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ATP translocator
Journal of Photochemistry
and Photobiology,
B: Biology, 4 (1989) 35 - 46
35
HAEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN II) PHOTOSENSITIZATION OF ISOLATED MITOCHONDRIA: INHIBITION OF ADP/ATP TRANSLOCATOR A. ATLANTE,
S. PASSARELLA+
and E. QUAGLIARIELLO
Dipartimento di Biochimica e Biologia Molecolare and Centro di Studio sui Mitocondri e Metabolismo Energetic0 CNR, Universitd di Bari, Via Amendola 165/A, 70126 Bari (Italy) G. MORENO and C. SALET Laboratoire de Biophysique, INSERM U. 201, CNRS UA. 481, Mu&urn National d’Hi.stoire Naturelle, 43 rue Cuvier, 75231 Paris CBdex 05 (France) (Received October 31,1988;
accepted January 15,1989)
Keywords: Haematoporphyrin derivative, photosensitization, ADP/ATP translocator, oxidative phosphorylation.
mitochondria,
Summary To gain further insight into the mechanism by which irradiation of mitochondria in the presence of haematoporphyrin derivative (Photofrin II) (PF II) causes impairment of mitochondrial oxidative phosphorylation, the rate of ADP/ATP exchange via the ADP/ATP translocator was measured fluorometrically in isolated rat liver mitochondria. In accord with noncompetitive inhibition, PF II photosensitization decreases the maximum rate (20.8 and 9.6 nmol ATP effluxed min-’ X mg protein in of exchange V,, the control and after 2 min irradiation, respectively) without changing the ADP affinity for the carrier (K, = 5 PM in both cases). Comparison of the rate of oxygen uptake by mitochondria stimulated by either ADP or by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) confirms that the adenine nucleotide carrier is a major target of photodynamic action which causes oxidative phosphorylation impairment.
1. Introduction Haematoporphyrin derivative (HPD) and its purer version, Photofrin II (PF II), are efficient photosensitizers used in photodynamic therapy of
+Author to whom correspondence loll-1344/89/$3.50
should be addressed. @ Elsevier Sequoia/Printed
in The Netherlands
36
various malignant tumours [l]. Although photoalteration of different cell organelles has been reported [2,3], mitochondria seem to be the primary target of PF II photodamage leading to cell lethality [4, 51. In fact, in tumoural cells in tissue culture [6 - 81 or in situ [9 - 111, depletion of the intracellular ATP pool was found, probably as a result of inhibition of mitochondrial functions owing to porphyrin-induced photosensitization. However, mitochondrial functions, such as respiration, coupling of oxidative phosphorylation and Ca2+ ion transport, have been shown to be impaired in isolated mitochondria irradiated in the presence of various porphyrins [12 - 141. Moreover, in tumour mitochondria, inhibition of mitochondrial components owing to HPD photosensitization has been observed for cytochrome c oxidase (E.C. 1.9.3.1) [15], succinic dehydrogenase (E.C. 1.3.99.1) [16], adenosine triphosphatase (EC. 3.6.1.3) [17,18], adenylate kinase (E.C. 2.7.4.3) [18,19] and monoamine oxidase (E.C. 1.4.3.4) [ 191. Recently the impairment of dicarboxylate, tricarboxylate and oxodicarboxylate carriers has also been reported in rat liver mitochondria irradiated with 365 nm light in the presence of PF II [20]. Nevertheless, the mitochondrial primary molecular target repsonsible for the impairment of the mitochondrial functions by porphyrin photosensitization is at present unknown. Thus, since most of the tested enzymes [ 15 - 181 play a major role in the main mitochondrial functions, i.e. electron transfer and oxidative phosphorylation, and in the light of previous results [13,14, 201, further investigation of the effect of PF II treatment of isolated mitochondria seems worthwhile. In particular, investigation of the effect of PF II treatment on the activity of the ADP/ATP carrier merits special attention as ATP cell availability is strictly related to the activity of this carrier. The ADP/ATP translocator proves to be a major target of photodamage induced by irradiation of isolated mitochondria with 365 nm light in the presence of PF II. As a result of ADP/ATP antiport inhibition and the impairment of oxidative phosphorylation, mitochondrial damage and cell death are likely to occur. 2. Materials and methods 2.1. Chemicals All the reagents were of the purest grade available and were used without further purification. All solutions were adjusted to pH 7.4 by addition of either Tris or HCI. Haematoporphyrin derivative (Photofrin II) was generously given by Photomedica, Inc., Raritan, NJ. 2.2. Isolation and irradiation of mitochondria Rat liver mitochondria (RLM) were isolated as previously described [21] and the final mitochondrial pellet was suspended in 0.25 M sucrose to give a concentration between 80 and 100 mg ml-’ of protein, as measured according to Waddel and Hill [22].
Mitochondria (0.5 mg protein ml-‘) were irradiated in a thermostatted (7’ = 30 “C) water-jacketted glass vessel fitted for oxygen measurements with a Clark electrode (Yellow Spring Instruments). The irradiation source was a Philips HPW 125 W lamp emitting at 365 nm. The power density at the level of the vessel was 30 W me2 as measured by a Black-Ray UV meter (J 221).
2.3. Fluorometric measurements of ADP/ATP efflux The measurements were made essentially as previously reported [23]. Briefly, mitochondria (0.5 mg ml-‘) were incubated in 2 ml of standard medium in the cuvette of an LS-5 Perkin-Elmer luminometer. An ATP detecting system (ADS), consisting of glucose (2.5 mM), hexokinase (E.C. 2.7.1.1.) (1 E.U.), glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49.) (0.5 E.U.) and NADP+ (0.2 mM), was added. NADPH formation in the extramitochondrial phase, which reveals ATP appearance due to externally added ADP, was followed fluorometrically with excitation and emission wavelengths of 334 nm and 456 nm respectively. The exchange reaction was started by adding ADP. The initial concentration of intramitochondrial pyridine nucleotide was equalized to zero. Checks were made to verify that in the absence of NADP+ no significant change of fluorescence occurred during the experiment. The rate of fluorescence increase expressed as nmol NADP reduced min-’ X mg protein obtained as a tangent to the initial part of the curve proves to be the rate of ADP/ATP exchange [23] (expressed as nmol ATP effluxed mini X mg protein). Calibration of NADPH fluorescence was made according to refs. 24 and 25.
2.4. Measurements of oxygen uptake Mitochondria (0.5 mg ml-‘) were incubated at 30 “C in a standard medium consisting of 87 mM sucrose, 60 mM KCl, 10 mM MgC12, 1.35 mM inorganic phosphate and 24 mM glycylglycine at pH 7.4 containing 2 pg rotenone. When present, PF II was added in the medium at the concentration of 6 pg ml-’ giving an optical density of 0.8 cm-’ at the irradiation wavelength of 365 nm. Experiments were typically performed under the following conditions: (a) untreated RLM kept in the dark; (b) untreated RLM irradiated; (c) PF II treated RLM kept in the dark; (d) PF II treated and irradiated RLM. Oxygen uptake was started by the addition of succinateTris (1.35 mM) followed by either ADP (1.35 mM) or FCCP (1.30 PM). The rate of oxygen uptake stimulated either by FCCP (V,,,,) or ADP (V,,,) was obtained as a tangent at the initial part of the curve and expressed as natoms O2 min-’ X mg protein. The respiratory control ratio (RCR), i.e. (the rate of oxygen uptake after either ADP or FCCP addition)/ (the rate of oxygen uptake before either ADP or FCCP addition), was also determined.
38
2.5. Measurement
of ATPase activity
ATPase activity was followed as in refs. 26 and 27 by measuring mitochondrial swelling which occurs when ATP hydrolysis is elicited in mersalyltreated mitochondria added with ATP. Mersalyl(20 nmol mg-’ protein) was added to the mitochondrial suspension after the treatment with PF II alone, light alone or PF II plus light. After 1 min, 2 mM ATP were added and, after 1 min, ATP hydrolysis was induced by adding 1.30 PM FCCP. 3. Results and discussion To investigate the effect of PF II photosensitization on the ADP/ATP carrier, an experimental approach was used which allows measurements of ADP uptake in exchange with intramitochondrial ATP (ADP/ATP antiport) in RLM without preventing mitochondrial respiration and ATP synthesis. This was done by continuously monitoring ATP efflux caused by ADP uptake and phosphorylation. As described in Scheme 1, ADP enters __.-.-._.-._.-.-.-.-.-.-._ EXTRAHITKHONDRIAL PHASE
MATRIX
GLUCOSE
Scheme 1.
mitochondria in exchange with endogenous ATP via the ADP/ATP translocator. Inside mitochondria, ADP is phosphorylated to ATP which in turn exchanges for further ADP. Outside mitochondria, ATP appearance is revealed by NADPH formation via hexokinase and glucose-6-phosphate dehydrogenase (see Section 2 and ref. 23). The ATP concentration in the phase outside mitochondria is negligible since no change in NADPH fluorescence is found. Following ADP (5 PM) addition, a rapid increase of fluorescence is observed, which shows the appearance of ATP outside mitochondria (Fig. l(A)). As expected [23], the inhibition of NADPH formation by carboxyatractyloside, a powerful inhibitor of the ADP/ATP translocator [28], was found, whereas no effect was observed in the presence of P,P,diadenosine [5-pentaphosphate] (ApsA), a specific inhibitor of adenylate kinase [29] (Fig. l(B)). In a previous paper [23], it has been reported that this method measures the rate of ADP/ATP exchange in mitochondria. However, it is possible that the observed inhibition of the rate of NADPH formation might also be
39
E
C
/ 102
:; AOSeDP
ADSAW
1
t
ADS PJF
Fig. 1. ADP/ATP carrier activity in control RLM and RLM treated with PF II and light. RLM (0.5 mg ml-i) were suspended at 30 “C in 2.0 ml of standard medium consisting of 87 mM sucrose, 60 mM KCI, 10 mM MgCls, 1.35 mM inorganic phosphate and 24 mM glycylglycine (pH 7.4), in the absence ((A), (B), (D)) or in the presence ((C), (E)) of PF II and irradiated for 2 min ((C), (D)). After ADS addition, the indicated compounds were added at the following concentration: 5 PM ADP; 10 @I ApsA; 0.5 @I carboxyatractyloside (CAT).
due to the added ADS and/or the intramitochondrial reactions directly related to oxidative phosphorylation. The first possibility was excluded by directly testing the effect of PF II plus light on the ADS both in the absence of mitochondria and in the presence of mitochondria added with ATP (not shown). The distinction between the translocation step and the intramitochondrial reactions will be made below. The nature of the inhibition was studied by investigating the dependence of the rate of fluorescence increase as a function of ADP concentration in control RLM and RLM treated with PF II and light (Fig. 2(A)). A purely non-competitive inhibition was found with a K, value (i.e. the ADP concentration which gives half-maximum rate of exchange) equal to 5 PM, and a Ki value (i.e. the irradiation time which gives 50% inhibition) equal to 100 s. As a result of PF II photodynamic action, a 54% decrease in the V of the transport was found following 2 min irradiation. When these ex;Trimental data were replotted as l/u, expressed as nmol NADP reduced min-’ X mg us. the irradiation time in a Dixon-like plot (Fig. 2(B)), straight lines were obtained. Statistical analysis shows that the intercepts to the ordinate axis, obtained by treating the experimental points according to linear regression, coincide perfectly with the corresponding experimental controls. This clearly shows that the measured inhibition is related to the
40
A
B 0.4
0.4 +
I
1 V
./
/
1
0.3
03
a2
02
01
01
.
P I
0
025 l/[ADPj IJIM-
0.50
A
P I 0
1 TIME
2 iminl
Fig. 2. Nature of the inhibition of the activity of the ADP/ATP carrier by PF II and irradiation. Experimental conditions as in Fig. 1. (A) Double reciprocal plot: dependence of the rate of exchange as a function of the ADP concentration was investigated in control RLM (0) and PF II-treated RLM irradiated for 30 s (A), 1 min (0) and 2 min (A), (B) Dixon plot: rate data of Fig. 2(A) replotted as a function of irradiation time. The ADP concentrations are 20 PM (o), 10 PM (A), 5 PM (X), 2.5 @I (A) and 2 PM (0). In both cases the rates of the uptake were obtained as a tangent to the initial part of the progress curve and expressed in nmol ATP effluxed min-’ X mg protein.
same parameter measured in the control, i.e. the rate of ADP/ATP exchange (see ref. 23). It should be noted that under these experimental conditions the energy for ATP synthesis is apparently given by the oxidation of endogenous substrates. In the light of the role played by the ADP/ATP carrier in the oxidative phosphorylation [ 301, the effect of PF II photosensitization on mitochondrial respiration was investigated to ascertain whether the inhibition of the ADP/ATP translocator could be considered as a primary cause of the impairment of oxidative phosphorylation [ 4, 5,121. As described in Scheme 2, succinate oxidation by isolated mitochondria stimulated by externally added ADP derives from several steps in which different possible targets of PF II photosensitization could be involved: (a) succinate transport across the inner mitochondrial membrane via the dicarboxylate translocator in exchange of endogenous phosphate; (b) succinate dehydrogenase activity; (c) electron n+ generation; (d) ADP transport flow along the respiratory chain and AE.C across the inner mitochondrial membrane via the ADP/ATP translocator; (e) A/J~+ utilization and ATP synthase activity. It should be noted that when oxygen uptake is measured after stimulation by an uncoupler, steps (d) and (e) are excluded. Most steps are inhibited in mitochondria after treatment with HPD and irradiation [ 15 - 181. For instance succinate dehydrogenase and cytochrome oxidase impairment could affect both electron flow in the respiratory chain and ATP synthesis [15,16]. Measurements of the rate of oxygen uptake stimulated by succinate plus rotenone were carried out under different experimental conditions.
41 OUT I
I
Pi
ATP .
\
IN OH-
ATP I
STEP
A
STEP
B
STEP
C
1
0
STEP
E
STEP
AOP
lAOP
1
Scheme 2.
In the first set of experiments, ADP was used to stimulate the oxygen uptake due to externally added succinate (1.35 mM), thus allowing for ATP synthesis (ATP conditions) (Fig. 3(A)). These conditions may reflect the physiological situation in duo. ADP increased the rate of oxygen uptake in untreated RLM: 206 natoms O2 min-’ X mg protein with an RCR value equal to 3(a). The same increase was observed upon exposure of RLM to either PF II (b) or 365 nm light (c), whereas about 60% inhibition of oxygen uptake (corresponding to an RCR value equal to 1.2) was found following irradiation for 30 s in the presence of PF II (d). In the second set of experiments, carried out with the same mitochondrial preparation, the uncoupler FCCP was used instead of ADP to stimulate succinate (1.35 mM) oxidation in the absence of ATP synthesis (uncoupling conditions) (Fig. 3(B)). Once more, PF II (b) or 365 nm light (c) alone did not significantly affect the rate of oxygen uptake compared with the control (a) (u = 234 natoms O2 min-’ X mg protein), but about 50% inhibition was observed when RLM were treated with PF II and irradiated for 30 s (d) (u = 110 natoms O2 min-’ X mg protein), the value of RCR being 1.6. These results confirm that besides the ADP/ATP carrier, other processes are also inhibited by PF II plus light. By plotting the percentage of stimulation of the rate of oxygen uptake by either ADP or FCCP us. irradiation time (Fig. 4), a lower photosensitivity of FCCP-stimulated respiration was found. However, it should be noted that, under uncoupling conditions, the absolute rates of oxygen uptake were in any case higher than those found under ATP
42
a
I
100 natoms
C
b
d
02
B
\
\
Fig. 3. Effect of PF II and light on the succinate oxidation by RLM stimulated by either ADP or FCCP. RLM (0.5 mg ml-‘) were suspended at 30 “C in 1.5 ml of the standard medium, in the absence ((a), (c)) or in the presence ((b), (d)) of PF II and irradiated (hy) for 30 s ((c), (d)). At the arrows, additions were made at the following concentration: 2 pg rotenone (ROT); 1.35 mM succinate (SUCC); 1.5 mM ADP (in (A)), 1.3 /.&I FCCP (in (B)). Numbers along the curves are the rates of oxygen uptake expressed in natoms 0s min-’ X mg protein.
0
10 IRRADIATION
30
20 TIME
ISI
Fig. 4. Dependence of stimulation of oxygen uptake vs. duration of the photodynamic treatment of RLM. Experimental conditions as in Fig. 3. Succinate (1.35 mM) oxidation was stimulated by the addition of either 1.35 mM ADP (0) or 1.30 FM FCCP (0) to control RLM and RLM treated with PF II and irradiated with different doses of light. Stimulation is given as the following ratio: (RCR of RLM treated with PF II and light -l)/(RCR of control RLM -1).
43
ATP
FCCP
1
1
ATP FCCP 1
I
ATP
1
FCCP 1
ATP
FCCP
I
I
110
112
1
:
C
0
Fig. 5. Effect of PF II plus light on the ATPase activity. ATP hydrolysis induced by externally added FCCP in control RLM and RLM treated with PF II and irradiated at 365 nm was followed as reported in Section 2. Irradiation times were 0 s (A), 10 s (B), 20 s (C) and 30 s (D). At the arrows, additions were made at the indicated concentration: 2 mM ATP; 1.3 PM FCCP. Absorbance changes at 546 nm were continuously recorded with a Perkin-Elmer Lambda 5 spectrophotometer.
conditions. As a result of these findings, steps other than those of ADP/ATP exchange via the specific translocator are excluded as being rate limiting in mitochondrial respiration following PF II photosensitization. In fact, if the dicarboxylate carrier, succinate dehydrogenase etc., were more sensitive to PF II plus light treatment than the ADP/ATP carrier, the higher sensitivity under ATP conditions would not be observed and the rate of oxygen uptake in the presence of PF II and 365 nm light under both ATP and uncoupler conditions would be the same. To assess whether ATPase is impaired under the reported experimental conditions, the sensitivity of mitochondrial ATPase to PF II and light was investigated by testing whether the photosensitization of RLM prevents FCCP-stimulated swelling of mersalyl-treated mitochondria added with 2 mM ATP (Fig. 5). This swelling has been reported to occur when phosphate and ADP formation (caused by ATP hydrolysis via ATPase stimulated by FCCP) takes place in the matrix of mitochondria in which the phosphate carrier is inhibited by its powerful inhibitor mersalyl [31]. No significant change in the rate of mitochondrial swelling with respect to the control (Fig. 5(A)) was found after irradiation for 10, 20 or 30 s (Figs. 5(B) - 5(D)), thus showing that there is no inhibition of mitochondrial ATPase under experimental conditions in which impairment of ADP/ATP translocator is found, as previously reported by Salet et al. [14].
44
4. Conclusion Evidence is given in this paper that photodynamic treatment of isolated mitochondria can inhibit the ADP/ATP carrier. As reported for dicarboxylate, tricarboxylate and oxodicarboxylate translocators, a non-competitive inhibition was found, showing that the photodynamic effect involves the carrier activity rather than the substrate affinity. Interestingly, the ADP/ATP translocator proves to be the mitochondrial molecular target whose inhibition by PF II photosensitization impairs ADP-stimulated succinate oxidation in RLM. How cell death in both normal and cancer cells depends on mitochondrial ADP/ATP translocator impairment remains to be established. However, as far as mitochondria are concerned it is worthwhile stressing that even if a number of enzymes are inhibited by porphyrin photosensitization [15 - 191, their inhibition plays a minor role in the impairment of oxidative phosphorylation. Finally, since mitochondria constitute an important if not a primary target of PF II cell photosensitization [ 32, 331, ADP/ATP translocator could be considered as a primary cellular target in studies designed to find out how cell death can occur in photodynamic therapy in duo.
Acknowledgments This work was partially supported by an exchange programme “Cooperation France-Italienne en Recherche Biomedicale INSERM-CNR”. The helpful cooperation of Dr. Angela Ostuni is gratefully acknowledged.
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45 9 T. L. Ceckler, R. G. Bryant, D. P. Penney, S. L. Gibson and R. Hilf, “P-NMR spectroscopy demonstrates decreased ATP levels in uiuo as an early response to photodynamic therapy, Biochem. Biophys. Res. Commun., 140 (1986) 273 - 279. 10 R. Hilf, S. L. Gibson, D. P. Penney, T. L. Ceckler and R. G. Bryant, Early biochemical responses to photodynamic therapy monitored by NMR spectroscopy, Photochem. Photobiol, 46 (1987) 809 - 817. 11 M. Chopp, H. Farmer, F. Hetzel and A. P. Shaap, in uiuo 31P-NMR spectroscopy of mammary carcinoma subjected to subcurative photodynamic therapy, Photochem. Photobiol., 46 (1987) 819 - 822. 12 S. Sandberg and I. Romslo, Porphyrin-sensitized photodynamic damage of isolated rat liver mitochondria, Biochim. Biophys. Actu, 593 (1980) 187 - 195. 13 C. Salet and G. Moreno, Photodynamic effects of hematoporphyrin on respiration calcium uptake in isolated mitochondria, Znt. J. Radiut. Biol., 39 (1981) 227 - 230. 14 C. Salet, G. Moreno and F. Vinzens, Effects of photodynamic action on energy coupling of Ca2+ uptake in liver mitochondria, Biochem. Biophys. Res. Commun., 115 (1983) 76 - 81. 15 S. L. Gibson and R. Hilf, Photosensitization of mitochondrial cytochrome c oxidase by hematoporphyrin derivative and related porphyrins in vitro and in uiuo, Cancer Res., 43 (1983) 4191 - 4197. 16 R. Hilf, D. B. Smail, R. S. Murant, P. B. Leakey and S. L. Gibson, Hematoporphyrin derivative-induced photosensitivity of mitochondrial succinate dehydrogenase and selected cytosolic enzymes of R3230AC mammary adenocarcinomas of rats, Cancer
Res., 44 (1984) 1483 - 1488. 17 D. S. Perlin, R. S. Murant, S. L. Gibson
and R. Hilf, Effects of photosensitization by derivative on mitochondrial adenosine triphosphatase-mediated and membrane integrity of R3230AC mammary adenocarcinomas, Cancer Res., 45 (1985) 4323 - 4328. N. K. Fu, S. Y. Yeh, C. Chang, X. H. Zhao and L. S. Chang, Photosensitization of mitochondrial adenosine-triphosphatase and adenylate kinase by hematoporphyrin derivative in vitro, Adu. Exp. Med. Biol., 193(1985) 161 - 167. R. S. Murant, S. L. Gibson and R. Hilf, Photosensitizing effects of photofrin II on the site-selected mitochondrial enzymes adenylate kinase and monoamine oxidase, Cancer Res., 47 (1987) 4323 - 4328. A. Atlante, G. Moreno, S. Passarella and C. Salet, Hematoporphyrin derivative (Photofrin II) photosensitization of isolated mitochondria: impairment of anion translocation, Biochem Biophys. Res. Commun., 141 (1986) 584 - 590. C. Salet, G. Moreno and F. Vinzens, Photodynamic effects induced by furocoumarins on a membrane system. Comparison with hematoporphyrin, Photochem. Photobiol., 36 (1982) 291- 296. W. J. Waddel and C. Hill, A simple ultraviolet spectrophotometric method for the determination of protein, J. Lab. Chn. Med., 48 (1956) 311 - 314. S. Passarella, A. Ostuni, A. Atlante and E. Quagliariello, Increase in the ADP/ATP exchange in rat liver mitochondria irradiated in uitro by helium-neon laser, Biochem. Biophys. Res. Commun., 156 (1988) 978 - 986. J. A. Gimpel, E. J. De Haan and J. M. Tager, Permeability of isolated mitochondria to oxaloacetate, Biochim. Biophys. Acta, 292 (1973) 582 - 591. S. Passarella, A. Atlante, M. Barile and E. Quagliariello, Anion transport in rat brain mitochondria: fumarate uptake via the dicarboxylate carrier, Neurochem. Res., 12 hematoporphyrin proton transport
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24 25
(1987) 255 - 264. 26 M. Klingenberg, R. Durand
and B. Guerin, Analysis of the reactivity of SH-reagents with the mitochondrial phosphate carrier, Eur. J. Biochem., 42 (1974) 135 - 150. 27 G. D’Arcangelo, M. Barile, S. Passarella and E. Quagliariello, Uncoupling of mitochondrial oxidative phosphorylation by hexetidine, Biochem. Biophys. Res. Commun., 147 (1987) 801 - 808. 28 F. K. LaNoue and A. C. Schoolwerth, Metabolite transport in mammalian mitochondria. In Bioenergetics, Elsevier, Amsterdam, 1984, pp. 221 - 261.
46 29 G. E. Lienhard and I. I. Secemski, Pi, P5-Di (adenosine-5’) pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase, J. Biol. Chem., 248 (1973) 1121 - 1123. 30 N. G. Forman and D. F. Wilson, Dependence of mitochondrial oxidative phosphorylation on activity of the adenine nucleotide translocase, J. Biol. Chem., 258 (1983) 8649 - 8655. 31 A. Fonyo, F. Palmieri, J. Ritvay and E. Quagliariello, Kinetics and inhibitor sensitivity of the mitochondrial phosphate carrier. In G. F. Azzone, M. E. Klingenberg, E. Quagliariello and N. Siliprandi (eds.), Membrane Proteins in Transport and PhosphoryZation, North-Holland, Amsterdam, 1974, pp. 283 - 286. 32 G. Moreno and C. Salet, Cytotoxic effects following micro-irradiation of cultured cells sensitized with haematoporphyrin derivative, Int. J. Radiat. Biol., 47 (1985) 383 - 386. 33 G. Singh, W. P. Jeeves, B. C. Wilson and D. Jang, Mitochondrial photosensitization by photofrin II, Photochem. PhotobioL, 46 (1987) 645 - 649.
and Photobiology,
B: Biology, 4 (1989) 35 - 46
35
HAEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN II) PHOTOSENSITIZATION OF ISOLATED MITOCHONDRIA: INHIBITION OF ADP/ATP TRANSLOCATOR A. ATLANTE,
S. PASSARELLA+
and E. QUAGLIARIELLO
Dipartimento di Biochimica e Biologia Molecolare and Centro di Studio sui Mitocondri e Metabolismo Energetic0 CNR, Universitd di Bari, Via Amendola 165/A, 70126 Bari (Italy) G. MORENO and C. SALET Laboratoire de Biophysique, INSERM U. 201, CNRS UA. 481, Mu&urn National d’Hi.stoire Naturelle, 43 rue Cuvier, 75231 Paris CBdex 05 (France) (Received October 31,1988;
accepted January 15,1989)
Keywords: Haematoporphyrin derivative, photosensitization, ADP/ATP translocator, oxidative phosphorylation.
mitochondria,
Summary To gain further insight into the mechanism by which irradiation of mitochondria in the presence of haematoporphyrin derivative (Photofrin II) (PF II) causes impairment of mitochondrial oxidative phosphorylation, the rate of ADP/ATP exchange via the ADP/ATP translocator was measured fluorometrically in isolated rat liver mitochondria. In accord with noncompetitive inhibition, PF II photosensitization decreases the maximum rate (20.8 and 9.6 nmol ATP effluxed min-’ X mg protein in of exchange V,, the control and after 2 min irradiation, respectively) without changing the ADP affinity for the carrier (K, = 5 PM in both cases). Comparison of the rate of oxygen uptake by mitochondria stimulated by either ADP or by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) confirms that the adenine nucleotide carrier is a major target of photodynamic action which causes oxidative phosphorylation impairment.
1. Introduction Haematoporphyrin derivative (HPD) and its purer version, Photofrin II (PF II), are efficient photosensitizers used in photodynamic therapy of
+Author to whom correspondence loll-1344/89/$3.50
should be addressed. @ Elsevier Sequoia/Printed
in The Netherlands
36
various malignant tumours [l]. Although photoalteration of different cell organelles has been reported [2,3], mitochondria seem to be the primary target of PF II photodamage leading to cell lethality [4, 51. In fact, in tumoural cells in tissue culture [6 - 81 or in situ [9 - 111, depletion of the intracellular ATP pool was found, probably as a result of inhibition of mitochondrial functions owing to porphyrin-induced photosensitization. However, mitochondrial functions, such as respiration, coupling of oxidative phosphorylation and Ca2+ ion transport, have been shown to be impaired in isolated mitochondria irradiated in the presence of various porphyrins [12 - 141. Moreover, in tumour mitochondria, inhibition of mitochondrial components owing to HPD photosensitization has been observed for cytochrome c oxidase (E.C. 1.9.3.1) [15], succinic dehydrogenase (E.C. 1.3.99.1) [16], adenosine triphosphatase (EC. 3.6.1.3) [17,18], adenylate kinase (E.C. 2.7.4.3) [18,19] and monoamine oxidase (E.C. 1.4.3.4) [ 191. Recently the impairment of dicarboxylate, tricarboxylate and oxodicarboxylate carriers has also been reported in rat liver mitochondria irradiated with 365 nm light in the presence of PF II [20]. Nevertheless, the mitochondrial primary molecular target repsonsible for the impairment of the mitochondrial functions by porphyrin photosensitization is at present unknown. Thus, since most of the tested enzymes [ 15 - 181 play a major role in the main mitochondrial functions, i.e. electron transfer and oxidative phosphorylation, and in the light of previous results [13,14, 201, further investigation of the effect of PF II treatment of isolated mitochondria seems worthwhile. In particular, investigation of the effect of PF II treatment on the activity of the ADP/ATP carrier merits special attention as ATP cell availability is strictly related to the activity of this carrier. The ADP/ATP translocator proves to be a major target of photodamage induced by irradiation of isolated mitochondria with 365 nm light in the presence of PF II. As a result of ADP/ATP antiport inhibition and the impairment of oxidative phosphorylation, mitochondrial damage and cell death are likely to occur. 2. Materials and methods 2.1. Chemicals All the reagents were of the purest grade available and were used without further purification. All solutions were adjusted to pH 7.4 by addition of either Tris or HCI. Haematoporphyrin derivative (Photofrin II) was generously given by Photomedica, Inc., Raritan, NJ. 2.2. Isolation and irradiation of mitochondria Rat liver mitochondria (RLM) were isolated as previously described [21] and the final mitochondrial pellet was suspended in 0.25 M sucrose to give a concentration between 80 and 100 mg ml-’ of protein, as measured according to Waddel and Hill [22].
Mitochondria (0.5 mg protein ml-‘) were irradiated in a thermostatted (7’ = 30 “C) water-jacketted glass vessel fitted for oxygen measurements with a Clark electrode (Yellow Spring Instruments). The irradiation source was a Philips HPW 125 W lamp emitting at 365 nm. The power density at the level of the vessel was 30 W me2 as measured by a Black-Ray UV meter (J 221).
2.3. Fluorometric measurements of ADP/ATP efflux The measurements were made essentially as previously reported [23]. Briefly, mitochondria (0.5 mg ml-‘) were incubated in 2 ml of standard medium in the cuvette of an LS-5 Perkin-Elmer luminometer. An ATP detecting system (ADS), consisting of glucose (2.5 mM), hexokinase (E.C. 2.7.1.1.) (1 E.U.), glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49.) (0.5 E.U.) and NADP+ (0.2 mM), was added. NADPH formation in the extramitochondrial phase, which reveals ATP appearance due to externally added ADP, was followed fluorometrically with excitation and emission wavelengths of 334 nm and 456 nm respectively. The exchange reaction was started by adding ADP. The initial concentration of intramitochondrial pyridine nucleotide was equalized to zero. Checks were made to verify that in the absence of NADP+ no significant change of fluorescence occurred during the experiment. The rate of fluorescence increase expressed as nmol NADP reduced min-’ X mg protein obtained as a tangent to the initial part of the curve proves to be the rate of ADP/ATP exchange [23] (expressed as nmol ATP effluxed mini X mg protein). Calibration of NADPH fluorescence was made according to refs. 24 and 25.
2.4. Measurements of oxygen uptake Mitochondria (0.5 mg ml-‘) were incubated at 30 “C in a standard medium consisting of 87 mM sucrose, 60 mM KCl, 10 mM MgC12, 1.35 mM inorganic phosphate and 24 mM glycylglycine at pH 7.4 containing 2 pg rotenone. When present, PF II was added in the medium at the concentration of 6 pg ml-’ giving an optical density of 0.8 cm-’ at the irradiation wavelength of 365 nm. Experiments were typically performed under the following conditions: (a) untreated RLM kept in the dark; (b) untreated RLM irradiated; (c) PF II treated RLM kept in the dark; (d) PF II treated and irradiated RLM. Oxygen uptake was started by the addition of succinateTris (1.35 mM) followed by either ADP (1.35 mM) or FCCP (1.30 PM). The rate of oxygen uptake stimulated either by FCCP (V,,,,) or ADP (V,,,) was obtained as a tangent at the initial part of the curve and expressed as natoms O2 min-’ X mg protein. The respiratory control ratio (RCR), i.e. (the rate of oxygen uptake after either ADP or FCCP addition)/ (the rate of oxygen uptake before either ADP or FCCP addition), was also determined.
38
2.5. Measurement
of ATPase activity
ATPase activity was followed as in refs. 26 and 27 by measuring mitochondrial swelling which occurs when ATP hydrolysis is elicited in mersalyltreated mitochondria added with ATP. Mersalyl(20 nmol mg-’ protein) was added to the mitochondrial suspension after the treatment with PF II alone, light alone or PF II plus light. After 1 min, 2 mM ATP were added and, after 1 min, ATP hydrolysis was induced by adding 1.30 PM FCCP. 3. Results and discussion To investigate the effect of PF II photosensitization on the ADP/ATP carrier, an experimental approach was used which allows measurements of ADP uptake in exchange with intramitochondrial ATP (ADP/ATP antiport) in RLM without preventing mitochondrial respiration and ATP synthesis. This was done by continuously monitoring ATP efflux caused by ADP uptake and phosphorylation. As described in Scheme 1, ADP enters __.-.-._.-._.-.-.-.-.-.-._ EXTRAHITKHONDRIAL PHASE
MATRIX
GLUCOSE
Scheme 1.
mitochondria in exchange with endogenous ATP via the ADP/ATP translocator. Inside mitochondria, ADP is phosphorylated to ATP which in turn exchanges for further ADP. Outside mitochondria, ATP appearance is revealed by NADPH formation via hexokinase and glucose-6-phosphate dehydrogenase (see Section 2 and ref. 23). The ATP concentration in the phase outside mitochondria is negligible since no change in NADPH fluorescence is found. Following ADP (5 PM) addition, a rapid increase of fluorescence is observed, which shows the appearance of ATP outside mitochondria (Fig. l(A)). As expected [23], the inhibition of NADPH formation by carboxyatractyloside, a powerful inhibitor of the ADP/ATP translocator [28], was found, whereas no effect was observed in the presence of P,P,diadenosine [5-pentaphosphate] (ApsA), a specific inhibitor of adenylate kinase [29] (Fig. l(B)). In a previous paper [23], it has been reported that this method measures the rate of ADP/ATP exchange in mitochondria. However, it is possible that the observed inhibition of the rate of NADPH formation might also be
39
E
C
/ 102
:; AOSeDP
ADSAW
1
t
ADS PJF
Fig. 1. ADP/ATP carrier activity in control RLM and RLM treated with PF II and light. RLM (0.5 mg ml-i) were suspended at 30 “C in 2.0 ml of standard medium consisting of 87 mM sucrose, 60 mM KCI, 10 mM MgCls, 1.35 mM inorganic phosphate and 24 mM glycylglycine (pH 7.4), in the absence ((A), (B), (D)) or in the presence ((C), (E)) of PF II and irradiated for 2 min ((C), (D)). After ADS addition, the indicated compounds were added at the following concentration: 5 PM ADP; 10 @I ApsA; 0.5 @I carboxyatractyloside (CAT).
due to the added ADS and/or the intramitochondrial reactions directly related to oxidative phosphorylation. The first possibility was excluded by directly testing the effect of PF II plus light on the ADS both in the absence of mitochondria and in the presence of mitochondria added with ATP (not shown). The distinction between the translocation step and the intramitochondrial reactions will be made below. The nature of the inhibition was studied by investigating the dependence of the rate of fluorescence increase as a function of ADP concentration in control RLM and RLM treated with PF II and light (Fig. 2(A)). A purely non-competitive inhibition was found with a K, value (i.e. the ADP concentration which gives half-maximum rate of exchange) equal to 5 PM, and a Ki value (i.e. the irradiation time which gives 50% inhibition) equal to 100 s. As a result of PF II photodynamic action, a 54% decrease in the V of the transport was found following 2 min irradiation. When these ex;Trimental data were replotted as l/u, expressed as nmol NADP reduced min-’ X mg us. the irradiation time in a Dixon-like plot (Fig. 2(B)), straight lines were obtained. Statistical analysis shows that the intercepts to the ordinate axis, obtained by treating the experimental points according to linear regression, coincide perfectly with the corresponding experimental controls. This clearly shows that the measured inhibition is related to the
40
A
B 0.4
0.4 +
I
1 V
./
/
1
0.3
03
a2
02
01
01
.
P I
0
025 l/[ADPj IJIM-
0.50
A
P I 0
1 TIME
2 iminl
Fig. 2. Nature of the inhibition of the activity of the ADP/ATP carrier by PF II and irradiation. Experimental conditions as in Fig. 1. (A) Double reciprocal plot: dependence of the rate of exchange as a function of the ADP concentration was investigated in control RLM (0) and PF II-treated RLM irradiated for 30 s (A), 1 min (0) and 2 min (A), (B) Dixon plot: rate data of Fig. 2(A) replotted as a function of irradiation time. The ADP concentrations are 20 PM (o), 10 PM (A), 5 PM (X), 2.5 @I (A) and 2 PM (0). In both cases the rates of the uptake were obtained as a tangent to the initial part of the progress curve and expressed in nmol ATP effluxed min-’ X mg protein.
same parameter measured in the control, i.e. the rate of ADP/ATP exchange (see ref. 23). It should be noted that under these experimental conditions the energy for ATP synthesis is apparently given by the oxidation of endogenous substrates. In the light of the role played by the ADP/ATP carrier in the oxidative phosphorylation [ 301, the effect of PF II photosensitization on mitochondrial respiration was investigated to ascertain whether the inhibition of the ADP/ATP translocator could be considered as a primary cause of the impairment of oxidative phosphorylation [ 4, 5,121. As described in Scheme 2, succinate oxidation by isolated mitochondria stimulated by externally added ADP derives from several steps in which different possible targets of PF II photosensitization could be involved: (a) succinate transport across the inner mitochondrial membrane via the dicarboxylate translocator in exchange of endogenous phosphate; (b) succinate dehydrogenase activity; (c) electron n+ generation; (d) ADP transport flow along the respiratory chain and AE.C across the inner mitochondrial membrane via the ADP/ATP translocator; (e) A/J~+ utilization and ATP synthase activity. It should be noted that when oxygen uptake is measured after stimulation by an uncoupler, steps (d) and (e) are excluded. Most steps are inhibited in mitochondria after treatment with HPD and irradiation [ 15 - 181. For instance succinate dehydrogenase and cytochrome oxidase impairment could affect both electron flow in the respiratory chain and ATP synthesis [15,16]. Measurements of the rate of oxygen uptake stimulated by succinate plus rotenone were carried out under different experimental conditions.
41 OUT I
I
Pi
ATP .
\
IN OH-
ATP I
STEP
A
STEP
B
STEP
C
1
0
STEP
E
STEP
AOP
lAOP
1
Scheme 2.
In the first set of experiments, ADP was used to stimulate the oxygen uptake due to externally added succinate (1.35 mM), thus allowing for ATP synthesis (ATP conditions) (Fig. 3(A)). These conditions may reflect the physiological situation in duo. ADP increased the rate of oxygen uptake in untreated RLM: 206 natoms O2 min-’ X mg protein with an RCR value equal to 3(a). The same increase was observed upon exposure of RLM to either PF II (b) or 365 nm light (c), whereas about 60% inhibition of oxygen uptake (corresponding to an RCR value equal to 1.2) was found following irradiation for 30 s in the presence of PF II (d). In the second set of experiments, carried out with the same mitochondrial preparation, the uncoupler FCCP was used instead of ADP to stimulate succinate (1.35 mM) oxidation in the absence of ATP synthesis (uncoupling conditions) (Fig. 3(B)). Once more, PF II (b) or 365 nm light (c) alone did not significantly affect the rate of oxygen uptake compared with the control (a) (u = 234 natoms O2 min-’ X mg protein), but about 50% inhibition was observed when RLM were treated with PF II and irradiated for 30 s (d) (u = 110 natoms O2 min-’ X mg protein), the value of RCR being 1.6. These results confirm that besides the ADP/ATP carrier, other processes are also inhibited by PF II plus light. By plotting the percentage of stimulation of the rate of oxygen uptake by either ADP or FCCP us. irradiation time (Fig. 4), a lower photosensitivity of FCCP-stimulated respiration was found. However, it should be noted that, under uncoupling conditions, the absolute rates of oxygen uptake were in any case higher than those found under ATP
42
a
I
100 natoms
C
b
d
02
B
\
\
Fig. 3. Effect of PF II and light on the succinate oxidation by RLM stimulated by either ADP or FCCP. RLM (0.5 mg ml-‘) were suspended at 30 “C in 1.5 ml of the standard medium, in the absence ((a), (c)) or in the presence ((b), (d)) of PF II and irradiated (hy) for 30 s ((c), (d)). At the arrows, additions were made at the following concentration: 2 pg rotenone (ROT); 1.35 mM succinate (SUCC); 1.5 mM ADP (in (A)), 1.3 /.&I FCCP (in (B)). Numbers along the curves are the rates of oxygen uptake expressed in natoms 0s min-’ X mg protein.
0
10 IRRADIATION
30
20 TIME
ISI
Fig. 4. Dependence of stimulation of oxygen uptake vs. duration of the photodynamic treatment of RLM. Experimental conditions as in Fig. 3. Succinate (1.35 mM) oxidation was stimulated by the addition of either 1.35 mM ADP (0) or 1.30 FM FCCP (0) to control RLM and RLM treated with PF II and irradiated with different doses of light. Stimulation is given as the following ratio: (RCR of RLM treated with PF II and light -l)/(RCR of control RLM -1).
43
ATP
FCCP
1
1
ATP FCCP 1
I
ATP
1
FCCP 1
ATP
FCCP
I
I
110
112
1
:
C
0
Fig. 5. Effect of PF II plus light on the ATPase activity. ATP hydrolysis induced by externally added FCCP in control RLM and RLM treated with PF II and irradiated at 365 nm was followed as reported in Section 2. Irradiation times were 0 s (A), 10 s (B), 20 s (C) and 30 s (D). At the arrows, additions were made at the indicated concentration: 2 mM ATP; 1.3 PM FCCP. Absorbance changes at 546 nm were continuously recorded with a Perkin-Elmer Lambda 5 spectrophotometer.
conditions. As a result of these findings, steps other than those of ADP/ATP exchange via the specific translocator are excluded as being rate limiting in mitochondrial respiration following PF II photosensitization. In fact, if the dicarboxylate carrier, succinate dehydrogenase etc., were more sensitive to PF II plus light treatment than the ADP/ATP carrier, the higher sensitivity under ATP conditions would not be observed and the rate of oxygen uptake in the presence of PF II and 365 nm light under both ATP and uncoupler conditions would be the same. To assess whether ATPase is impaired under the reported experimental conditions, the sensitivity of mitochondrial ATPase to PF II and light was investigated by testing whether the photosensitization of RLM prevents FCCP-stimulated swelling of mersalyl-treated mitochondria added with 2 mM ATP (Fig. 5). This swelling has been reported to occur when phosphate and ADP formation (caused by ATP hydrolysis via ATPase stimulated by FCCP) takes place in the matrix of mitochondria in which the phosphate carrier is inhibited by its powerful inhibitor mersalyl [31]. No significant change in the rate of mitochondrial swelling with respect to the control (Fig. 5(A)) was found after irradiation for 10, 20 or 30 s (Figs. 5(B) - 5(D)), thus showing that there is no inhibition of mitochondrial ATPase under experimental conditions in which impairment of ADP/ATP translocator is found, as previously reported by Salet et al. [14].
44
4. Conclusion Evidence is given in this paper that photodynamic treatment of isolated mitochondria can inhibit the ADP/ATP carrier. As reported for dicarboxylate, tricarboxylate and oxodicarboxylate translocators, a non-competitive inhibition was found, showing that the photodynamic effect involves the carrier activity rather than the substrate affinity. Interestingly, the ADP/ATP translocator proves to be the mitochondrial molecular target whose inhibition by PF II photosensitization impairs ADP-stimulated succinate oxidation in RLM. How cell death in both normal and cancer cells depends on mitochondrial ADP/ATP translocator impairment remains to be established. However, as far as mitochondria are concerned it is worthwhile stressing that even if a number of enzymes are inhibited by porphyrin photosensitization [15 - 191, their inhibition plays a minor role in the impairment of oxidative phosphorylation. Finally, since mitochondria constitute an important if not a primary target of PF II cell photosensitization [ 32, 331, ADP/ATP translocator could be considered as a primary cellular target in studies designed to find out how cell death can occur in photodynamic therapy in duo.
Acknowledgments This work was partially supported by an exchange programme “Cooperation France-Italienne en Recherche Biomedicale INSERM-CNR”. The helpful cooperation of Dr. Angela Ostuni is gratefully acknowledged.
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