THN JOURNAL OF BIOLOGICAL CAEMIBTRY Vol. 249, No. 11, Iseue of June 10, pp. 3823-3337, 1974

P&&d in U.S.A.

Rhodamine 6G

A POTENT INHIBITOR OF MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION*

(Received for publication, November 12, 1973)

ADRIAN R. L. GEAR

From the Department of Biochemistry, The University of Virginia, Charlottesville, Virginia ,%Y?901

SUMMARY

The lipophilic dye, rhodamine 6G, has been shown to be a potent inhibitor of oxidative phosphorylation, with a Ki of about 3 w, corresponding to 1.2 nmoles of dye per mg of mitochondrial protein. ATP-supported Cazf accumulation was blocked, but not -that driven by succinate oxidation. Concentrations of rhodamine 6G above 20 PM slowly un- couple respiration and inhibit respiration-dependent Ca2+ uptake. Neither the dinitrophenol-stimulated ATPase nor uncoupled respiration of intact mitochondria were inhibited by the dye, even at 50 PM. Arsenate-stimulated respiration, on the other hand, was partially blocked by 7.5 PM dye, but energy-linked phosphate accumulation was unaffected. Be- low 10 PM rhodamine 6G, no mitochondrial swelling, loss of matrix protein, or endogenous I(+, Cazf, or Mg2+, was ob- served. The dye was bound very tightly to mitochondria, being present at 1.3 and 0.8 nmoles per mg of protein for the inner and outer membranes, respectively. Binding was also monitored by direct measurement of H+ release into the suspending medium in the presence of rhodamine 6G. Some 0.6 to 0.75 mole of Hf was ejected per mole of dye added, of which only about 50% was firmly bound. A red shift occurred on binding, the X,,, increasing from 527 to 535 nm. The related compound, rhodamine B, a free acid and

uncharged at pH 7, was completely without influence on mitochondrial energy-linked functions.

Kinetic studies on the rate of ADP-stimulated Hf uptake in the presence and absence of rhodamine 6G yielded non- linear, Lineweaver-Burk plots and were of noncompetitive type. The K,,, for ADP decreased from 56 to 29 PM in the presence of 4 PM rhodamine 6G. When the data is expressed as a Hill plot, straight lines are obtained, with the value of e, the interaction coe5cient, increasing from 1.28 to 1.72 after dye addition.

Rhodamine 6G did not inhibit the Mg2+ATPase in soni- cated submitochondrial particles, unlike aurovertin. It did, however, block adenine nucleotide binding to intact mito- chondria, both for [ICJATP and [14C]ADP. Inhibition by 10 PM dye for low levels of added nucleotide was identical with that caused by 10 PM atractyloside, but was only very slight at higher nucleotide concentrations (0.2 mM).

The results of this study support the conclusion that rhodamine 6G blocks the adenine nucleotide translocase apparently being similar to atractyloside and bongkrekic acid. However, in sharp contrast to these inhibitors, rhodamine 6G did not inhibit the 2,4-dinitrophenol- stimulated ATPase of intact mitochondria. Consequently, uncouplers destroy the dye’s ability to interfere with the translocase, and this suggests a lipid binding to be involved in the action of rhodamine 6G.

This paper describes the action of the lipophilic dye, rhodamine 6G on mitochondrial energy-linked functions. The compound is positively charged at pH 7, compared to its close relative,

* This research was supported by the United States Public Health Service National Institutes of Health Grant GM-01814, as well as a subgrant from Institutional Grant (Gu-2551) to the University of Virginia by the National Science Foundation.

Rhodamine B rhodamine B, which is not, due to the presence of a free carboxyl group. Some studies on rhodamine 6G by Huang el al. (1) pointed to specific binding with well characterized liposome preparations. In addition, Levshin and Baranova (2) have described the detailed titration behavior of rhodamine 6G. These observations helped prompt the present investigation of lipophilic dyes like rhodamine 6G on energy-linked functions of

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mitochondria, especially since phospholipids probably play a key role in energy transduction (3).

It was found that the dye strongly inhibits both oxidative phosphorylation and ATP-supported ion transport, while having little action on respiration driven cation uptake by mitochondria. The Ki for inhibition of oxidative phosphorylation was about 3 PM. Interestingly, 2,4-dinitrophenol-stimulated ATPase was not inhibited at all. Concentrations above 10 PM began to un- couple respiration and inhibit respiration-driven cation transport. Based upou these studies as well as ou ATPase activity and arsenate-stimulated respiration, it was concluded that rhodamine 6G probably acts on the adenine nucleotide translocase. Direct measurement of this revealed very strong inhibition of adenine nucleotide binding at low, but not at high nucleotide levels. Lack of inhibition of the Mg2fATPase in “inside-out” submito- chondrial particles also points to nucleotide translocase involve- ment. It was also evident that a uet positive charge is essential for inhibition, since rhodamine B, which is uncharged at pH 7, was completely without effect on oxidative phosphorylation.

EXPERIMEKTAL PROCEDURES

Chemicals

Rhodamine GG was kindly supplied and purified chromato- graphically by Dr. C. Huang of the I)epartment of Biochemistry, University of Virginia. Rhodamine B was obtained from the Aldrich Chemical Co. and also made up to 0.2 rnM in deionized water. Oligomycin and atractyloside were supplied by the Sigma Chemical Co., St. Louis, MO., and dissolved in ethanol at 2 mg per ml. Lubrol WX, a nonionic detergent, came from I.C.I. Organic.3 Inc., Provincetown, R.I. Gramicidin, digitonin, and m-chlorocarbonylcyanide phenylhydrazone were from Mann Re- search Chemicals. 46CaC12, [idC]ADP, and [idC]ATP were ob- tained from Amersham-Searle, Chicago, 111. All other reagents were of AR grade, or the highest purity commercially available.

Apparatus An oxygen electrode (Yellow Springs Instrument Co.) in a

water-jacketed, closed chamber (Gilson Electronics, Madison, Wis.), was used to monitor mitochondrial respiration and oxida- tive phosphorylation. Hydrogen ion movements, both for ion uptake and oxidative phosphorylation and ATPase studies, were followed with a microcombination electrode (Thomas, 4858-L15) coupled to a Beckman Expandomatic pH meter and a Honeywell 194, or Sargent SLR 10” recorder. Potassium movements were monitored with a Beckman cation No. 39047 electrode used in conjunction with an Orion No. 90-02 reference electrode, and dis- played with the above recording system.

Animals

Male albino Sprague-Dawley rats were obtained from Carworth Inc., New York, N.Y. or from Charles River Breeding Labora- tories, Wilmingt,on, Mass.; Wistar strain rats were obtained from Mann Research in Puerto Rico (now Purina-Ralston Laboratory Animals, Vincent Town, N.J.). The animals weighed from 150 t,o 300 g and were kept on normal rat chow until killed.

TABLE I Comparison of phosphate release and H+ ejection during

assay of 2,4-dinitrophenol-stimulated ATPase

Rat liver mitochondria, 2.5 mg per ml, were incubated in a medium containing 80 mM NaCl, 10 mM MgC12, 10 mM sodium succinate, and 5 mM Tris-Cl, pH 7.4. One minute after the addi- tion of ATP at 0.25 mM, 5 X 10-c M 2,4-dinitrophenol was used to initiate ATPase action. Recording of pH was continuous and every 10 s up to 1 min, l-ml aliquots were withdrawn for phos- phate analysis. After that, phosphate analyses were done every minute up to 5 min. Results are expressed as the mean ratio of H+ to PO4 over the two time periods and include standard devia- tions.

Initial 1 min Final 4 min

H+/PO, 1.003 =t 0.148 1.033 f 0.072

branes, the mitochondria were subjected to digitonin treatment (6). High amplitude, respiration-dependent swelling was moni- tored spectrophotometrically by following the extinction changes at 520 nm with a Gilford recording spectrophotometer (7). Res- piration and ATP-supported calcium uptake were determined by pH and isotope procedures previously described (8). Oxidative phosphorylation was studied both by pH and polarographic tech- niques (5).

ATPase Assay by Monitoring pH-ATPase activity whether 2,4-dinitrophenol or Mg2+-stimulated, was monitored by following pH changes instead of by the more common assay of phosphate or ADP. The pH technique is particularly suited for measuring rapid, initial rates of ATP hydrolysis, but becomes less desirable for reactions longer than 10 min; mainly, because of the slow pH drift, which has to be allowed for. This problem can be minimized by increasing the buffering power of the medium.

In order to validate the use of H+ for assaying ATPase activity, the following experiments were performed. First, 2,4-dinitro- phenol-stimulated ATPase was followed over 5 min. During the 1st min, five samples were withdrawn for phosphate analysis, and then one sample every minute for 4 min. The results of experi- ments on three different mitochondrial preparations are given in Table I. Very good agreement between the two parameters, H+ and Pod, is evident.

A second experiment was carried out, using a pa-stat titrator (Radiometer, Copenhagen) to show that the small pH changes during a pH experiment do not lead to false H+ movements. The latter might conceivably arise due to pK effects and change in buffering power with pH. In any event the total change in pH during an actual ATPase assay was never more than 0.05 pH unit. The result of comparing acid production with a pH-stat device and directly from pH changes, is shown in Fig. 1. It is clear that good agreement exists up to the 5-min period. In actual practice, ATPase activity is always estimated from the initial rate; that is during the first 30 s.

These two experiments then provide a firm basis for using the pH procedure to monitor ATPase activity.

ADP-ATP Binding---This was measured by Millipore filtration (9). All measurements were at 25” unless otherwise indicated. Protein was estimated by an ultraviolet absorption method (10) with bovine serum albumin being used as standard. Other ex-

Methods perimental details are given in t,hL legends.

Mitochondrial Isolation-Rat liver mitochondria were isolated RESULTS

by differential centrifugation of liver homogenates in 0.25 M

sucrose (4) and stored on ice at 50 me of orotein oer ml. “Heavv” Action of Rhodamine 6G on Oxidative Phosphorylation <, . and “light” mitochondrial fractions were combined for the present investigations. All mitochondrial preparations were checked for When rhodamine 6G is added to coupled mitochondria, the

structural integrity using the criterion of respiratory control. efficiency of ADP phosphorylation is drastically reduced, A This was determined polarographically with sodium succinate as typical oxygen electrode trace is illustrated in Fig. 2. Addition substrate in the medium described by Gear and Lehninger (5). Specific variations in substrate, or ionic content, are described

of 150 nmoles of ADP to mitochondria respiring with succinate as

later for the appropriate experiments. Experiments were always substrate increased the rate of oxygen consumption from 49 to

completed within 3 to 4 hours from carrying out the respiratory 177 ng atoms/M mg of protein. When 3 PM rhodamine 6G was

control measurement. present State 3 respiration was inhibited to 132 ng atoms per min., For binding studies to the inner and outer mitochondrial mem- while the State 4 respiration was increased from 46 to 65 ng atoms

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250

200

2 150

0 E c

; 100

'u a, 2

+ = 50

0 L

0 1 2 3 4 5

Time after DNP addition (mln)

FIG. 1. Comparison of ATPase activity monitored by direct pH recording, or by following the amount of alkali needed to main- tain constant pH (pH-stat). The medium (final volume 2.0 ml) contained 80 mM NaCl, 10 mM MgCb, 10 mM sodium succinate, 5 mM Tris-Cl, pH 7.4, and 4.5 mM sodium phosphate. At zero time, 0.1 ml of a stock mitochondrial suspension containing 5 mg of mitochondrial protein, was mixed with the medium. One minute later, ATP at 0.25 mM was added, followed by dinitrophenol (DNP) a further minute later at 5 X 10e6 M. pH or pa-stat trac- ing was then followed for 5 min. Absolute acid ejection was then obtained by int,ernal standardization or alkali titration. Results are expressed as nanomoles of H+ ejected per min.

per min. Also evident in this experiment is the lack of effect on the normal uncoupling action of 50 FM dinitrophenol.

In Fig. 2 the striking ability of 5 PM rhodamine 6G to inhibit ADP-stimulated proton uptake by 80 y0 is shown in a pH record- ing. These results then demonstrate that low concentrations of the dye markedly reduced the rate of ATP formation by coupled mitochondria. The effect of increasing concentrations of rhodamine 6G on State 3 and State 4 respiration, up to 10 pM, is shown in Fig. 3. Concentrations above 3 pM slightly uncoupled respiration and a Ki of about 2 C(M rhodamine 6G for inhibition of oxygen uptake can be derived from Fig. 3. Also shown are data on the influence of higher concentrations of rhodamine 6G on State 4 respiration. Here rates induced by 30 pM dye approached maximum rates of uncoupling caused by 50 pM 2,4-dinitrophenol.

Effect on Respiration and ATP-supported Accumulation of Calcium

When 10 pM rhodamine 6G was added to a succinate-contain- ing medium (5), it only had a negligible effect on both the rate and net ejection of H+ elicited during Ca2f accumulation (Fig. 4). Lower concentrations (5 m) often stimulated the rate of H+ ejection by some 10 to 40%, although this effect appeared to depend on the mitochondrial preparation being tested. Higher dye concentrations, however, did significantly begin to inhibit H+ ejection, probably as a result of the uncoupling action men- tioned earlier. On the other hand, ATP-supported Ca2f accumu- lation (11) was strongly inhibited by 10 pM rhodamine 6G (Table II). A Ki of 12 PM can be calculated from this data for inhibi- tion of the rate of Hf ejection, and may be contrasted with the

RLM

I

100 ng atoms 0, \

control f5uM Rh-6-G

ADP

f 132

-lm,“-

FIG. 2. Typical oxygen electrode trace illustrating the influence of rhodamine 6G (RLB-G) on State 3 and State 4 respiration. The medium (final volume 1.5 ml) contained 80 mM NaCl, 10 mM MgC12, 10 mM sodium succinate, 5 mM Tris-Cl, pH 7.4, and 4.5 mM sodium phosphate, pH 7.4. At zero time, 0.05 ml of a stock suspension of mitochondria in 0.25 M sucrose, containing 2.5 mg of mitochondrial protein (RLM) was mixed with the me- dium. ADP (150 nmoles) was then injected into the closed system at the point indicated. After State 4 respiration resumed, rhodamine 6G (3 PM) was added, followed by a second addition of ADP (150 nmoles), and finally, 2,4-dinitrophenol (DNP) at 50 pM was injected. The results are expressed as nanogram atoms of oxygen consumed per min. Temperature, 25”. Influence of rhodamine 6G on ADP-stimulated H+ uptake is shown in inset. Conditions were as above, except final volume was 2.0 ml. Rat liver mitochondria (5 mg of protein, RLAf) was added first, fol- lowed by ADP, 400 nmoles. A second trace was carried out with rhodamine 6G present at 5 PM throughout. The system was cali- brated at the end of each trace by internal standardization.

x ‘,“[ , I , ,h 0 1 2 3 4 5 6 7 8 9 10

Rh-6-G (~JM)

FIG. 3. The effect of low rhodamine 6G (RAG-G) concentrations on State 3 and State 4 respiration. Experimental details were as in Fig. 2. Rhodamine 6G was added to the system before the beginning of each concentration run. Also shown is the uncou- pling action of 2,4-dinitrophenol for comparison at higher rhoda- mine 6G concentrations (inset).

lower Ki for oxidative phosphorylation. Monitoring actual Cazf uptake with 4jCaC12 confirmed the above results obtained by con- tinuous pH recording.

Mitochondrial Swelling

Mitochondria behave as osmometers swelling both in re- sponse to changes in external tonicity, as well as to internal ones

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PH 7.35

RLM

400

n moles Hf

control t IOPM Rh-6-G

FIG. 4. The influence of rhodamine 6G (R&6-G) on respiration- dependent calcium accumulation by mitochondria. The medium (final volume 2.0 ml) contained 80 mM NaCl, 5 mM sodium suc- cinate, and 5 mM Tris-Cl, pH 7.4. Rat liver mitochondria (RLM) were added (5 mg of protein) at the times shown, followed by 400 nmoles of calcium chloride. Khodamine 6G was present for the second trace at 10 pM before the mitochondrial addition. Acid movements were quantitatcd by.internal standardization.

TABLE II

Eject of rhodamine 6G on ATP-supported Ca2+ uptake

This was monitored by the pH recording apparatus described under “Experimental Procedures.” The following medium (10) was used, final volume 2.0 ml: ATP, 0.5 mM; MgC12, 10 mM; NaCl, 80 mu; NaF, 10 mM; and Tris-chloride, 10 mM, pH 7.4. The mito- chondria, 5 mg of protein, were preincubated at 25” for 3 min to oxidize endogenous substrates. Then 1 min before the addition of 400 nmoles of CaC12, (80 nmoles per mg of protein), Antimycin A at 0.5 pg per ml, and Rotenone at 0.5 pM were added to block any residual, respiration-dependent calcium uptake. All values are expressed as nanomoles per mg of mitochondrial protein.

Rhodamine 6G Net rate of H+ ejection Net H+ ejection

PM nmoles/min

0 111.6 5 93.4

10 65.4 20 27.2 50 15.2

69.6 50.0 30.2

9.2 2.8

generated by the active accumulation of ions (12, 13). Rhoda- mine 6G was, therefore, tested for its influence on the energy- linked uptake of phosphate (7). The results (Fig. 5) show that the dye had little effect on both the rate and magnitude of swell- ing, (p > 0.01). Also shown is a trace revealing lack of ability to act as a simple detergent in promoting mitochondrial frag- mentation in 0.25 M sucrose. It may be noted that phosphate addition caused a greater, sudden initial drop in light scattering when rhodamine 6G was present. What this means is not clear. A rapid, but only partial swelling could have occurred. In order to confirm further that rhodamine 6G does not damage mito- chondria structurally, three batches of stock mitochondria were diluted to 2.5 mg of protein per ml of 0.25 M sucrose. The first was untreated, the second contained rhodamine 6G at 10 pM,

while the third was treated with Lubrol WX at 0.3 mg per mg of mitochondrial protein. After mixing and standing for 10 min at 25”, the tubes were centrifuged at 15,000 x g for 10 min, and the protein content of the supernatants was estimated.

The results revealed a 12% protein loss with rhodamine 6G,

Rh-6-G

T + -TAf------

1 0.1 OD

1

-1 ml”+

FIG. 5. Energy-dependent accumulation of phosphate moni- tored by light scattering; the influence of 5 PM rhodamine 6G (Rh-6-G). The system included (3.0.ml final volume) 0.125 M KC1 and 0.02 M Tris-Cl, pH 7.4. Mitochondria were added to the cuvettes (0.5 mg of protein), and after 2 min phosphate (POJ was injected to make its concentration 10 mM. Light scattering changes at 520 nm were then followed for 3 to 4 min. The third trace represents a run in 0.25 M sucrose, with rhodamine 6G (5 FM)

being added at the point shown. Temperature, 25”.

compared with 14% for the control, while the detergent Lubrol caused a massive 64% loss.

Arsenate-stimulated Respiration

In order to define more accurately a possible site or sites of action, it was decided to test whether arsenate-stimulated respira- tion could be inhibited by rhodamine 6G. Ter Welle and Slater (14, 15) and Ernster et al. (16) have carried out detailed studies on the uncoupling action of arsenate on mitochondrial respiration. Based upon these studies, arsenate appears to react with a hypo- thetical, nonphosphorylated intermediate. The resultant arse- nate derivative may either combine with ADP to form ADP- arsenate, which hydrolyses rapidly; or, it may undergo slow hy- drolysis in the absence of ADP, both of which events lead to in- creased rates of respiration.

Using the conditions described by Cross and Wang (17) where 20 mM arsenate significantly stimulated respiration, rhodamine 6G was tested for its abilit,y to inhibit arsenate-stimulated res- piration. The traces illustrated in Fig. 6 demonstrate that 7.5 pM dye strongly inhibited phosphorylating respiration in the presence of arsenate. Significantly, however, arsenate-stimu- lated respiration was only partially inhibited by 7.5 mM rhoda- mine 6G, and 15 pM still only inhibited the reaction by about 50% as compared with the normal, phosphate inhibition (17). These concentrations produced essentially complete inhibition of ADPstimulated respiration (Fig. 3). Also, shown is that the ability of 2,4-dinitrophenol to uncouple respiration remains unaffected by rhodamine 6G.

The significance of these findings, together with some addi- tional experiments to be described below, will be considered in the discussion.

Binding of Rhodamine 6G to Mitochondria

Several types of experiments were carried out to determine the site and extent of dye binding to mitochondria. First, since the compound is positively charged at pH 7.4, it might be ex- pected to elicit H+ ion release on binding. This possibility was tested in the following experiment.

When mitochondria are added to a cation-containing medium,

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RLM hiM

-4

t 100

ng atoms 0,

1 +1 ml,“-,

380 \

FIG. 6. The influence of rhodamine 6G (Rh-6-G) on arsenate- stimulated respiration. The medium (final volume 1.5 ml) con- tained 80 rnM NaCl, 10 mM MgC12, 10 mM sodium succinate, 1 mM EDTA, and 10 mM Tris-Cl, pH 7.4. Rat liver mitochondria (RLM) equivalent to 2.5 mg of protein were injected into the oxygen-electrode system as shown. After a short time, arsenate (final concentration 20 mM) was injected, followed by phosphate (20 mM) and 300 nmoles of ADP. Rhodamine 6G (7.5 MM) was then added, followed by a second addition of 300 nmoles of ADP. Finally, 2,4-dinit,rophenol (50 PM) was injected into the system. For the second trace, the various concentrations of added sub- stances were the same, except that rhodamine 6G was injected before the phosphate. Results are expressed at per cent oxygen consumed per min. Temperature, 25”.

TABLE III H+ ejection during rhodamine 6G binding to mitochondria

Rat liver mitochondria were added (5 mg of protein) to make a total volume of 2.0 ml containing increasing NaCl concentra- tions, from 0 to 320 mM. The osmotic strength was maintained at 160 mosM by the inclusion of suitable concentrations of sucrose, except for the final 320 mM NaCl vial. The net ejection of H+ was monitored by the sensitive pH recording system described under “Experimental Procedure” and in Ref. 5. A second series of traces was then obtained where the medium contained 10 pM

rhodamine 6G. Results are expressed as nanomoles of H+ ejected per mg of mitochondrial protein, and include the extra H+ ejected elicited by the presence of rhodamine 6G at the ratio of 4 nmoles per mg of mitochondrial protein (10 @M).

I N&l, IIIM

1 0 1 5 1 20 1 so 1 32lJ

+ rhodamine 6G. 3.0 12.8 16.8 17.7 19.2 - rhodamine 6G.. 2.6 11.6 13.8 15.5 18.2 Extra ejection. 0.4 1.2 3.0 2.2 1.0 -

H+ ions are ejected into the medium (5), corresponding to the actual amount of cation bound. Measurement of proton release is thus a sensitive means of following a cation binding. Rhoda- mine 6G was therefore added to a sodium-containing medium at pH 6.6 and the proton ejection monitored as a function of salt concentration. The data (Table III) illustrate that there was indeed an “extra” release of protons caused by the presence of 10 pM rhodamine 6G. Interestingly, the net release was dependent

TABLE IV Measurement of rhodamine 6G binding to mitochondria after

treatment with lubrol or digilonin

A mitochondrial suspension, 1 ml, 50 mg of protein per ml, was added to 0.4 ml of 0.2 mM rhodamine 6G; that is to give a ratio of 1.6 nmoles of rhodamine 6G per mg of protein. To one set of tubes was added Lubrol WX at the ratio of 0.3 mg per mg of pro- tein; to another set was added digitonin to make the final digitonin to protein ratio equal to 0.1 (6). A third set served as control. The tubes were mixed slowly at 0” for 15 min, diluted to 10 ml with 0.25 M sucrose, and then centrifuged at 100,000 X g for 90 min. The extinction of the final supernatants was then mea.+ ured at 527 nm and compared with that of 0.4 ml of 0.2 mM rhodamine 6G diluted also to 10 ml. The results are expressed as per cent of rhodamine 6G originally added.

Initially in supernatant.. Correct for control.

14 20 0 6

on ionic strength, being at a maximum at 200 mM NaCl, and low at both 0 and 320 mM N&l. Another noteworthy fact is that the extra ejection of H+ at 20 mM NaCl of 3.0 nmoles per mg of mitochondrial protein, and 2.2 nmoles at 80 mM NaCl approaches a 1: 1 molar ratio of the dye added, versus protons ejected into the medium; the dye being present at the ratio of 4 nmoles per mg of protein.

Second, a direct estimation of the amount bound to whole mito- chondria, as well as to the inner and outer membranes, was at- tempted. Rhodamine 6G was added to a mitochondrial suspen- sion at the ratio of 1.6 nmoles of dye per mg of protein. This corresponds to slightly less than the biding suggested by the earlier experiment (Table III). The pertinent experimental details are given in the legend to Table IV. It appears that two forms of dye binding may exist in mitochondria, “tight” and “loose” forms. The disruption of mitochondria with the non- ionic detergent, Lubrol WX, released 52% of the added dye. Centrifugation of the control mitochondria in the absence of de- tergent causes 14y0 of the dye to appear in the supernatant. Consequently, 38% of the rhodamine 6G may be considered loosely bound to inner and outer mitochondrial membranes, while 100 minus 52, or 48% appears to be firmly bound in the presence of detergent. The differences between the control and Lubrol- treated, and digitonin-treated preparations allow one to calculate that 32y0 and 6% were bound loosely to the inner and outer mitochondrial membranes, respectively. The ratio between these quantities, 5.2, is identical with the relative amounts of pro- tein and lipid associated with these membranes (6), and the loosely bound form is probably complexed with lipid, or specific membrane proteins. The two types of binding just discussed may well be related to the presence of at least two relaxation timesi for dye binding to pure, phospholipid vesicle preparations m3).

A third experiment was performed to measure the quantity of dye tightly bound to purified inner and outer mitochondrial membranes. These were isolated by exposure of mitochondria to digitonin (6). After addition of 1.6 nmoles of rhodamine 6G per mg of original mitochondrial protein, 1.3 and 0.8 nmoles of dye per mg of membrane protein were associated with the inner

1 C. Huang, personal communication

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Rh-6-G grat-$cidin

4-l mind I FIG. 7. The influence of rhodamine 6G (Rh-B-G) on the potas-

sium permeability of mitochondria. To the medium (final volume 4.0 ml) of 0.25 M sucrose lightly buffered with 0.5 mM N-2-hydroxy- ethylpiperazine-N’-2-ethanesulfonic acid, pH 7.4, were added 10 mg of rat liver mitochondria. The potassium electrode arrange- ment is described under “Experimental Procedure.” Rhodamine 6G was added 2 min later at a final concentration of 10 pM, followed by gramicidin at 1 ag per mg of mitochondrial protein. KCl, 560 nmoles, was finally injected to serve ae internal standard. Tem- perature, 25’.

and outer membranes, respectively. A spectral scan of both membrane preparations revealed a red shift of 8 nm after dye binding from the normal X,,, of 527 to 535 nm. A similar bind- ing experiment with rhodamine B, which earlier was shown to have no influence on mitochondrial energy-linked functions, gave no significant spectral shift; the X,,, for free rhodamine B was 553 nm, while the maximum for the membrane-associated dye was 552 nm.

Possible Interaction with Adenine Nucleotides

Since rhodamine 6G has its major effect on energy-coupled reactions involving adenine nucleotide transport, direct spectro- scopic testing of a possible interaction of ADP with dye was considered. Using split cuvettes in a Cary 14 double beam spectrophotometer, and the appropriate controls, a complete absence of spectroscopic interaction was noted.

Direct Measurement of Ion Movements

A potassium electrode was used to monitor K+ movements from mitochondria in the presence of rhodamine 6G. The data (Fig. 7) show that addition of 10 PM dye failed to cause any K+ release, in sharp contrast with the effect of gramicidin. A second experiment where loss of intramitochondrial Ca2f and Mg”+ was checked by atomic absorption spectrophotometry again demonstrated that the ionic content of mitochondria was not influenced by concentrations of dye which nearly block oxi- dative phosphorylation completely. These results confirm that rhodamine 6G does not behave like uncouplers such as 2,4-di- nitrophenol or gramicidin, which are known to induce mitochon- drial ion movements.

Direct ATPase Activity of Rhodamine 6G

An important point to test was whether ATPase activity was inhibited in a manner analogous to oligomycin, which could lead to blocking of both ATP formation and ATP-driven ion uptake, or whether it stimulated ATPase activity with a concomitant decrease in the rate of ATP formation. In Table V results are presented of a pH experiment where ATPase activity was moni- tored from 0 to 50 PM rhodamine 6G. In the same table the in-

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TABLE V

Effect of rhodamine 6G on ATPase activity ADP, 500 nmoles, was added to a standard phosphorylation

medium containing 5 mg of rat liver mitochondrial protein, and the following: sodium succinate, 5 mM; NaCl, 80 mM; MgCl*, 10 mM; sodium phosphate, 4.5 mM; pH 7.4; Tris-Cl, 5 mM, pH 7.4, final volume 2.0 ml. One minute after phosphorylation was complete, as monitored by pH recording techniques, rhodamine 6G was injected at the concentrations indicated, and finally 2,4- dinitrophenol at 5 X lo-& M was added. The calcium-stimulated ATPase wae measured by the same procedure and is described fully in Table I. ATPase values are expressed as nanomoles of H+ released per min per mg of protein.

I I Rhodamine 6G Intrinsic ATPase 2,,4-Dinitrophenol- Induced ATPase

CM

0 0

5 2.2 10 7.0 20 11.2 50 52.6

50.8 114.2 53.8 102.6 47.0 49.4 45.0 31.2 45.2 4.0

fluence of 50 I.~M 2,4-dinitrophenol as well as the Ca2f-induced ATPase may be noted.

The ability of rhodamine 6G itself to stimulate ATPase ac- tivity only becomes significant above 20 PM, while it is clear that at concentrations near 50 PM, it caused almost complete inhibi- tion of oxidative phosphorylation. 2,4-Dinitrophenol is still able to stimulate extra ATPase activity by the same amount as in the absence of dye. The ATPase activity of rhodamine 6G is strongly reminiscent of its uncoupling ability demonstrated earlier (Fig. 3).

Injtuence of pH on Action of Rho&mine 6G

The data given in Fig. 8 illustrate that the inhibitory action of 3 pM dye on State 3 respiration is maximal at about pH 7.5. On the other hand, the uncoupling action, or ability to stimulate State 4 respiration, only became evident above pH 6.7 and in- creased dramatically by pH 8.5, such that respiratory control was almost completely absent at this pH.

Kinetic Analysis of Rhodamine 6G on Oxidative Phosphorylation

A series of three pH experiments was carried out where ADP concentration was varied from 20 to 400 PM to obtain data for Lineweaver-Burk plots in the presence and absence of 4 pM rho- damine 6G. Fig. 9 shows that linear, double reciprocal plots are not obtained, indicating some degree of interactions for the rate-limiting step of oxidative phosphorylation (19), which is thought to be the actual translocation of adenine nucleotides across the inner membrane. Rhodamine 6G is clearly a non- competitive inhibitor with a Ki calculated to be 3.2 m, and decreasing the K, for ADP from 56 I.IM to 29 PM in the presence of 4 PM dye. The data are also shown as a Hill plot (Fig. 10) to gain an idea as to the inhibitory mechanism of rhodamine 6G. The observation that f is greater than 1 and equal to 1.28 indi- cates that some positive cooperativity occurs which is accen- tuated in the presence of rhodamine 6G, where +j = 1.72. Work of Weideman et al. (20) has provided evidence for two binding sites involved in adenine-nucleotide translocation. The present research thus suggests that one of these sites may be a positive allosteric modifier, and that rhodamine 6G in some manner in- creases the extent of interaction, while inhibiting the over-all reaction. This point will be considered further in the discussion.

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FIG. 8 (ZefL). The influence of pH on the ability of rhodamine 6G (R/z-6-G) to inhibit State 3 respiration and stimulate State 4 respiration. The system (1.5 ml final volume) was the same as for Fig. 1, except that the nature of the buffer was varied. Tris- Cl, 5 mM was used for pH 8.5, 8.0, and 7.5; sodium phosphate, 5 mM, for pH 7.0; and histidylhistidine, 5 mM, for pH 6.5 and 6.0. After the completion of the first State 3 to State 4 transition,

TABLE VI

Influence of rhodamine 6G on Mg2+ATPase in sonicaled mitochondria

Rat liver mitochondria (50 mg of protein per ml) were sonicated in 15-s bursts for a total time of 2 min at 0”. The resulting sus- pension, 0.1 ml, was then added to 1.9 ml of the standard, Mg2+- containing phosphorylating medium (Table V). One minute later 0.25 mM ATP, pH 7.4, was added to the medium and ATPase activity monitored by the pH recording technique. When tested, rhodamine 6G (10 PM) was added to the medium before the son- icated mitochondria. In all, four controls and six runs with rhodamine 6G were carried out, using two different mitochondrial preparations. Results are expressed as nanomoles of H+ ejected per mg of protein at 25”.

Control 10 PM rhodamine 6G

203 f 25 278 f 38 p < 0.005

Analysis for Specific Site of Action

Mg2+-stimulated ATPase-In order to determine whether rho- damine 6G might act directly at the level of Fl, the ATPase (3), its influence on the Mg *+-stimulated ATPase in sonicated mitochondria was tested. The results in Table VI show that the initial rate of ATP hydrolysis, as evidenced by II+ ejection, was in fact stimulated by 10 pM rhodamine 6G, from a control value of 203 nmoles of Hf per min per mg of protein to 278 in the presence of dye (p < 0.005). One interesting observation was that the subsequent rate was slower in the presence of dye. At 2 min it was about 70% that of the control. It is known that

ADP is a competitive inhibitor of the ATPase, and these findings suggest that rhodamine 6G may enhance the initial binding of ATP, and also that of ADP, the competitive inhibitor.

ADP and ATP Binding-The above experiment, by exclusion, points to rhodamine 6G probably having its effect via the adenine nucleotide translocase. Consequently, ADP and ATP binding (9) in the presence and absence of 10 pM atractyloside, or rho-

rhodamine 6G (3 PM) was added and a second transition was initi- ated by 150 nmoles of ADP. Temperature, 25”.

FIG. 9 (center). Lineweaver-Burk plots of oxidat.ive phos- phorylation rates, with the influence of 4 PM rhodamine 6G. Con- ditions were as described for Fig. 2.

FIG. 10 (right). Hill plots derived from monitoring rates of ox- idative phosphorvlation in the nresence and absence of 4 j.bM (Rh- 6-G). The data are derived from those for Fig. 9.

i

0 1 2 3 4 5

Tfme (mln)

FIG. 11. Influence of rhodamine 6G on ADP and ATP binding at low external nucleotide concentrations. The basic binding medium and procedure are exactly as described in Ref. 9. 0.1 ,uCi per ml of either [%]ADP- or [ldC]ATP (2rM)-initiated binding 100-J aliquots were withdrawn for Millipore filtration at various intervals up to 5 min. When present, rhodamine 6G was added at 10 /IM before the labeled nucleotide, as was 10 pM atractyloside. After drying, the filters were counted by liquid scintillation. Temperature 0”. 0, control; l , rhodamine 6G; A, atractyloside.

damine 6G, was measured over a 5-min period. The results of an experiment for low levels (2 PM) of [“C]ADP and [W]ATP are given in Figs. 11 and 12. Like atractyloside, rhodamine 6G was a powerful inhibitor of binding for both ATP and ADP. It is noteworthy that the dye was significantly less potent in block- ing ATP binding than ADP. This distinction parallels that noted earlier, where the Ki for ADP-stimulated oxygen uptake (Fig. 2) was some 5-fold less than that for ATP-driven Cazf uptake (Table II).

When 10 pM rhodamine 6G was tested for inhibition at a lOO-

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0 1 0 1 2 3 4 5

Time (men)

FIG. 12. Influence of rhodamine 6G on ADP and ATP binding at low external nucleotide concentrations. The basic binding medium and procedure are exactly as described in Ref. 9. 0.1 &i per ml of either [i%Z]ADP- or [“C]ATP (2pM)-initiated binding IOO-~1 aliquots were withdrawn for Millipore filtration at various intervals up to 5 min. When present, rhodamine 6G was added at 10 pM before the labeled nucleotide, as was 10 pM atractyloside. After drying, the filters were counted by liquid scintillation. Temperature 0”. 0, control; l , rhodamine 6G; A, atractyloside.

1500

2°oo a

”

,” 0 c

m 500

k Q !

vu

0 - 0 1 2 3 4 5

TlfTl‘Z mill)

FIG. 13. Influence of rhodamine 6G on ADP and ATP binding at high external nucleotide concentrations. The procedure is identical with that described for Figs. 11 and 12, except that 0.2 mM cold nucleotide was present for 2 min before adding the radio- active nucleotides (0.1 &i per ml). 0, control; 0, rhodamine 6G; A, atractyloside.

fold higher nucleotide concentration, namely 200 PM, very little influence on final net binding was seen (Figs. 13 and 14). A couple of observations may be noted. At the earliest binding times practicable with the Millipore filtration technique (9), 5 to 10 s, ATP binding was actually enhanced by about 50% in the presence of rhodamine 6G. On the other hand, ADP binding was still inhibited (Fig. 13), but not nearly so drastically as at the low external nucleotide concentrations (Fig. 11). These results suggest that rhodamine 6G may modulate the rate of nucleotide binding or exchange, at high levels, without influenc- ing the final net binding.

600 m c

72 ‘: 400 m

0 1 2 3 4 5

Time (mln)

FIG. 14. Influence of rhodamine 6G on ADP and ATP binding at high external nucleotide concentrations. The procedure is identical with that described for Figs. 11 and 12, except that 0.2 mM cold nucleotide was present for 2 min before adding the radio- active nucleotides (0.1 &i per ml). 0, control; l , rhodamine 6G; A, atractyloside.

DISCUSSION

The results just presented show that the lipophilic dye, rho- damine 6G, at concentrations near 3 PM powerfully inhibited mitochondrial oxidative phosphorylation and ATP-supported calcium accumulation. In particular, only those reactions in volving adenine nucleotides were blocked by low dye concentra- tions. At higher concentrations, near 20 PM, respiration-de- pendent uptake of calcium became inhibited at the same time that State 4 respiration began to increase. At concentrations below 10 pM, rhodamine 6G did not cause any mitochondrial swelling, loss of matrix protein, or endogenous K+, Ca2+, or Mg*+. The 2,4-dinitrophenol-stimulated ATPase was not inhibited by the dye, even up to 50 pM, nor was the Mgr+ATPase in sonicated mitochondria. Its own ability to stimulate ATPase activity only became apparent above 10 PM, as might be expected from the uncoupling action on mitochondrial respiration seen at the higher dye concentrations. Rhodamine 6G was able partially to inhibit arsenate-stimulated respiration, while having no in- fluence on 2,4-dinitrophenol-uncoupled respiration. Finally, the dye was a potent inhibitor of adenine nucleotide binding at low external concentrations.

The mechanisms by which agents block State 3 respiration may be related to which reactions they inhibit. Thus, oligo- mycin, aurovertin and dicyclohexylcarbodiimide specifically

prevent energy transfer at the level of the ATPase itself, while translocation of the needed substrates, phosphate and ADP, can be, respectively, blocked by compounds such as mercurials and atractyloside (21, 22). One of the enigmas in rationalizing such effects lies in the enormous differences in chemical structure between compounds. Thus, for example, dicyclohexylcarbo- diimide is a relatively simple organic compound totally distinct from oligomycin B, which is a complex carbohydrate, (mol wt 804) with three conjugated double bonds, a separate unsaturated carbonyl structure, four secondary hydroxyl groups, and prob- ably a lactone (23).

Recently, reports have appeared describing the effects of other inhibitors on State 3 respiration. Wikstriim and Saris (24) re-

vealed in a careful study that hydroxylamine at rather high concentrations, 1 to 10 mM, effectively blocked phosphorylating

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respiration while only slightly inhibiting State 4 respiration. Based upon the use of different electron acceptors, they concluded that hydroxylamine was specific for Site 3, and that since 2,4- dinitrophenol-stimulated ATPase was not inhibited, the site of hydroxylamine action was before that of oligomycin. In this respect, hydroxylamine is similar to azide which also inhibits State 3 respiration at the terminal site of the electron transport chain (25, 26).

A study of robenzidene (27), an anticoccidial agent, has shown this compound to be a powerful inhibitor of phosphorylating respiration. At 20 pM, State 4 respiration was only slightly stimulated. Its action was similar to oligomycin in inducing respiratory control in submitochondrial particles, as well as inhibiting uncoupler-stimulated ATPase in intact mitochondria and submitochondrial particles. These observations led the authors to propose a very similar inhibitory mechanism for robenzidene as for oligomycin and dicyclohexylcarbodiimide; that is, at the level of the ATPase itself.

Another compound, tetradifon, and acaricide (28) have re- cently been shown to behave almost as robenzidene (29, 30), and thus resemble oligomycin in being able to block State 3 respira- tion, 2,4dinitrophenol.-stimulated ATPase, Mg*+ATPase, and the I’i-ATP exchange reaction. It was without action on the purified ATPase and therefore probably requires a lipoprotein complex and the oligomycin sensitivity-conferring protein (31, 32) for being able to inhibit the ATPase.

Some studies on bilirubin by Mustafa and King (33) are also of interest to the present one on rhodamine 6G. Evidence was presented that the well known toxic effects of the bile pigment could be rationalized in terms of its ability to act as a powerful inhibitor of oxidative phosphorylation. Bilirubin, 17 pM, com- pletely abolished any mitochondrial respiratory control, as well as having some direct uncoupling activity. The authors sug- gested on the basis of binding studies that bilirubin inhibits energy coupling by its ability to bind to lipid and thus interfere with the role of lipid in energy transduction.

At this point it is perhaps pertinent to compare the structure of some of the compounds just discussed.

It is interesting to contrast rhodamine 6G with the inhibitors mentioned above. Half-maximal inhibition of oxidative phos- phorylations occurred at 2 to 3 PM rhodamine 6G, or equivalent to almost 1 nmole per mg of mitochondrial protein. This is some IO-fold lower than the half-maximal concentrations needed for tetradifon (29) and robenzidene (27). The main distinction between these three inhibitors lies in the fact that rhodamine 6G did not inhibit the 2,4-dinitrophenol-stimulated ATPase. An- other activity not considered for the other two compounds is that of energy-linked ion uptake. Here rhodamine 6G had no effect on respiration-driven CaZ+ uptake, while strongly blocking ATP-supported accumulation at the same dye concentration.

In terms of the chemical-coupling concept of oxidative phos- phorylation (34), electron transport is envisaged to generate a nonphosphorylated, high energy intermediate, which is then phosphorylated for final reaction with ATP. Such a concept has been used by Cross and Wang (17) to explain why phosphate inhibits arsenate-stimulated respiration, and allows for the differ- ential effects of aurovertin and oligomycin (11, 3537). It also provides a framework for attempting to rationalize the mecha- nism of rhodamine 6G inhibition.

The first important observation is that at concentrations which almost completely block oxidative phosphorylation, rho- damine 6G neither inhibits or stimulates respiration, nor does it affect 2,4-dinitrophenol-stimulated respiration or respiration driven calcium uptake. Thus, the site of action must lie beyond the formation or stability of the hypothetical [X - I] chemical intermediate. The next step in a chemical coupling scheme consists of the formation of the phosphorylated (or arsenylated) derivative [X - Pi]. Here the experiment where 7.5 pM rho- damine 6G did partially inhibit the rate of arsenate-stimulated respiration does indeed suggest that this step is affected by rhodamine 6G. However, two other observations run counter to this. First, phosphate remained completely effective for inhibiting the arsenate-stimulated respiration; and second, res- piration-dependent uptake of phosphate monitored by light scattering (7) was unaffected by rhodamine 6G. Thus, actual inhibition of this reaction, either the enzyme, or of phosphate or

Tetradifon

C)--N=C=N~

Dicyclohexylcarbodiide

(ECD)

Robenzidene

Rhodamine 6-G

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arsenate, transport itlto the> mitocholltlriolr nppcars llot to bc valid for rhotlarnillc~ 6G.

Thcsc c~onsitlerations point to a site of action very c~losc to the first reactions of osidative ~)hosl)hor~latioll, Ilwmcly, the ATl’ase itself, or the xdrnintl Ilucleotidc translocasc. The csgeriments on the 2,4-tlillitroplirriol- and 1lg*+-stinlulatecl .2Tl’ases, as well as on a&nine nucleotitle bin(ling help ill this analysis. The fact that IO PM rhodamine 6G ciitl llot dramatically inhibit the M$+ATl’ase in sonicatcd particles, which arc “inside out” (3), and thcreforr not colistrained by a nucleotide permeability barrier, would suggest the translocase to be involved in inhibition. Indeed, this conclusion appears to have been amply confirmed by direct experiment (Figs. 11 and 12) where rhodamine 6G was almost as potent as atractyloside in blocking ADl’ or ATI’ bind- ing. However, st>veral facts clearly differentiate rhodamine 6G from bongkrekic acid or atractyloside (38-40). First, rhodamine 6G did not inhibit the 2,4-dinitrophenol-stimulated ATI’ase of intact mitochondria which requires an active translocase, in contrast with atractyloside and bongkrekic acid, which do ill- hibit (41, 42j. Second, higher concentrations of ADP or ATI’ caused loss of the rhodamine 6G inhibition on net nucleotide binding, while atractyloside was still a moderately effective ill- hibitor. Rhodaminc 6G did, however, have some inHuence 011

the rate of nucleotide exchange. Finally, the dye influenced the kinetics of the Mg2+-stimulated ATl’ase in sonicated mitochorl- dria, whereas bongkrckic acid and atractyloside have 110 effect (41).

Rhodaminc 6G is thus somewhat different from either of the translocase blockers, or the direct inhibitors of ATl’ase such as oligomycin or aurovertin. The classical chemical reactions out- lined earlier are, therefore, inadequate to provide a satisfying explanation for the mode of action of rhodamine 6G. The lipid- soluble nature of the dye perhaps emphasizes the necessary role of lipid-protein interactions for energy transduction. Such inter- action could be disturbed by rhodamine 6G, and reflected in its effects on the adenine nucleotide binding changed characteristics for the Mg*+ATl’ase and its interesting influence in the Hill plot for increasing the interaction constant for ADP. Adenine nu- cleotides then form a common theme for all of rhodamine 6G effects, with perhaps the most plausible explanation involving changed binding at the level of the translocase.

Acknowledgments-The author is very grateful to Dr. C. Huang for providing the original stimulus and supply of rhodamine 6G, and also to Dr. James W. Ogilvie for his kindly interest and critical judgment, especially with regard to the kinetic studies. Dr. Michaeleen I?. Lee also is to be thanked for many useful discussions. Finally, I wish to acknowledge the cheerful and capable technical help of Mr. John T. Spears.

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Adrian R. L. GearPHOSPHORYLATION

Rhodamine 6G: A POTENT INHIBITOR OF MITOCHONDRIAL OXIDATIVE

1974, 249:3628-3637.J. Biol. Chem. 

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