulation of a cellular function. However, it is becoming abundantly clear that these drugs are not as specific as once thought (13, 31). The results presented show that trifluoperazine is a potent inhibitor of electron transport and phosphorylation in plant mitochondria. In contrast to the inhibition of rat liver mitochondria (15), this drug inhibited both ADP- and uncoupler-stimulated respiration in mung bean mitochondria when either succinate, malate, or exogenous NADH were used as substrates. The finding that respiration supported by any of these substrates was inhibited to a similar extent suggests a common inhibitor-binding site, possibly located in the bei complex. This is substantiated by the observed inhibition of succinate:ferricyanide oxidoreductase (Fig. 2) and the relative insensitivity of substrate oxidation in S. guttatum mitochondria (Table I) to the phenothiazine. In these mitochondria substrate oxidation occurs via the alternative oxidase and thus bypasses the beI complex. Inhibition of ascorbate/TMPD oxidation in mung bean mitochondria suggests that cytochrome oxidase is a further site of inhibition. Whether Complex I is another site of inhibition, as suggested by others (14,31), is uncertain. Although malate oxidation by S. guttatum mitochondria was insensitive to the phenothiazine, it is important to remember that in such tissues NAD-linked substrates are oxidized in a rotenone insensitive fashion. Thus, insensitivity to trifluoperazine merely confirms that the bq complex is a prime site of inhibition but does not exclude a further site at Complex I. Other workers (16) have suggested that trifluoperazine acts at two sites in porcine skeletal muscle mitochondria, one at the bci complex and the other between cytochrome c and cytochrome oxidase. Recently, CaM has been implicated in the regulation of the external NADH de’ hydrogenase of plant mitochondria (10). This is based upon the observation that the dehydrogenase is stimulated by mi-

BY TRIFLUOPERAZINE

293

cromolar free Ca’+ concentrations (30). However, trifluoperazine inhibits the OXidation of exogenous NADH by mung bean mitochondria even in the presence of excess EGTA, whereas trifluoperazine will only inhibit CaM-mediated events in the presence of Ca’+. Furthermore, NADH oxidation via the alternative oxidase in S. guttatum mitochondria was relatively insensitive to the drug, suggesting that inhibition is mainly due to an interaction with the electron transport chain rather than the dehydrogenase. Since trifluoperazine inhibited ADPstimulated respiration it was also conceivable that it would inhibit the isolated ATPase. Membrane-bound and soluble ATPases were indeed found to be potently inhibited by micromolar concentrations of the phenothiazine. As both forms of the enzyme were inhibited to similar extents, the site of inhibition is probably on F1, although the exact site is at present uncertain. The inhibition observed does not appear to involve an interaction with CaM since it was not affected by an excess of EGTA, by changes in Ca2+ concentration, nor by exogenous CaM. Similar findings were reported for inhibition of the ATPase of rat liver mitoplasts (15) although trifluoperazine was a more potent inhibitor of this enzyme. It may therefore be concluded that trifluoperazine is a potent inhibitor of both electron transport and phosphorylation in plant mitochondria. The inhibition does not appear to be due to an interaction between phenothiazine and CaM or a membrane anesthetic effect. The discovery of CaM as a ubiquitous Ca2+ messenger necessitates that studies on the involvement of CaM in cellular function use precise probes of the protein’s effects. Evidently, considerable care needs to be exercised in interpreting the nature of reactions that are inhibited by phenothiazines and related compounds (cf (10, 11)). ACKNOWLEDGMENTS This work was supported by grants from the SERC and ARC (A. L. Moore), an SERC CASE award (P. P. J. Dunn.), and a grant from Unilever Research.

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