Release of Ca2+ from heart and kidney mitochondria by peripheral-type benodiazepine receptor ligands

hr. J. Biochem. Vol. 23, No. 2, pp. 207-213, 1991 Printed in Great Britain. All rights reserved

0020-711X/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc

RELEASE OF Ca2+ FROM HEART AND KIDNEY MITO~HONDRIA BY PERIPHERAL-TIE BENZODIAZEPINE RECEPTOR LIGANDS RAFAELMORENO-UNCHEZ,‘* CONCEPCI~NBRAVO,’ JOSEFINA GUTIBRREZ,~AMY H. NEWMAN* and PETER K. CHIANG’

‘Departamento

de Bioquimica, Institute National de Cardiologia, Mexico, D.F. 014080, Mexico [Fax (905) 573-09261

2Walter Reed Army Institute of Research, Washington, DC 20307, U.S.A. (Received 14 May 1990) Abstract-1. The effect of the henzodiazepines Ro5-4864, AHN 086 and clonazepam on the release of CaZ+ from rat heart and kidney mitochondria was studied. 2. The peripheral-type benzodiazepines RoS-4864 and AHN 086 induced Ca2+ release which was

blocked by Mg2+ whereas the central-type benzodiazepine clonazepam was ineffective. 3. An associated collapse of membrane potential and swelling were also induced by AHN 086 in the presence of Ca*+. 4. However, no oxidation of pyridine nucleotides or increased rate or respiration were observed. 5. Release of S13+ was induced by AHN 086 in the absence of inorganic phosphate but not in its presence. 6. These data are discussed in the context of the current hypotheses on the mechanism of mitochondrial Ca*+ release.

INTRODUCTION

Peripheral type benzodiazepine receptors (PBR) have been characterized in central and peripheral tissues using radioligand binding and autoradiographic techniques (Braestrup and Squires, 1977a,b; De Souza et al., 1985; Anholt et at., 1985, 1986). The PBR are clearly different from the central ~~odiazepine receptors (CBR) and are not responsible for the mediation of anxiolytic and anticonvulsant activity of classical benzodiazepine drugs such as diazepam, flunitrazepam or clonazepam (Braestrup and Squires, 1977a,b; Tallman et al., 1980; Marangos et al., 1982; Schoemaker et al., 1983). The PBR are not associated with GABA-regulated anion channels and are found in discrete localization that differ from those of the CBR (Tallman et al., 1980; Marangos, 1982; Schoemaker, 1983; Anholt et ai., 1985, 1986). The ~~odi~~ine Ro5-4864 (~~hlorodiazepam) binds with nanomolar affinity to the PBR which contrasts with its micromolar affinity to CBR, and does not display classical benzodiazepine pharmacology (Braestrup and Squires, 1977a,b; Tallman et al., 1980; Marangos et al., 1982; Schoemaker et al., 1983). Subcellular localization and fractionation studies of the PBR suggest that mitochondria are the primary organelle that these receptors are associated with (Doble et al., 1985; Basile and Skolnick, 1986; O’Beirne and Williams, 1988; Antkiewi~-Mi~haluk et al., 1988; Mukherjee and Das, 1989). Based on the evidence

that the PBR ligands

*To whom all correspondence

inhibited

0, con-

should be addressed.

sumption of neuroblastoma cells it has been hypothesized that they play a role in cellular energy metabolism (Larcher et al., 1989). Indeed, compounds that bind to PBR inhibit oxidative phosphorylation of isolated mitochondria (Hirsch et al., 1989a; Newman et al., 1989) with potencies that correlate well with binding affinities (Hirsch et al., 1986). An association of the PBR with voltage sensitive calcium channels, in heart (Taft and Delorenzo, 1984; Mestre et al., 1985; Holck and Gsterrieder, 1985) and a lower affinity (micromolar) PBR may exist at the calcium channel (Taft and De Lorenzo, 1984). In addition, a recent study using the irreversible PBR ligand, AHN 086 (Lueddens et al., 1986; Newman et al., 1987), suggests that there may be a functional association between PBR and voltage operated calcium channels in the spon~eously beating guinea pig atria but not in the ileum (Bolger et al., 1989). It has been reported that diazepam, a centrally acting benzodiazepine, inhibits the Na+- induced release of Ca*+, in heart and brain mitochondria, with an IC, of 40 PM (Matlib and Schwartz, 1983). It should be noted that this effect could not be blocked by the CBR antagonist Rol5-1788, suggesting that this effect was not mediated through classical CBR. Since diazepam binds to PBR with nanomolar affinity (Braestrup and Squires, 1977a,b) and in view of the incomplete understanding of the physiological function of the PBR, it was of interest to study the effect of selective PBR ligands on mitochondrial Ca2+ transport. The present study examines the effects of the PBR ligand Ro5-4864 and AHN 086 on Ca2+ efflux in rat heart and kidney mitochondria.

207

RAFAEL MORENO-SANCHEZ et al.

208 MATERIALS AND METHODS

Preparation af mifochondria

Dr P. Skolnick, NIDDK, NIN, Bethesda. Md, Arsenazo III and Ruthenium Red were purchased from Sigma Chemical, and further purified.

Heart mitochondria were prepared from Wistar rats weighing 150-250 g by a nagarse method (Moreno-SBnchez

and Hansford, 1988). Kidney mitochondria were prepared as described previously for liver mitochondria (MorenoS&chez, 1985) except that the medium was slightIy modified f250 mM sucrose, 10 mM Hepes, 1mM EGTA, pH 7.4) and the aIbumin concentration during the incubation of the mitochondrial pellet was 1% (w/v). Mitochondrial protein concentration was determined by a biuret method in presence of 0.066% (W/Y)deoxycolate and using bovine serum albumin as standard. Ca ‘+ and Sr 2+ transport Uptake of 45CaZ+was done essentially as described previously (Moreno-Sgnchez, 1983). Mitochondria (0.2-0.3 mg protein/ml) were incubated in t ml of a standard medium comprising: I20 mM KCI, 25 mM MOPS, 5 mM K-phosphate, 10 mM succinate, 2.5 PM rotenone, pH 7.2 at 25°C. For the experiments performed with kidney mitochondria, the standard medium was supplemented with 0.3 mM ATP. After 1min of incubation, 50 p M 45CaC12(4000-7000 cpm/ nmol) was added. Then at predetermined times an aliquot of O.?ml was withdrawn and filtered through Millj~re filters of 0.45 pm of pore diameter. The filters were washed with 10 ml of ice-cold 150 mM KC1 and counted for radioactivity after solubilization with 7.5 ml of a scintillation liquid. Measurement of Ca*+ and Sr2+ fluxes was carried out by using the metallochromic indicator Arsenazo III (Scarpa et af., t978). M~t~hond~a were incubated in 2ml of standard medium in the presence of 50 p M Arsenazo III and the changes in absorbance difference at 685-675 nm were measured in an Aminco DW-2C spectrophotometer equipped for continuous gassing with 100% 4, thermostated to 25°C and under gentie stirring. Determination of the fransmembrune potential Mitochondria were incubated with 10pM safranine in 2 ml of standard medium and the absorbance difference at 51 l-533 nm was followed by dual spectrophotometry (Akerman and Wikstrom, 1976). Defermi~at~a~of fire redox state af pyrid~~-n~c~eof~des This parameter was determined spectrophotometrically by the absorbance difference at 340-370 nm of mitochondria incubated in 2 ml of standard medium under smooth stirring, continuous gassing with 100% O2 and 25°C. Determinaf~a~of mitacho~d~~l sweikag Mitochondria were incubated in 2 ml of standard medium and the change in absorbance at 520 nm was registered in a spectrophotometer.

RESULTS

E#ect ~~~e~z~d~aze~~~e~ on ~~ta~~~~dr~~~Ca2+ e#hAnalysis of Tabte 1 shows that AWN 086 induced a significant Ca*+ efflux from heart mitachondria that

was independent from the type of substrate used to support Ca*+ accumulation. The slightly higher release of Ca2+ induced by AHN 086 or Ro5-4864 in the presence of pyruvate + mafate might be related to the reported specific interaction of PBR with the pyruvate dehydrogenase enzyme complex (Daval et al., 1989). Ro5-4864 induced some release of Ca*+

with succinate as substrate when inorganic phosphate was omitted from the incubation medium: 11 and 17% Calf released for two different preparations. The percentage of Ca’+ release induced by AHN 086 was not modified by the omission of inorganic phosphate. The activation of the mitochondrial Ca*+ release appeared to be specific for PBR ligands since clonazepam did not modify the levels of accumulated Ca*+ (Table 1). An unspecific membrane perturbation by PBR ligands can also be discarded since clonazepam, a structurally similar benzodiazepine that binds with high affinity to CBR but does not recognize the PBR, at the same concentration, was unabIe to induce Ca’” release (Table 1). Release of Ca2+ was also specifically induced by PBR ligands from kidney mitochondtia incubated with succinate + rotenone: 63 + 12% Ca*+ released (E = 8) for 25 PM AHN 086 and 54 f i 7% (n = 6) for 25 FM Ro5-4864, vs 8 4 10% (n i= 6) for 25 p M clonazepam. Figure 1 shows the effect of increasing concentrations of AHN 086 on Ca2+ release in heart mitochondria oxidizing succinate, A near complete release of accumulated Ca2+ was induced with 50 p M AHN 086, whereas 50% release was accomplished with 18 pM AHN 086 for a mitochondrial protein concentration range of 0.2-0.3 mg/ml. This range of AHN 086 concentrations is significantly higher than that needed for binding of AHN 086 to classical peripheral receptors (Luddens et al., 1986). This suggests that other mitochondrial sites different to Table 1. Ca’+ release induced by benzodiazepines ~t~hond~a

Mitochondria were incubated in 3 ml of air saturated standard medium at 25X’. O2 uptake was recorded by means of an oxygen electrode (Yellow Springs Instrument Co.). The solubility of 0, in equilibrium with air was taken to be 420 ng atoms/ml at 25°C and 2240 m altitude. Presentatjo~ of data and statistical analysis The values shown in this paper represent means & SD with the number of preparations assayed indicated in parentheses. Where a significant difference was found a pairedsample Student’s f-test was used. Chem~eais AHN 086 was synthesized as described previously (Newman et al., 1987); fresh ethanolic solutions of this compound were used thoroughly this study. Ro5-4864 HCl was synthesized by A. H. N. Clonazepam was a gift from

+AHN 086

+ Clonazepam

% Ca2+ releasMi

Substrate Succ (+ Rote) Pyr+ Ma1 ATP (i-Rote)

+ Ro5-4864

in rat heart

54 f 9 (4)* 79+ 15(3)* 51 f 13(3)*

4 * 7 (3) IS k E(3)**

Oi:8(4) 7 + 6 (3)

M~t~bond~a were incubated in standard medium for I min before addition of 50 pM %a2*. After 4 min 25 p M of the indicated benzodiazepine was added. The amount of %a’” retained into mitochondria was measured by filtration of an aliquot 5 min latex as described in the Materials and Methods section. Symbols: Succ + rote, 10 mM succinate + 2.5 rM rotenone; &r + Mal. 5 mM pyruvate + 1mM malate; ATI’, 3mM, the amount of accumulated Cal+ aRer 9 min was: 69 f f4 nmoi/mg protein (n = 6) for succ + rot: 53 f 17 nmol/ mg protein (n = 3) for Pyr + mal; and 29 f 5 nmoljmg protein (n = 3) for ATP + rot. These values were of a similar magnitude for the 5 min paint. *P < 0.001 vs control; +*Pc 0.05.

209

Ca*+ release and henzodiazepines

looIi

0

I

I

I

I

t

10

20

30

40

50

ptd

CAHN

0863

Fig. I. Effect of different AHN 086 concentrations on the release of Ca*+ from heart mitochondria. The experimental conditions described in the legend of Table 1 were used, exceut that the indicated concentrations of AHN 086 were add&. The oxidizable substrate was succinate (+ rotenone). The dashed line represents a non-linear regression analysis for hyperbolic curve of the statistical means of each AHN 086 concentration used. Each point shows mean + SD of 4 different mitochondrial preparations. The amount of accumulated Ca*+ after 9 min was of 48.5 f 13 nmol/mg protein (n = 4).

PBR are involved in the benzodiazepine-induced Ca*+ release In an attempt to block the AHN 086-induced Ca2+ release several agents were tested (Table 2). Among the agents assayed only Mgr+ (5 mM) showed an inhibitory effect on the Ca*+ release induced by AHN 086. Higher concentrations of Mg*+ were not assayed. Other agents such as tetracaine, dithiotreitol and Ruthenium Red potentiated the release of Ca*+ induced by AHN 086. Agents as verapamil, ADP (+oIigomycin), 1 mM cysteine, 1 mM procaine, 5 PM La3+ or 0.5 mM diltiazem exhibited no effect on the AHN 086~induced Ca*+ release. The PBR antagonist, PK 11195 (50 PM) did not prevent the release of Ca*+ induced by 25 PM AHN 086. This observation is consistent with previous findings Table 2. Effect of various agents on Car+ release induced by AHN 086 in rat heart mitochondria

Agent AHN 086 1 mM Mg’+ +AHN 5 mM Mgr+ + AHN

5mM M2+ 0. I mM Verapamil-BAHN

0.1 mM Verauamil I mM ADP + 10 PM oligomycin- AHN I mM tetracaine + AHN I mM tetracaine 1mM dithiothreitol - AHN

1 mM dithiothreitol 0.5gM Ruthenium Red + AHN 0.5 gM Ruthenium Red

% Ca2+ released

Eflect of benzodiazepines on several mitochondrial parameters

The addition of AHN 086 to mitochondria loaded with Ca2+ induced a complete and fast collapse of the membrane potential (Fig. 2, trace d). However, AHN 086 did not modify the membrane potential in absence of Ca*+ (result not shown, but similar to Fig. 2, trace a). The addition of 25 FM Ro5-4864 did not affect the membrane potential in heart mitochondria; however, after a delay it induced a collapse of the membrane potential in kidney mitochondria (data not shown). Thus the sensitivity of the membrane potential to Ro5-4864 correlated well with the effect of RoS-4864 on Ca2+ release in both heart and kidney mitochondria. The collapse of the membrane potential induced by AHN 086 was prevented by the Ca*+ uniport inhibitors Ruthenium Red and Md+ (Fig. 2, traces a and b respectively). Because AHN 086 induced release of Ca*+ in the presence of Ruthenium Red, but did not, in the presence of Mg2+ (see Table 2), the results of Fig. 2 suggest that the collapse of the membrane potential was a consequence of an acceleration in the cycling of Ca*+ i.e.

58~11(11) 52 f 8 (4)

22 f 10 (7). 17 f i0(4j**

Table 3. Effect of pH on Car+ release induced by AHN 086 in rat heart mitochondria

46It4(3) 2 (21 \-,

65 (2) 82 f 5 (3)* 4 + 6 (3) 86 * 2 (4).

28(2) 75* 12(3)* 27 f 17 (6)***

The same experimental conditions described in the legend of Table 1 were used. The indicated concentrations of the agents were added 15sec before the addition of 25pM AHN 0.86. The amount of accumulated Ca*+ after 9min was of

61.3f 19nmol/mg protein (n = 13). *P < 0.01 vs AHN 086 alone; l*P < 0.05 vs control; ***P -c 0.001 vs control.

(Lueddens et al., 1986; McCabe et al., 1989) where PK 11195 did not block the irreversible binding of AHN 086. Mg*+ alone induced a slight spontaneous release of Ca*+ (Table 2). It is well established that M$+ competitively blocks the Ca2+ uniport, and that the level of intramitochondrial Ca*+ is determined by the rates of influx and efflux (Nicholls and Akerman, 1982). Thus, it would appear that 5mM Mg2+ was able to fully inhibit the Ca*+ release induced by AHN 086, i.e. 17% Ca2+ released with M$+ alone vs 22% with Mg*+ plus AHN (Table 2). Table 2 also shows that dithiothreitol and Ruthenium Red induce Ca2+ release. This latter data could account for the observed higher AHN 086-induced Ca2+ release in presence of these agents. The Ca*+ release induced by AHN 086 was strongly dependent on the pH value of the incubation medium (Table 3): AHN 086 was more effective at more acidic pH values. At this range of pH values a possible involvement of histidine residues in the action of AHN 086 has been suggested (Lueddens er al., 1986). However, in contrast to the present data, binding of AHN 086 to specific receptors was increased by increasing pH (Lueddens et al., 1986).

PH

+AHN 086 % Ca’+ released

6.7 7.2 7.7

79 +z 10 (5). 56 f 10 (5) 23 + 1I (5)’

Mitechondria were incubated as described in the legend of Table 1 except that the pH value of the medium was as indicated. The accumulated Ca*+ was 21 f 9 nmol/mg at pH6.7. 39k6 nmol/mg at pH 7.2, and 40.2nmol/ mg at pH 7.7. lf < 0.001 vs pH 7.2.

RAFAELMORENO-SANCHEZ et

210

al.

Mlt RR or Mg2+

Mit

AAbs \

L

0.02

200sec

(

Fig. 2. Effect of AHN 086 on the mitochondrial membrane potential in presence of several agents. Mitochondria (0.4mg protein/ml) were incubated with 1OpM safranine and the change in the absorbance difference at 51 l-533 nm was measured as described in the Materials and Methods section. Where indicated, Ca2+ was added to a final concentration of 50 PM. Other additions were 25 PM AHN 086, 0.5 PM Ruthenium Red (trace a), 5 mM Mg2+ (trace b), 1 mM tetracaine or 1 mM ADP + 10 PM oligomycin (trace c). Trace d was performed with AHN alone. CCCP, 1 FM. a Ca*+ efflux pathway being stimulated by AHN 086

and blocked by high M$+, but not by Ruthenium Red. The rate of respiration in presence of Ca’+ was 26 + 3% (n = 3) inhibited by AHN 086 in heart mitochondria, and 30 f 4% (n = 3) in kidney mitochondria. AHN 086 induced an extra swelling of mitochondria incubated in the presence of Ca2+ and inorganic phosphate (Fig. 3, trace c) which was inhibited by Ruthenium Red or Mg’+ (trace b). When phosphate or Ca*+ were omitted, no swelling induced by AHN 086 was observed (data not shown). However, Ca*+ was still released by AHN 086 in the absence of phosphate. This would indicate that Ca2+ release and mitochondrial swelling are not strict consequences of a general increase in inner membrane permeability as previously suggested (Bratrice et al., 1980, 1984), but two events which can be separated. The redox state of the pyridine-nucleotides was not modified by AHN 086 in presence of Ca2+ in heart mitochondria (not shown), neither by AHN 086 or Ro5-4864 in kidney mitochondria (not shown). Eflect of AHN 086 on eflux of 5” A comparative study on the effect of AHN 086 on Ca*+ and Srr+ fluxes in heart mitochondria is shown in Fig. 4. Trace A shows that AHN 086 induced a fast and complete release of Ca*+ in presence of phosphate; this result correlated with the data obtained by the filtration technique (see Table 1). However, AHN 086 was not able to induce Sr*+ release [Fig. 4(B)] in the presence of 5 mM inorganic phosphate; but AHN 086 could induce S13+ release from mitochondria incubated in the absence of phosphate

L

5min

Fig. 3. Effect of AHN 086 on mitochondrial swelling. Heart mitochondria (0.54 mg protein/ml) were incubated in 2 ml of standard medium and the changes in absorbance at 520 nm were measured. In trace a, only Ca*+ was added; in trace b, Ca*+, Ruthenium Red or Mg*+, and AHN 086 were added; in trace c, Ca*+ and AHN 086 were added. Additions: Ca*+, 50pM; AHN 086, 25yM; RR, 0.5pM Ruthenium Red; Mgr+, 5 mM; CCCP, 1 pM. RR or M&+ were added 15 set before AHN 086.

[Fig. 4(C)]. Sti’ appeared not to be irreversibly sequestered by inorganic phosphate in the mitochondrial matrix since the uncoupler CCCP induced a fast and complete release of ST*+ [Fig. 4(B)]. Similar experiments performed with higher concentrations of Sr*+ (125 PM) showed identical results to those of Fig. 4. DISCUSSION

The pharmacological action of benzodiazepines on mitochondrial Ca*+ release appeared to be specific for the PBR ligands since clonazepam, a central-type benzodiazepine, was unable to induce Ca*+ release. Specific receptors for peripheral-type benzodiazepines have been identified in mitochondria, mainly in the outer membrane fraction (Doble et al., 1985; Anholt et al., 1986; Basile and Skolnick, 1986; Antkiewicz-Michaluk et al., 1988). However, due to its effect on Ca*+ release, an interaction of peripheral-type benzodiazepines with the inner membrane or the mitochondrial matrix must not be discarded. To this regard it must be pointed out that binding of RoS-4864 to specific receptors from different membrane preparations was inhibited by the mitochondrial matrix pyruvate dehydrogenase enzyme complex (Daval et al., 1989). Evidence recently published (Mukherjee and Das, 1989) indicates that the PBR are located in the inner membrane of guinea pig lung mitochondria. Nevertheless the physiological role mitochondrial PBR remains unclear. The present studies reveal an interaction of PBR ligands with mitochondrial Ca*+ and Srr+ transport systems which might be mediated through a PBR mechanism. However, a specific involvement of PBR in the release of Ca2+ and Srr+ induced by AHN 086 and Ro5-4864 is still uncertain: the concentrations of AHN 086 required were 2-3 orders of magnitude higher than that needed for specific binding of AHN 086 to PBR (Lueddens et al., 1986). At these high concentrations

211

Cal+ release and bcnzodiazcpines

AHN

AHN

CCCP I

A 665-675nm AAbr-0.04

5min

Fig. 4. Effect of AHN 086 on Cal+ and S?+ fluxes in heart mitochondria. Mitochondria (0.55mg protein/ml) were incubated with arsenazo III and the changes in absorbance difference at 685-675 nm were measured as described in the Materials and Methods section. Other experimental conditions were: (A)

50 pM Cal+; (9) 50 PM Sti+; (C) 50 PM Sr’+ but phosphate was omitted. Abbreviations: AHN, 25 PM AHN 086; CCCP, 1 PM. there is nonspecific covalent binding to mitochondrial proteins (McCabe et al., 1989) by AHN 086. On the other hand the release of Ca2+ and collapse of membrane potential induced by AHN 086 and in a lesser magnitude by RoS-4864 could account for the reported effects of peripheral-type benzodiazepines on heart (Mestre et al., 1985; Grupp et al., 1987; Bolger ef al., 1989; Elgoyhen and Adler-Graschinsky, 1989) and renal function (Basile et al., 1988). The mechanism by which AHN 086 induces mitochondrial Ca*+ release is not completely clear at this moment. There are three models proposed in the literature to explain the mechanism of Ca2+ release as induced by several different agents: (a) activation of the inner membrane phospholipase A, by accumulated Ca2+, and decrease of the acyltransferase activity induced by the Ca2+-releasing agent. This results in a net increase of membrane lysophospholipids, and hence on increase in membrane permeability (Beatrice er al., 1984); (b) modification of the redox state of the inner membrane thiol groups by the Ca’+-releasing agent either by direct interaction or by inducing oxidation of pyridine-nucleotides which, in turn, avoids maintainance of the membrane thiol groups in a reduced state (Vercesi, 1984; Chavez and Holguin, 1988); (c) stabilization of the cytosolic conformation of the translocase of ATP/ADP by the Ca’+-releasing agent which induces an increase in membrane permeability (Le Quoc and Le Quoc, 1988). The data of the present work do not address adequately to any of the above-mentioned models of Ca2+ release. The phospholipase A,/acyltransferase (Beatrice et al., 1984) and the membrane thiol groups hypotheses (Vereesi, 1984; Chavez and

Holguin, 1988) postulate that an increased oxidation of pyridine-nucleotides should trigger the release of Ca2+. In the former hypothesis the redox state of pyridine-nucleotides is associated with the acyltransferase activity through the action of transhydrogenase and glutathione reductase. These latter enzymes would preserve, in a reduced state, essential thiol groups of the acyltransferase (Beatrice et al., 1984). However, AHN 086 was able to induce release of Ca2+, collapse of transmembrane potential and mitochondrial swelling, but did not modify substantially the redox state of pytidine-nucleotides. According to the phospholipase A,/acyltransferase hypothesis, our findings would be explained through an inhibition of the glutahione reductase activity; this would affect the acyltransferase activity remaining without modification the redox state of pyridine nucleotides. Measurements of glutathione reductase activity in the supematant of sonicated mitochondria (and centrifuged at lO5,OOOg), showed to be not affected by 25 PM of each benzodiazepine used in this work. A direct interaction of AHN 086 [the ethylisothiocyanate derivative of Ro5-4864, see Lueddens et al. (1986)] with essential thiol groups of specific enzymes (i.e. acyltransferase) or inner membrane proteins may be involved in the mechanism of Ca2+ release since isothiocyanate compounds react very quickly with thiols. However, ,313’ release was not induced by AHN 086 (in the presence of inorganic phosphate), as it expected from a genera1 increase in membrane permeability. The prior additon of dithiothreitol (1 mM), a thiol reducing agent, did not prevent the release of Ca2+ nor the collapse of membrane potential induced by AHN 086 [see Beatrice et al. (1984)). The third hypothesis of Ca’+ release postulates that incubation of mitochondria with ATP or ADP induces a stabilization of the matrix conformation of the translocator of ATP/ADP and hence a decreased

RAFAELMORENO-!XNCHEZ et al.

212

inner membrane permeability (Le Quoc and Le Quoc, 1988). However, AHN 086 was also able to induce release of Ca*+ from mitochondria in the presence of adenine nucleotides (ADP + oligomycin in heart and ATP in kidney mitochondrial). The effect of AHN 086 on Sr*+ release was dependent on the presence of inorganic phosphate. The diminution of free Sr*+ concentration in the mitochondrial matrix, due to the formation of soluble Sr-phosphate complexes, could be involved in the lack of action on Sr*+ release by ANH 086 when inorganic phosphate was added. This suggests that the efflux pathway induced by AHN 086 has a lower affinity for Sr*+ than for Ca*+. Alternatively a mechanism involving release of Ca*+ through two different pathways has been also proposed (Chavez et al., 1989). In the context of the present data AHN 086 could be activating the two Ca*+ efflux pathways, but the pathway specific for Sti+ would be antagonized by inorganic phosphate. This is in line with recent reports indicating that mitochondrial swelling promoted by Ca*+ (but not Sti’) and inorganic phosphate is associated with an increase in K+ permeability (Halestrap, 1989) or an opening of a pore in the inner membrane (Crompton et al., 1988). Acknowledgements-The authors thank helpful discussions with Dr E. Chavez. The collaboration of Mr Florencio

Hernandez in drawing the figures and Mrs Conception Rivera in typing the manuscript are acknowledged.

Braestrup C. and Squires R. F. (1977b) Specific benzodiazepine receptors in rat brain characterized by high affinity [3H]diazepam binding. Proc. natn. Acad. Sci. U.S.A. 74, 3805-3809.

Chavez E. and Holguin J. A. (1988) Mitochondrial calcium release as induced by Hg2+. J. biol. Chem. X3,3582-3587. Chavez E., Zazueta C., Diaz E. and Holguin J. A. (1989) Characterization by Hg2+ of the two different pathways for mitochondrial Ca2+ release. Biochim. Biophys. Acta 986, 27-32

Crompton M., Ellinger H. and Costi A. (1988) Inhibition by ciclosporina A of a Ca”+-dependent pore in heart mitochondira activated by inorganic phosphate and oxidative stress. Biochem. J. 255, 357-360. Daval J. L., Post R. M. and Marangos P. J. (1989) Pyruvate dehydrogenase interactions with peripheral-type benzodiazepine receptors. J. Neurochem. 52, 1 l&l 16. DeSouza E. B., Anholt R. R. H., Murphy K. M. M., Snyder S. H. and Kuhar M. J. (1985) Peripheral-type benzodiazepine receptor in endocrine organs: autoradiographic localization in rat pituitary, adrenal and testis. Endocrinology 116, 567-573.

Doble A., Benavides J., Ferris O., Bertrand P., Menanger J., Vaucher N., Burgevin M. C., Uzan A., Gueremy C. and Le Fur G. (1985) Dihydropyridine and peripheral type benzodiazepine binding sites: subcellular distribution and molecular site determination. Eur. J. Pharmac. 119, 153-167.

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Grupp I. L., Frevich J. F. and Matlib M. A. (1967) Benzodiazepine RoS-4864 increases coronary flow. Eur. J. Pharmac. 143, 1433147.

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