Mitochondrial sulfhydryl groups under oligomycin-inhibited, aging, and uncoupling conditions: Beneficial influence of cardioprotective drugs

ARCHIVES OF BIWHEMISTRY AND BIOPHYSICS Vol. 266, No. 1, October, pp. X3-88, 1988

Mitochondrial Uncoupling JiiRGEN

Sulfhydryl Conditions:

Groups under Oligomycin-Inhibited, Aging, and Beneficial Influence of Cardioprotective Drugs

FIJCHS, HANS-,JOACHIM FREISLEBEN,’ AND GUIDO ZIMMER

(;ustnll-EmMen-Z~ntrurn

LIJISE

der Biologischen C’hemie, Universitiit Frankfurt, 6 Frmkfwt mm Mniw, Federal Republic of Gmmmy

Received December

MATNKA,

Theodor-Stern-Kai

7,

22, 1987, and in rcviscd form May lo,1988

Uncoupling, oligomycin-inhibited, and aging/swelling conditions comprise three models for mitochondrial dysfunction. In these models, the effects of cardioprotective agents on rat heart mitochondrial membrane -SH reactivity have been studied. For -SH detection two different chromophores were used: dithionitrobenzoate (Nbs,) and monobromobimane (MB). The objective of this study is to reveal the influence of three cardioprotective substances against the loss of membrane -SH reactivity: (i) The thiol reagent !&mercaptopropionylglycine (MPG) prevents the decrease of thiols caused by carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), aging, and oligomycin measured with MB and Nbss, and the diminution by oleate detected with MB. The small amount of MPG (6 nmol/mg protein), necessary for the protection, agrees with oligomycin sensitivity of the -SH groups concerned. (ii) The active metabolite of molsidomine, 3-morpholinosydnonimine (SIN-l), protects against the decrease of thiols by FCCP, oleate, and aging monitored with MB. In the case of oligomycin -SH groups accessible to Nbsa are protected. (iii) Another antianginal drug, isosorbidedinitrate (ISDN) does not protect membrane thiol groups. In contrast to SIN-l, ISDN probably requires enzymatic activation. It is suggested that MPG as well as SIN-l may help to rcstitute the original -SH status of the mitochondrial membrane. (c)1988AcademicPress,Inc.

During hypoxia mitochondria suffer from a decay in structure and function: coupling degree and ATP levels are reduced; membrane potential is decreased; and mitochondrial membrane-bound lipids and -SH groups are oxidized. Hypoxia (ischemia), swelling, and aging conditions have often been considered as representing similar stages of mitochondrial loose coupling. A substantial decrease of protein -SH groups was found to be connected with myocardial damage during ischemia (1). Recoupling may occur with restitution of physiological -SH status (2). We have shown that uncoupling of oxidative phosphorylation and inhibition of mitochondrial functions by oligomycin di1 To whom correspondence

minished protein -SH reactivity in rat heart mitochondria (3). On the other hand, cardioprotective drugs, like MPG’ and SIN-l have been reported to unmask -SH groups (4, 5), whereas nitroesters proved ineffective, or contrarily used up thiols (4). MPG improves mitochondrial structure and function in cardioplegic canine hearts in the reperfusion phase (6). Furthermore, MPG reverses changes in rat heart and liver mitochondria induced by partial uncoupling and aging: condensed forms of mitochondria appear in the preparation ’ Abbreviations used: MPG, 2-mercaptopropionylglycine; SIN-l, 3-morpholinosydnonimine; ISDN, isosorbidedinitrate; FCCP, carbonylcyanide-p-triAuoromethoxyphenylhydrazone; Nbsz, S,S’-dithiohis(2-nitrobenzoic acid); MB, monobromobimane.

should be addressed. 83

0003-986X38 Copyright All rights

$3.00

0 1988 by Academic Press, Inc. of reproduction in any form reserved

84

FUCHS

(7). In isolated perfused rat hearts replenishment of CP and ATP levels is improved during postischemic reperfusion (8). In bovine heart oligomycin-sensitive ATPase MPG (3.5-7 nmol/mg protein) increases protein bands at 28-31 kDa from higher molecular mass aggregates via -SH/-S-S interchange. Concomitantly ATPase activity is decreased and oligomycin sensitivity as well as ATP-Pi exchange are increased (9, 10). Since recoupling has been found to be correlated with the unmasking and mobilization of -SH groups (ll), in the present paper we investigate, whether MPG, SIN-l, or ISDN protect mitochondrial membrane thiols. To this end the classical uncoupler FCCP was applied as well as the very weak uncoupler oleate and the inhibitor oligomycin. As a correlate to structural damage mitochondria were exposed to aging and swelling conditions. MATERIALS

AND

METHODS

Reagents. The formulas of the substances used in this work are shown in Scheme 1. FCCP was purchased from Boehringer, Mannheim, GFR. 5,5’-Dithiobis(Z-nitrobenzoic acid) (Nbs,), oleic acid, and oligomycin were products of Sigma, Munich. The monobromobimane was obtained from Calbiochem, Frankfurt am Main; MPG from Fresenius AG, Bad Homburg, GFR; SIN-l from Cassella AG, Frankfurt am Main; and ISDN from Pharma Schwarz, Monheim, GFR. All other substances were purchased from Merck, Darmstadt, GFR, or from BoehringerMannheim, analytical grade or for biochemical purposes. Preparation of mitochwzdria. Rat heart mitochondria were isolated from male Wistar rats according

SCHEME

work.

1. Formulas

of substances

used in this

ET AL. to a procedure described by Mela and Seitz (12), omitting the nagarse treatment. Isolation medium A: The isolation medium was composed of 0.225 M mannitol, 75 mM sucrose, and 1 mM EGTA, pH 7.4, with Tris. Incubation medium B for the evnluation of functimal parameters. The incubation medium was composed of 250 mM sucrose, 10 mM Tris-HCl, 10 mM K2HPOd, 1 mM EDTA, and 10 mM substrate (cu-ketoglutarate, @-hydroxybutyrate, malate, and pyruvate, each 2.5 mM). Photometric -SH group determination with Nbs,. For determination of reactivity of -SH groups 0.5 mM Nbs, (13) in 2 ml (glutamate buffer (0.01 M glutamic acid, 0.01 M Tris, pH 7.4) together with 10 mM substrate (see incubation medium B) was used. Sequence of additions: (a) Buffer with substrate and 1 mg of mitochondrial protein, incubated for 1 min at 25°C. (b) Subsequently (if present) uncoupler or inhibitor (c)or drug and afterwards the uncoupler or inhibitor were added and, after each addition incubation was carried on for 1 min. (d) Thereafter, 0.5 mM Nbsz was added, and after further incubation for 2 min, measurements were taken at 412 nm with a Zeiss PMQ II spectrophotometer. A control cuvette without Nbs, was set to 100% transmission. Fluorometric SHgrvup determination with trromobimanes. About 50 ~1 of rat heart mitochondrial suspension containing 0.1 mg of protein was pipetted into 2 ml glutamate buffer, and 10 mM substrate was added (see incubation medium B). Buffer, substrate, sequences of additions, and incubation times were as described under Photometric -SH group determination with Nbsz. MB (14) was added at a 10 pM final concentration. Measurements were done in a Perkin-Elmer LS-3 spectrofluorometer at 25°C. The values were taken at 380/480 nm 2 min after addition of the fluorochrome. Aging conditions. Aging was carried out at 40°C for 20 min in 2 ml glutamate buffer, thereafter Nbsz or MB were added as described above under photometric (1 mg of protein) and fluorometric (0.1 mg of protein) SH group determinations. Swelling conditions. Swelling was performed at 4O”C, up to 70 min (see Table I): 0.1-0.2 mg of mitochondrial protein was pipetted into 2 ml isolation medium A with or without addition of MPG, SIN-l, or ISDN. Decrease of optical density was measured at 546 nm in an Eppendorf spectrophotometer. Concentrations of reagents and drugs. FCCP, 0.2 KM; oleate, 50 PM; oligomycin, 5 pg/ml; SIN-l and ISDN, 10 /IM; MPG, 6 nmol/mg of protein. The MPG concentration was chosen to be low enough (6 nmol MPG per 70 nmol total mitochondrial membrane protein -SH groups) to make sure that the substance did not react with chromophores directly. MPG-treated mitochondria were centrifuged in several cases and the pellets were investigated: there were no differences

PROTECTION

OF MITOCHONDRIAL

SULFHYDRYL

85

GROUPS

in -SH reactivity detectable in comparison with uncentrifuged mitochondria, indicating that there was no residual MPG reacting with the chromophores in the supernatant. Stntistics. For statistical evaluation Student’s t test was used; significance is denoted in the figures. RESULTS

For evaluation of -SH protective activities of the antianginal drugs SIN-l and MPG the classical uncoupler FCCP as well as the weak uncoupler oleate were chosen. Oligomycin was previously found to decrease mitochondrial thiol groups (15) and to prevent completely the unmasking due to ADP (16, 17). For this reason oligomytin was also applied. The aging and swelling procedures were used as a corollary for tissue lesion. Figures 1 and 2 reveal a significant decrease of -SH reactivity monitored by Nbs, and MB in FCCP- and oligomycin-treated and in aged mitochondria; mitochondria treated with oleate only exhibit a significant decrease of -SH groups titrated with MB. On the basis of these results further experimentation using the -SH protective agents was devised. MPG causes significant (P cO.01) protection against the actions of the uncouplers FCCP and oleate, against aging and oligomycin blockade. This is found with both -SH chromophores applied (Fig. 1). In Fig. 2 protection of thiol reactivity by SIN-l is revealed: against FCCP, oleate,

”

27

D.00,

5

30 00,

10

30 001

9

20 001

5

29 001

6

25 001

5

29

6

20

30

001

FIG. 1. Protection of mitochondrial -SH groups by MPG. (B) Nbs,; (m) MB. The empty columns represent the MPG-treated samples. Ordinate: percentage of the -SH groups of the controls (~100%). Abscissa: number of experiments (n) and significance (P). The weak uncoupler oleate did not exhibit any changes in thiols monitored by Nbsz, and therefore not shown.

”

27

0’

001

6

30 001

10

30 001

17

20 001

5

29 001

9

24 001

5

29

5

20

30

ns

FIG. 2. Protection of mitochondrial -SH groups by SIN-l. @) NbsP; (m) MB. The empty columns represent the SIN-l-treated samples. Ordinate: percentage of the -SH groups of the controls (=lOO%). Abscissa: number of experiments (n) and significance (P). The weak uncoupler oleate did not exhibit any changes in thiols monitored by Nbse, and therefore not shown.

and aging with MB and Nbsz; against oligomycin only with Nbsz. No significant protection is found with MB after inhibition by oligomycin. ISDN does not exhibit any thiol protective activity (not shown). SIN-l diminishes swelling under our conditions by 25%, and MPG only by 10% (Table I). DISCUSSION

Oligomycin-inhibited, uncoupled, and aged/swollen mitochondria exhibit states of impaired oxidative phosphorylation mimicking conditions of hypoxia/ischemia (2, 18). Uncouplers of oxidative phosphorylation generally decrease -SH reactivity in rat heart mitochondria (3). Similar to FCCP and oligomycin, aging conditions decreased Nbs2 and monobromobimane accessible -SH groups (Fig. 1). Therefore, the decrease in -SH reactivity is not specific for uncouplers. In all cases, MPG restores -SH reactivity to or above the control values (Fig. 1). SIN-l generally is quantitatively less effective and does not protect against loss of -SH reactivity by oligomycin in a hydrophobic environment monitored with MB (Fig. 2). ISDN does not influence -SH reactivity or swelling (Table I). For possible mechanisms of action of the cardioprotective agents MPG, SIN-l

86

FUCHS TABLE

I

SWELLINGOFMITOCHONDRIAUNDERDRASTIC CONDITIONSIN THE PRESENCEOF SIN-l, MPG, AND ISDN

AOLT~ Minutes:

30

40

70

Control

0.08

0.11

0.17

SIN-l MPG

0.07

0.07

0.08

0.13

ISDN

0.09

0.09 0.12

0.15 0.17

Percentage of decrease of swelling’ 0 25 10 0

a AODHs = difference in optical density at 546 nm; representative experiments. bDetermined at the end of each experiment (70 min), averaged from all determinations (n = 5). For further details see Materials and Methods.

and ISDN the following points should be considered: Restitution of a physiological -SH/-S-S- status may be essential for cellular function (19). (i) Free radical scavenging potency has been found for SIN-l and MPG; the latter compound, like other thiols, acts only in presence of metal ions such as Fe2+‘3+ or Co’+ (20). The ensuing point (ii) may depend on a radical transition of reactive -SH groups to disulfide bonds. (ii) -SH/-S-S- interchange occurs as a direct interaction between disulfides and thiols, in which both membrane-bound and low-molecular-weight sulfides participate. Evidence for a direct -SH/-S-S- interchange is available only for MPG and not for SIN-l. (iii) The low redox potential of MPG, -0.25 V (21), facilitates the reduction of disulfide bonds into reactive thiols (see ii) including the physiologically active couple GSSG/GSH. (iv) The antianginal compound, molsidomine and its metabolite SIN-l-although not being thiol reagents-significantly increase reactive -SH groups in rat heart mitochondrial membranes. “Nitrates” like ISDN or ISMN did not show similar effects (4). (v) The endothelium-derived relaxing factor has recently been suggested to be

ET AL

identical to NO (22) which is also supposed to be the active principle of SIN-l and of the nitroesters (23). SIN-1A and NO/HNO which probably comprise the active metabolite of molsidomine are formed from SIN-l in a nonenzymatic step (Scheme 2). Bioactivation of nitrates like ISDN requires enzymatic reduction to NO/HNO via binding to -SH groups (4, 24). Uncmpling,

Aging, and Swelling

Protection to loss of -SH groups caused by FCCP, aging, and oligomycin differs between MPG and SIN-l. In total, MPG prevails. The very weak uncoupler oleate only decreases thiols accessible to MB. Oleate-sensitive -SH groups are almost equally well preserved by MPG and SIN-l. Thus, it is not generally the hydrophobic environment of the -SH groups, but rather the way of masking which determines the interaction with SIN-l. Swelling of mitochondria, furthermore, does not quantitatively reflect the protective effects of SIN-l and MPG on mitochondrial thiols: In this model, SIN-l prevails. The significant recoupling and protective activities against uncouplers especially of MPG suggest that polar groups in different regions of the membrane, including thiol groups, might participate also in the uncoupling event.

SCHEME2. Biotransformation

of molsidomine.

PROTECTION

Oligomycin-Sensitive

OF

MITOCHONDRIAL

-SH Groups

In liver mitochondria, it was found previously that MPG at 3.5-7 nmol/mg protein brought about an increase of mitochondrial reactive -SH groups by 30% (25). This effect was completely prevented by oligomycin. On the other hand 9-10 nmol/mg protein of MPG counteracted the blockade of oligomycin (25). In hydrophobic environment MPG thus is able to unmask thiols which were masked by oligomycin. MPG improves functional parameters of damaged mitochondria, i.e., ADP/O ratio, oxidative phosphorylation rate, and respiratory control index (5,26), and increases ATP contents during hypoxia (7) and reperfusion (8). ADP stimulates mitochondrial ATP synthesis and simultaneously unmasks membrane thiol groups (15-17) in an oligomycin-sensitive way. Release of mitochondrial membrane -SH groups under similar conditions has also been found to occur in the adenine nucleotide translocator (27); however, oligomycin sensitivity of unmasked -SH groups, as found in (16,17), contradicts an attribution of these thiols solely to the AdN translocator which is only marginally affected by oligomytin (28). This also applies to the phosphate carrier (25, 29). Moreover, oligomycin, in addition to respiratory inhibitors has been used in assays of other anion carriers (30). Dicarboxylate and tricarboxylate carriers were found not to be inhibited by oligomycin (31). The oxoglutarate carrier also is not oligomycin sensitive (32), as far as malate-2-oxoglutarate exchange diffusion is concerned. For analysis of the pyruvate carrier, oligomycin as well as antimycin and rotenone has been used in the assays (30). Moreover, the concentrations of Nethylmaleimide necessary for inhibition of the glutamate carrier are higher than the anticipated total of available membrane -SH groups (33, 34). Similar considerations apply for the glutamate-aspartate exchange diffusion system (35). All these observations essentially preclude participation of presently known translocator -SH groups in the efficiency of the studied drugs on oligomycin-induced -SH mask-

SULFHYDRYL

87

GROUPS

ing/unmasking. On the other hand, our calculations on the total amount of carrier -SH groups reach a value of less than 20% of the totally available -SH groups. This coincides with the work of Deber et al. (36), which reveals that in currently known proteins noncarrier -SH groups are available at least 2 times as much as carrier thiols. Recently, it has been found that oligomycin completely blocks 9-aminoacridine fluorescence quenching in proteoliposomes (ammonia-EDTA/NaBr extracted submitochondrial particles) which were treated with factor B (37). This efficiency of oligomycin may be compared with the positive influence of the thiol activating drugs on recoupling of loosely coupled or uncoupled mitochondria. The proton F, channel is specifically sensitive to coupling factor B, which, in turn, could be reactivated by thiol reagents. This would coincide with the small amount of MPG necessary for titration of the oligomycin-sensitive range of thiol groups (25). DCCD or oligomycin may disturb essential interactions between F. (Fl) subunits (38). Although the interaction of oligomycin with the proton channel differs from that of DCCD (39), oligomytin seems to hinder binding of DCCD (38). Contrary to its probably superficial binding site (39), it also influences hydrophobic membrane domains (3). In any case, thiol reagents (thiol blockers as well as thiol activators) react in a concentration-dependent way with the same components of the proton conduction pathway affected by oligomycin (40). Inhibition by oligomytin and thiol reagents was found additive under certain experimental conditions (40) also in inhibition of OS-ATPase activity (9). Decrease of membrane thiols seems to occur in diverse mitochondrial dysfunctions. Restitution of the physiological -SH/-S-Sstatus may be essential for cellular function and postischemic/posthypoxic cellular survival. ACKNOWLEDGMENT Support of this work by the Deutsche gemeinschaft is gratefully acknowledged.

Forschungs-

88

FUCHS REFERENCES

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