Placenta (1992), 13, 535-544
Effect of Calcium on Mitochondria From Human Term Placenta J. P. GARDURO, M. T. ESPINOSAGARCfA, J. P. PARDO & F. MARTfNEZ” Depatiamento de Bioyuimica, Fact&d de Medicina, Unizvrsidad National Atrtdnoma de .W.ico, Apdo. Postal 70-159, 04jI0, Mtkico D. F. u To whom correspondence should be addressed Paper accepted 9.3.1992
SUMMARY We describe here the effects offree Cal’ on several functions of mitochondrza from human term placenta.In thepresenceof0. I PMfree Ca2+, an inhibit0 y efect on both ADP-induced respiration and succinate-DCPIP reductase actizity was observed. At the same Ca2+ concentration, ATPase activity as well as various segments of the respiratory chain were stimulated. How,a’er, a higher free Ca2+ concentration (0,3 ,UUM) was needed to stimulate progesterone synthesis. Our results sugest that Ca2f plays an important role in the metabolicfunctions of mitochondria from human term placenta.
INTRODUCTION The importance of Ca2+ in the regulation of numerous cellular functions is well established (Lehninger, Carafoli and Rossi, 1961; Thorn et al, 1978; Haksar and Peron, 1973). Intracellular Ca2+ is critical in muscular contraction, hormone release and other cellular events (Carafoli, 1988). In the case ofplacenta, Ca2+ is fundamental, since during gestation there is a very active transport of Ca2+ to the fetus, mainly for bone ossification. Indeed a 2-month human fetus contains 0.032 g of elemental calcium, which rises to 20.4 g in an &month fetus (Comar, 1956). The total Ca2+ in the fetus derives from maternal circulation, either directly or after temporary storage in microsomal vesicles in the placenta (Brunette, 1988; Shennan and Boyd, 1987). Ca2+ transfer takes place via an active process in which a specific ATPase activity is involved (Brunette, 1988; Shennan and Boyd, 1987; Abramovich et al, 1987; Treinen and Kulkarni, 1986; Tuan and Kushner, 1987). It is known that mitochondria have the capacity to accumulate large amounts of Ca2+ (Lotscher et al, 1980), and that this modifies the activity of intramitochondrial enzymes involved in the tricarboxylic acid cycle (Hansford, 1985) and oxidative phosphorylation (Moreno-Sanchez, 1985a). Recently, it was reported that human placental mitochondria 0143-4004/92/060535
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contain high amounts of Ca2+ (Martinez, Chavez and Echegoyen, 1987). However, its role in mitochondria from human term placenta has not been studied. In order to get insight into a possible regulation of mitochondrial activity by Ca2+ in human term placenta, we studied the effect of external physiological free Ca2’ concentrations upon respiration, electron transport activity in several segments of the respiratory chain, the activities of both the ATP/ADP carrier and the ATPase, as well as progesterone synthesis.
MATERIAL
AND METHODS
Isolation of mitochondria Normal human term placentae were obtained from the delivery room, transported to the laboratory, and processed in not more than 30 min after delivery. They were washed with cold 0.25 M sucrose/l mM EDTA, pH 7.4. The cotyledons were processed at 4°C to isolate the mitochondrial fraction as reported previously (Martinez, Chavez and Echegoyen, 1987). Protein content was measured by the Lowry method (1951). Submitochondrial particles were obtained as reported previously (Martinez, Chavez and Echegoyen, 1987). Ca’+-EGTA buffer and Ca*+ determination The concentration of free Ca2+ in the incubation medium was adjusted to the desired level by the Cazf -EGTA buffer system using the computer program described by Fabiato (1988). Arsenazo III was used to quantify the total Ca2+ content of mitochondria (Scarpa et al, 1978). Oxygen consumption The incubation medium contained 20 mu MOPS, 120 mu KCl, 2 mu EGTA, 2 mu HsP04,lO mu succinate, and 0.1 per cent BSA, adjusted to pH 7.2 with KOH (medium A) in a final volume of 3 ml. Mitochondria (1 mg/ml) were added to this medium and, after 1 min of incubation, respiration was stimulated with 300 nmol of ADP. Only mitochondria with respiratory control higher than 3.5 were used. Electron transport chain activity The different segments of the respiratory chain were studied using medium A. The corresponding substrate and specific inhibitors for each segment were added to this medium. NADH-DCPIP reductase activity was assessed in medium A plus 100,~~ KCN (inhibitor of site 3), 1 pugantimycin (inhibitor of site 2), and 200 PM NADH using 0.125 mg of protein per ml. The reaction was initiated with 160 ,uMdichlorophenol indophenol (DCPIP) and its reduction was followed in a spectrophotometer Aminco DW-2a using a wavelength of 600 nm. A molar extinction coefficient of 2l/mM/cm was used to calculate the activity. Succinate-DCPIP reductase activity was determined in medium A plus 5 PM rotenone (inhibitor of site. l), 100 ,uM KCN, 1.1 mu phenazine methosulphate (PMS), 20 mu succinate, and 0.5 g of protein per ml. The activity was initiated with 80,~~ DCPIP, and its reduction was followed at 600 nm. NADH-cytochrome c reductase was assayed in medium A plus 100 ,uM KCN, 5 ,uM NADH, 3 mg of protein. The reaction was initiated by the addition of 0.5 mg of cytochrome c and its reduction followed spectrophotometrically at 550 run. A molar extinction coefficient of 19/m~/cm was used to calculate the activity. Succinate-cytochrome c reductase was quantified using medium A plus 5 ,ug rotenone, 100 PM KCN, 20 mu succinate, 2 mg of protein per ml, and 0.5 mg of cytochrome c.
Gardtrtio et al: Effect of Cazi in Placental .Mitochondria
Cytochrome oxidase was measured in an oxymeter in 3 ml of medium antimycin, 5 rnzl ascorbate, 50 ,UM TMPD, and 1 mg of protein per ml.
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ATPase activity and Adenine Nucleotide Translocase ATPase activity of placental mitochondria was studied in the presence of different concentrations of Ca*+ in medium A, plus 50 ,UMdinitrophenol (DNP) and 1 mg of protein in a final volume of 1 ml at 30°C. ATPase activity was started by the addition of 2.5 rn,M ATP; after 10 min incubation the reaction was stopped with 6 per cent trichloroacetic acid, and the suspension centrifuged. Phosphate released was quantified in the supernatant by the method of Sumner (1944). The activity of the ATP/ADP carrier was assessed in medium A as reported previously (Martinez, Chavez and Echegoyen, 1987), using [adenosine-8-“C] diphosphate with a specific activity of 100 000 ct/min/mol, and [2,3,8-3H]-ATP with a specific activity of 300 000 ct/min/mol. Assay of progesterone Synthesis of progesterone by mitochondria after state 3 and 4 was determined by radioimmunoanalysis after its extraction in diethyl ether, as described before (Sufi, Donalson and Jefcoate, 1983). The antibody displayed negligible (< 1 per cent) cross-reaction with other steroids.
RESULTS When mitochondria from human term placenta were exposed to increasing concentrations of free extramitochondrial Ca*+, an inhibition of the respiratory activity stimulated by ADP (state 3) was observed with approximately 0.1 ,LLMCa*+ [Figure 1 (a)]. In the fully inhibited state, oxygen uptake was close to the non-stimulated respiration (state 4). The rate of state 4 respiration was not affected by different concentrations of free Ca*+. It has been reported that Mg*+ inhibits competitively the transport of Ca*+ in mitochondria (Robertson, Potter and Rouslin, 1982). Figure l(b) shows the percentage of mitochondrial respiration in the presence of 2 rn,q Mg*+ and different free Ca*+ concentrations. In the presence of M$+, higher concentrations of Ca*+ (0.3 ,LL_M) were required to produce an inhibition of the rate of state 3 respiration, suggesting that Mg*+ antagonized the effect of Ca*+. This conclusion derives from the analysis of the last point where the slope changes from uninhibited to inhibited respiration. As a result of the inhibition of respiration by Ca*+ , in the absence of Mg*+ the respiratory control dropped as the concentration of free Ca*+ was raised (data not shown). However, the respiratory control without Ca*+ (lop9 M) in the presence of succinate was higher (at least 3.5 times) than those (around 3) reported previously (Olivera and Meigs, 1975; Klimek, Boguslawski and Zelewski, 1979; Negrie et al, 1979; Illsley, Coade and Harkness, 1985). The inhibition of respiration induced by Ca*+ could be due to an effect on the electron transport chain. Accordingly, we studied the effect of Ca*+ on several segments of the reductase respiratory chain, and observed that Ca*+ increased the activity ofNADH-DCPIP in placental submitochondrial particles [Figure 2(a)]. In contrast, the succinate-DCPIP reductase activity was inhibited by 0.3 ,,WVof Ca*+ [Figure 2(b)]. NADH-DCPIP reductase and succinate-DCPIP reductase activities were studied in submitochondrial particles, since
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Figure 1. Effect of free Ca2+ on respiration of mitochondria from human term placenta. The incubation conditions were: 20 mMMOPS, 120 mu KCl, 2 ~IIJVI phosphate-T& (pH 7.2), 2 mMEGTA, 10 mu succinate, and 0.1 per cent bovine serum albumin, adjusted to pH 7.2 with KOH. Mitochondrial protein (3 mg) were added to a tinal volume of 3 ml at 25°C. Respiration was started with 300 nmol ADP. Results show the effects of increasing free Ca” on respiration in the absence of Mgaf (a) or the presence of 2 mM Mgri (b). -O--, state 3; -0-, state 4. Bars indicate the standard deviation of three experiments in duplicate from three different placentae.
the artificial electron acceptor DCPIP is not freely transported across the inner mitochondrial membrane. NADH-cytochrome c reductase and succinate-cytochrome c reductase activities [Figures 3(a) and (b), respectively] were stimulated by increasing free Ca’+ concentration. On the other hand, the activity of cytochrome oxidase was not affected by Ca2+ (data not shown). The effect of Ca2+ on other mitochondrial functions was also studied. Translocation of adenine nucleotide by the ADP/ATP carrier was not modified by Ca2+ (data not shown). ATPase activity increased when free Ca2’ varied from 0.1 to 1.0 ,uM; at higher concentrations, the activity decreased. Progesterone biosynthesis takes place in mitochondria from human placenta. At 0.3 ,kM, Ca2+ stimulated the synthesis of this hormone (50 per cent with respect to the amount formed with 1 no; Figure S), whereas 2 mu Mgaf abolished this stimulation (data not shown). These results suggest that Ca2+ could be involved in the regulatory mechanism of progesterone production, as occurs in placenta from bovine (Shemesh, Hansel and Strauss III, 1984).
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Figure 2. Activities of NADH-DCPIP reductase and succinate-DCPIP reductase from submitochondrial particles in the presence of free Ca’+. Activities were determined as mentioned under Material and Methods. Activity of NADH-DCPIP reductase (a) or succinate-DCPIP reductase (b) was started by addition of the artificial electron acceptor, 2,6 dichlorophenol indophenol (DCPIP) in the presence of increasing concentrations of free Ca2+. Panel (b), 100 per cent ofactivity was considered taking into account the highest reduction value ofDCPIP (4.7 to 5.7 nmol DCPIP/mg/min) obtained from six experiments. Bars in panel A indicate the standard deviation of three experiments.
DISCUSSION As noted, Ca*+ is a regulatory cation of several cellular and mitochondrial functions. However, no studies on .the role of Ca*+ have been performed in human placental mitochondria. In this report, we show that Ca2+ decreased oxygen consumption supported by succinate and stimulated by ADP. The effect on electron transport might be due to the inhibition of succinate dehydrogenase, since succinate-DCPIP was the only segment inhibited by Ca*+. To this regard, controversial data are described in the literature: succinate dehydrogenase is not known to be stimulated by Ca*+ (Hatefi, 198S), and Johnston and Brand (1987) reported no changes in the activity of succinate dehydrogenase with different concentrations of Ca2+ in rat liver mitochondria. Contrary to these results, Moreno-Sanchez
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Figure3.
Effect of increasing free Ca*+ concentrations on NADH-cytochrome c reductase and succinatecytochrome c reductase activities. The activity of NADH-qtochrome c reductase (a) or auccinate-cytokome c reductase (b) was started by the addition of 0.5 mg of cytochrome c. Results show the mean of three experiments. Bars indicate the standard deviation.
(1985b) found that, at higher Ca2+ COnCentMiOnS (5 X 1oe6 M), the ITSpiratOIy rate in the presence of succinate was inhibited, supporting the results presented in this paper. The increase of activity in individual electron transport components could be explained based on the experimental conditions used, since the activity of the respiratory chain is related to the rate-limiting step that controls the overall process (Tager et al, 1983); on the other hand, the use of specific inhibitors to explore only a section of the electron transport chain allows us to study the specific effect of Ca2+ on the activity of the segment (johnston and Brand, 1987; Moreno-Sanchez, 1985b). Furthermore, some of the respiratory chain segments and ATPase activities were stimulated significantly by Ca2’ concentrations in the range of 0.1-0.3 m. This indicates that under phys comiitims, Ca2+ could regulate the respiratory activity, as has been described (Can&&, $982; Hansford, 1985;Crompton, 1990).
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Figure 4. ATPase activity in the presence of extramitochondrial free Ca2+ concentrations. ATPase activity was determined in Medium A, described under Material and Methods, in the presence of 2,4 dinitrophenol(50/.& and 1 mg of protein in a final volume of 1 ml at 30°C. Bars indicate the standard deviation, n = 3.
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Figure.% Effect of free Ca2+ on the biosynthesis of progesterone in mitochondria from human term placenta. Mitochondria were recuperated after that the oxygen consumption was stimulated with ADP. Mitochondria were treated with diethyl ether to determine the amount ofprogesterone, as described under Material and Methods. The results are the mean of three experiments. Bars indicate the standard deviation.
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Regarding inhibition of respiration, it has been reported that oxidative phosphorylation is inhibited in mitochondria with a high content of endogenous Ca2+, i.e., mitochondria from tumor cells (Thorne and Bygrave, 1974; Abou-Khalil, Abou-Khalil and Yunis, 1981; Villalobo and Lehninger, 1980), and it has been proposed that the potential factor, which allows tumor mitochondria to accumulate large amounts of Ca2’, is their elevated level of membrane cholesterol (Parlo and Coleman, 1984; Murphy and Fiskum, 1988). In steroidogenic mitochondria, such as adrenal (della-Cioppa et al, 1986) and placental (unpublished data), the concentration of cholesterol is around 3Opg/mg (compared to 5 pugof cholesterol/ mg in rat liver mitochondria; Daum, 198.5), suggesting that this membrane composition, which resembles that of tumor mitochondria, is responsible for the large accumulation of Ca’+. The phenomenon described in this paper may be of importance for the overall functions of placental mitochondria, particularly considering that Ca’+ increases progesterone synthesis. It is well known that mitochondria from steroidogenic tissues, such as placenta, have another electron transport chain coupled to cytochrome P-450,, (Mason and Boyd, 1971; Meigs and Ryan, 1968), which is responsible for the cleavage of the side chain of cholesterol to yield pregnenolone. In a second step, progesterone is formed through the A5-ene-3/3hydroxysteroid dehydrogenase-isomerase complex localized in the mitochondrial matrix (Rabe et al, 1985; Boguslawski, 1983). 0 ur results show that the synthesis of progesterone was stimulated by Ca2+ at concentrations that decreased respiratory activity. It is possible that Ca2+ could regulate the distribution of substrates between the respiratory chain and the electron transport chain coupled to cytochrome P450. It has been described that metabolites from the tricarboxylic cycle are involved in the production of NADH and NADPH (Klimek et al, 1976), which are the substrates for both electron transport chains. It is also well known that Ca2+ regulates the dehydrogenase activity of several enzymes of this cycle. In support of the role of Ca*+ in the biosynthesis of progesterone it has been shown that, in placental explants, extracellular Ca*+ is apparently necessary for progesterone production in fizstimulation (Kasugai et al, 1987). In bovine placenta, Ca*+ increases the biosynthesis of progesterone by a pathway different from the classical &IMP-induced pathway (Shemesh et al, 1988), suggesting that a similar mechanism could exist in human placental mitochondria.
ACKNOWLEDGEMENTS The authors thank Dr Armando Gomez-Puyou for his helpful criticisms of the manuscript, and Federico Martinez, Jr for the illustrations. This work was partially supported by Grant IN200189 IFC/UNAM from the Direction General de Asuntos de1 Personal Academic0 de la Universidad National Autonoma de Mexico.
REFERENCES Abou-Khalil, S., Abou-Khalil, W. H. & Yunis, A. A. (1981) Inhibition by Ca2+ of oxidative phosphorylation in myeloid tumor mitochondria. Archives ofBiochen&y and Biophysics,460,460-464. Abramovich, D. R., Da&e, C. G., Elcock, C. SKPage, K. R. (1987) Calcium transport across the isolated dually perfused human placental lobule._?oumulofPhysielogy, 382,397-410. Fksgwlawski, W. (1983) Cytochrome P450 and steroidogenic activities of the human placental mitochondria. 3oournalof&mid Biochemistry,18,771~775. Brunette, M. G. (1988) Calcium transport through the placenta. CanadianJournal ofPhysiologyand Pbannacology, f&281-298. Garafdi, E. (1982) The transport of calcium across the inner membrane of mitochondria. InMembmne Tmnspon of
Calcislm(Ed.) Carafii, E. pp. 109-139, New York Academic Press. Carafoli, E. (1988) Membrane transport of cakium: an overview. Methods in Enzymology, 157,3-l 1.
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Comar, C. L. (1956) Radiocalcium studies in pregnancy. Annals oj‘Thelrw YorkAcadeq ofsciences, 64, 28 l-298. Crompton, M. (1990) Role of mitochondria in intracellular calcium regulation. In Intrucelhdar Calcbrm Re~u/at/oN, (Ed.) Bronner, F. pp. 181-209, New York: Willey-Liss. Daum, G. (1985) Lipids of mitochondria. Biochivu’ca et Bioph~~~ica .-lcta, 822, l-12. della-Cioppa, G., Muftly, K. E., Yanagibashi, K. & Hall, P. F. (1986) Preparation and characterization of submitochondrial fractions from adrenal cells. .Mole&r and CeUular Endocrinology, 48, 11 l-120. Fabiato, A. (1988) Computer programs for calculating total from specified free or free form specified rota1 ionic concentrations in aqueous solutions containing multiple metals and ligands. Vf&ocis in Euq~o/o,y~, 157, 378416. Haksar, A. & Peron, F. G. (1973) The role of calcium in the \teroidogenic response of rat adrenal cells to adrenocorticotropic hormone. Biochirnictr et Biophysics Acta, 313, 363-371. Hansford, R. G. (1985) Relation between mitochondrial calcium transport and control of energ! metabolism. R~renv itr PhyioloAq: Biochenzistv and PharmacoloR): 102, l-72. Hatefi, Y. (1985) Tbe mitochondrial elecrron transport and oridative phosphonlation system. .drrv~o/ R&w oj Rioriwrtris!,,, 54, 101 S-1069. IllsleJ-, N. P., Coade, S. B. & Harkness, R. A. (1985) H uman placental mitochondrial respiration and its regulation b>-adenine nucleotides. Placenta, 6, 187-198. Johnston, J. D. & Brand, M. D. (1987) Stimulation of the respiration rate of rat liver mitochondria b!- whmicromolar concentrations of extramitochondrial Ca2+. Biorhpmi~,irl_?/~,trn2n/, 245, 217-222. Kasugai, %I., Kato, H., Iriyama, H., Kato, M., Minagawa, T. & Tomoda, Y. (1987) The roles of CaL’ and adenosine 3’,5’-monophosphate in the regulation ofprogesteronc production b!- human placental tissue..~ow/rr/ ~!f‘C%‘ttiide-c\;ain cliayagc reaction in the mito~hondria from human tern‘ 362-37’. Lehninger, A. L., Carafoli, E. & Rossi, C. S. (1961) EnerR -linLed ion mowments in mitochondrial systems. -I&crriiw in &~JNIU/U~, 29, 259-320. of mitocbondria Liitscher, H. R., Winterhalter, K. H., Carafoli, E. & Richther, C. (1980) Th e energ-state during the transport of Ca*+. E/lrc~peun.~~nrrma/afBiochemist~’,110, 211-216. Low-n;, 0. H., Rosebrough, N. J., Farr, A. L. & Randal, R. J. (195 1) Protein measurement with the Folin phenol reagent. TlreJof/,unrc~l ofBiological Chetvistty, 193, 265-275. Martinez, F., Ch&ez, E. & Echegoyen, S. (1987) Decreased exchanRc ofadenine nucleotides in human placental mitochondria. l~~k~nrrrti,,~~al.~a’urrr~aiafBin~henrist~~, 19, 275-279. Mason. 1. I. 61 Bovd. G. S. (1971) The cholesterol side-chain cleakane en/\-me s\strm in mitochondria of hunun termilacenta. E;rropecrrr~fo~rmcrl;?f.Bio~hl,trrist~, 21, 308-321. L .IIeigs, R. A. & Ryan, K. J. (1968) C!-tochrome P450 and steroid hios!ntbrsi\ in the human placenta. Bio&irnrici c’f Bir/pir,),si~~a .qcta, 165, 476-482. Moreno-Sinchez, R. (1985a) Regulation of oxidatixe phosphqlation in mitochondria b! external free Ca-‘+ concentrations. Thc,~oumal qfBio/ogita/ Chemistq: 260, 402%4034. Moreno-SBnchez, R. (1985b) Contribution ofthe translocator ofadenine nucleotides and the .%TP wnthase to the control of oxidatixc phosphoq lation and arsem-lation in liter mitochondria. Th“.?o/rrrra/ ofBiolq&a/ C’hen~~~~, 260, 1255+-12560. Murphy, .I. N. & Fiskum, G. (19X8) Abnormal Ca” transport characteristics of hepatoma mitochondria and endoplasmic reticulum. In Adxw~~x in E.~peinrmtcrl.lledici~~~~ and Bioloa: Cellular Calcirrrr~Rqulatiotf, ~~olume 232, (Ed.) Pfeiffer. D. R.. hlcllillin. I. B. & Little. T. S. New k-ork: Plenum Press. NCgrid, C., Triadou, N., Michei,“O., Bouhnik, J. & Michel, R. (1979) Oxidative phosphovlation reactions and cholesterol h! dro\? lation mechanisms in human term placental mirochondria..7~r/rlm/ r!~.~tc,rn;~/Bk~.~?ef~f;s/~,. 11, 1135-11~0. Olivera, A. i\. &-Me&s, R. A. (1985) \;Iitochondria from human rerm placenta. I. Isolation and assay conditions ior rxidati\ c phospho?lation. Biochintira PIBiopbysica .&a, 376, 426135, Parlo, R. A. & Coleman, P. S. (19%) Enhanced rate of citrate exF,ort from cholesterol-rich hepatonra mitocbondria: the truncated Krebs c\cle and other metabolic ramilications of mitochondrial membrane cholesterol. The.~ouma/ ofBiological Ch&risrr): 259, 9997-10003. Rabe, T.. Gresel, L. & Runnebaum, B. (1985) Regulation ofplacental progesrerone synthesis in vitro b! natwall! occurring steroids..~owwl ofSteroid Biochemistry, 22, 657-661. Robertson. S. P., Potter, .I. D. & Rouslin, W. (1982) The Ca” and &lg2’ dependence of CaZ’ uprake and respiratory f&ion of porcine heart mitochondria. TheJoumal ~fBiolqi~a~ Chen&fry, 257, 1743-1748. Scarpa, A., Brinley, F. J., Tiffert, T. & Dubyak, G. R. (1978) \letallochromic indicators of ionized calcium. inrra~~ ~!fflimYork.4uuletny of Sciences, 307, 86-l 12. Shemesh, M., Hansel, W. & Strauss, F. J. III (1984 Calcium-dependent, qclic nucleotide-independent stcroidogenesis in the bovine placenta. Proreedings ofthe L;?riona/,-lcadeq~ qfSciences ofthe b-X.4,81, 6403-6407. Shemesh, M., Strauss, J. F. III, Hansel, W., Shore, L. & Izhar, M. (1988) Control of bovine placental
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progesterone synthesis: roles of cholesterol availability and calcium-activated systems. Jownal c!f‘Steroid Biochemistry, 29,21-25. Sherman, D. B. & Boyd, C. A. R. (1987) Ion transport by the placenta: a review of membrane transport systems. Biochimica et Biophysics Acta, 906,437-457. Sufi, S. B., Donalson, A. & Jefcoate, S. L. (1983) Method Manual of Matched Assay Reagent Programme 7th Ed. Special Programme of Research in Human Reproduction, World Health Organization, Geneva, Switzerland. Sumner, J. B. (1944) A method for the calorimetric determination of phosphorus. Science, 100,413-414. Tager, J. M., Wanders, R. J. A., Groen, A. K., KUM, W., Bohnensack, R., Kiister, U., Letko, G., Biihme, G., Duszynski, J. & Wojtczak, L. (1983) Control of mitochondrial respiration. FEBS Letters, 151, 1-9. Thorn, N. A., Russell, T. J., Pedersen, T. C. & Treiman, M. (1978) Calcium and neurosecretion. Annals of The NeD York Academy ofSciences, 307, 618-639. Thorne, R. F. W. & Bygrave, F. L. (1974) The role of mitochondria in modifying the cellular ionic environment. Calcium-induced respiratory activities in mitochondria isolated from various tumor cells. Biochemical_?wrnal, 144,551-558. Treinen, K. A. & Kulkarni, A. P. (1986) High-affinity, calcium-stimulated ATPase in brush border membranes of the human term placenta. Placenta, 7,365-373. Tuan, R. S. & Kushner, T. (1987) Calcium-activated ATPase of the human placenta: identification, characterization, and functional involvement in calcium transport. Placenta, 8,53-64. Villalobo, A. & Lehninger, A. L. (1980) Inhibition of oxidative phosphorylation in ascitic tumor mitochondria and cells by intramitochondrial Ca2+. TheJournal ofBiological Chemistry, 255, 2457-2464.
Effect of Calcium on Mitochondria From Human Term Placenta J. P. GARDURO, M. T. ESPINOSAGARCfA, J. P. PARDO & F. MARTfNEZ” Depatiamento de Bioyuimica, Fact&d de Medicina, Unizvrsidad National Atrtdnoma de .W.ico, Apdo. Postal 70-159, 04jI0, Mtkico D. F. u To whom correspondence should be addressed Paper accepted 9.3.1992
SUMMARY We describe here the effects offree Cal’ on several functions of mitochondrza from human term placenta.In thepresenceof0. I PMfree Ca2+, an inhibit0 y efect on both ADP-induced respiration and succinate-DCPIP reductase actizity was observed. At the same Ca2+ concentration, ATPase activity as well as various segments of the respiratory chain were stimulated. How,a’er, a higher free Ca2+ concentration (0,3 ,UUM) was needed to stimulate progesterone synthesis. Our results sugest that Ca2f plays an important role in the metabolicfunctions of mitochondria from human term placenta.
INTRODUCTION The importance of Ca2+ in the regulation of numerous cellular functions is well established (Lehninger, Carafoli and Rossi, 1961; Thorn et al, 1978; Haksar and Peron, 1973). Intracellular Ca2+ is critical in muscular contraction, hormone release and other cellular events (Carafoli, 1988). In the case ofplacenta, Ca2+ is fundamental, since during gestation there is a very active transport of Ca2+ to the fetus, mainly for bone ossification. Indeed a 2-month human fetus contains 0.032 g of elemental calcium, which rises to 20.4 g in an &month fetus (Comar, 1956). The total Ca2+ in the fetus derives from maternal circulation, either directly or after temporary storage in microsomal vesicles in the placenta (Brunette, 1988; Shennan and Boyd, 1987). Ca2+ transfer takes place via an active process in which a specific ATPase activity is involved (Brunette, 1988; Shennan and Boyd, 1987; Abramovich et al, 1987; Treinen and Kulkarni, 1986; Tuan and Kushner, 1987). It is known that mitochondria have the capacity to accumulate large amounts of Ca2+ (Lotscher et al, 1980), and that this modifies the activity of intramitochondrial enzymes involved in the tricarboxylic acid cycle (Hansford, 1985) and oxidative phosphorylation (Moreno-Sanchez, 1985a). Recently, it was reported that human placental mitochondria 0143-4004/92/060535
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contain high amounts of Ca2+ (Martinez, Chavez and Echegoyen, 1987). However, its role in mitochondria from human term placenta has not been studied. In order to get insight into a possible regulation of mitochondrial activity by Ca2+ in human term placenta, we studied the effect of external physiological free Ca2’ concentrations upon respiration, electron transport activity in several segments of the respiratory chain, the activities of both the ATP/ADP carrier and the ATPase, as well as progesterone synthesis.
MATERIAL
AND METHODS
Isolation of mitochondria Normal human term placentae were obtained from the delivery room, transported to the laboratory, and processed in not more than 30 min after delivery. They were washed with cold 0.25 M sucrose/l mM EDTA, pH 7.4. The cotyledons were processed at 4°C to isolate the mitochondrial fraction as reported previously (Martinez, Chavez and Echegoyen, 1987). Protein content was measured by the Lowry method (1951). Submitochondrial particles were obtained as reported previously (Martinez, Chavez and Echegoyen, 1987). Ca’+-EGTA buffer and Ca*+ determination The concentration of free Ca2+ in the incubation medium was adjusted to the desired level by the Cazf -EGTA buffer system using the computer program described by Fabiato (1988). Arsenazo III was used to quantify the total Ca2+ content of mitochondria (Scarpa et al, 1978). Oxygen consumption The incubation medium contained 20 mu MOPS, 120 mu KCl, 2 mu EGTA, 2 mu HsP04,lO mu succinate, and 0.1 per cent BSA, adjusted to pH 7.2 with KOH (medium A) in a final volume of 3 ml. Mitochondria (1 mg/ml) were added to this medium and, after 1 min of incubation, respiration was stimulated with 300 nmol of ADP. Only mitochondria with respiratory control higher than 3.5 were used. Electron transport chain activity The different segments of the respiratory chain were studied using medium A. The corresponding substrate and specific inhibitors for each segment were added to this medium. NADH-DCPIP reductase activity was assessed in medium A plus 100,~~ KCN (inhibitor of site 3), 1 pugantimycin (inhibitor of site 2), and 200 PM NADH using 0.125 mg of protein per ml. The reaction was initiated with 160 ,uMdichlorophenol indophenol (DCPIP) and its reduction was followed in a spectrophotometer Aminco DW-2a using a wavelength of 600 nm. A molar extinction coefficient of 2l/mM/cm was used to calculate the activity. Succinate-DCPIP reductase activity was determined in medium A plus 5 PM rotenone (inhibitor of site. l), 100 ,uM KCN, 1.1 mu phenazine methosulphate (PMS), 20 mu succinate, and 0.5 g of protein per ml. The activity was initiated with 80,~~ DCPIP, and its reduction was followed at 600 nm. NADH-cytochrome c reductase was assayed in medium A plus 100 ,uM KCN, 5 ,uM NADH, 3 mg of protein. The reaction was initiated by the addition of 0.5 mg of cytochrome c and its reduction followed spectrophotometrically at 550 run. A molar extinction coefficient of 19/m~/cm was used to calculate the activity. Succinate-cytochrome c reductase was quantified using medium A plus 5 ,ug rotenone, 100 PM KCN, 20 mu succinate, 2 mg of protein per ml, and 0.5 mg of cytochrome c.
Gardtrtio et al: Effect of Cazi in Placental .Mitochondria
Cytochrome oxidase was measured in an oxymeter in 3 ml of medium antimycin, 5 rnzl ascorbate, 50 ,UM TMPD, and 1 mg of protein per ml.
537
A plus 1 ,LL~
ATPase activity and Adenine Nucleotide Translocase ATPase activity of placental mitochondria was studied in the presence of different concentrations of Ca*+ in medium A, plus 50 ,UMdinitrophenol (DNP) and 1 mg of protein in a final volume of 1 ml at 30°C. ATPase activity was started by the addition of 2.5 rn,M ATP; after 10 min incubation the reaction was stopped with 6 per cent trichloroacetic acid, and the suspension centrifuged. Phosphate released was quantified in the supernatant by the method of Sumner (1944). The activity of the ATP/ADP carrier was assessed in medium A as reported previously (Martinez, Chavez and Echegoyen, 1987), using [adenosine-8-“C] diphosphate with a specific activity of 100 000 ct/min/mol, and [2,3,8-3H]-ATP with a specific activity of 300 000 ct/min/mol. Assay of progesterone Synthesis of progesterone by mitochondria after state 3 and 4 was determined by radioimmunoanalysis after its extraction in diethyl ether, as described before (Sufi, Donalson and Jefcoate, 1983). The antibody displayed negligible (< 1 per cent) cross-reaction with other steroids.
RESULTS When mitochondria from human term placenta were exposed to increasing concentrations of free extramitochondrial Ca*+, an inhibition of the respiratory activity stimulated by ADP (state 3) was observed with approximately 0.1 ,LLMCa*+ [Figure 1 (a)]. In the fully inhibited state, oxygen uptake was close to the non-stimulated respiration (state 4). The rate of state 4 respiration was not affected by different concentrations of free Ca*+. It has been reported that Mg*+ inhibits competitively the transport of Ca*+ in mitochondria (Robertson, Potter and Rouslin, 1982). Figure l(b) shows the percentage of mitochondrial respiration in the presence of 2 rn,q Mg*+ and different free Ca*+ concentrations. In the presence of M$+, higher concentrations of Ca*+ (0.3 ,LL_M) were required to produce an inhibition of the rate of state 3 respiration, suggesting that Mg*+ antagonized the effect of Ca*+. This conclusion derives from the analysis of the last point where the slope changes from uninhibited to inhibited respiration. As a result of the inhibition of respiration by Ca*+ , in the absence of Mg*+ the respiratory control dropped as the concentration of free Ca*+ was raised (data not shown). However, the respiratory control without Ca*+ (lop9 M) in the presence of succinate was higher (at least 3.5 times) than those (around 3) reported previously (Olivera and Meigs, 1975; Klimek, Boguslawski and Zelewski, 1979; Negrie et al, 1979; Illsley, Coade and Harkness, 1985). The inhibition of respiration induced by Ca*+ could be due to an effect on the electron transport chain. Accordingly, we studied the effect of Ca*+ on several segments of the reductase respiratory chain, and observed that Ca*+ increased the activity ofNADH-DCPIP in placental submitochondrial particles [Figure 2(a)]. In contrast, the succinate-DCPIP reductase activity was inhibited by 0.3 ,,WVof Ca*+ [Figure 2(b)]. NADH-DCPIP reductase and succinate-DCPIP reductase activities were studied in submitochondrial particles, since
Placenta (1992), Vol. 13
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9
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0 -log
[Ca’+]
6
5
I 5
CM)
I
I
I
I
-9
0
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[Caa+] (MI
Figure 1. Effect of free Ca2+ on respiration of mitochondria from human term placenta. The incubation conditions were: 20 mMMOPS, 120 mu KCl, 2 ~IIJVI phosphate-T& (pH 7.2), 2 mMEGTA, 10 mu succinate, and 0.1 per cent bovine serum albumin, adjusted to pH 7.2 with KOH. Mitochondrial protein (3 mg) were added to a tinal volume of 3 ml at 25°C. Respiration was started with 300 nmol ADP. Results show the effects of increasing free Ca” on respiration in the absence of Mgaf (a) or the presence of 2 mM Mgri (b). -O--, state 3; -0-, state 4. Bars indicate the standard deviation of three experiments in duplicate from three different placentae.
the artificial electron acceptor DCPIP is not freely transported across the inner mitochondrial membrane. NADH-cytochrome c reductase and succinate-cytochrome c reductase activities [Figures 3(a) and (b), respectively] were stimulated by increasing free Ca’+ concentration. On the other hand, the activity of cytochrome oxidase was not affected by Ca2+ (data not shown). The effect of Ca2+ on other mitochondrial functions was also studied. Translocation of adenine nucleotide by the ADP/ATP carrier was not modified by Ca2+ (data not shown). ATPase activity increased when free Ca2’ varied from 0.1 to 1.0 ,uM; at higher concentrations, the activity decreased. Progesterone biosynthesis takes place in mitochondria from human placenta. At 0.3 ,kM, Ca2+ stimulated the synthesis of this hormone (50 per cent with respect to the amount formed with 1 no; Figure S), whereas 2 mu Mgaf abolished this stimulation (data not shown). These results suggest that Ca2+ could be involved in the regulatory mechanism of progesterone production, as occurs in placenta from bovine (Shemesh, Hansel and Strauss III, 1984).
Gardutio et al: Effect of Ca 2+ in Placental .Mitorhondria
539
80
-log
[Cap+]
(M)
(b)
60 I 8
I 7 -log
[Co”]
I 6
I 5
(M)
Figure 2. Activities of NADH-DCPIP reductase and succinate-DCPIP reductase from submitochondrial particles in the presence of free Ca’+. Activities were determined as mentioned under Material and Methods. Activity of NADH-DCPIP reductase (a) or succinate-DCPIP reductase (b) was started by addition of the artificial electron acceptor, 2,6 dichlorophenol indophenol (DCPIP) in the presence of increasing concentrations of free Ca2+. Panel (b), 100 per cent ofactivity was considered taking into account the highest reduction value ofDCPIP (4.7 to 5.7 nmol DCPIP/mg/min) obtained from six experiments. Bars in panel A indicate the standard deviation of three experiments.
DISCUSSION As noted, Ca*+ is a regulatory cation of several cellular and mitochondrial functions. However, no studies on .the role of Ca*+ have been performed in human placental mitochondria. In this report, we show that Ca2+ decreased oxygen consumption supported by succinate and stimulated by ADP. The effect on electron transport might be due to the inhibition of succinate dehydrogenase, since succinate-DCPIP was the only segment inhibited by Ca*+. To this regard, controversial data are described in the literature: succinate dehydrogenase is not known to be stimulated by Ca*+ (Hatefi, 198S), and Johnston and Brand (1987) reported no changes in the activity of succinate dehydrogenase with different concentrations of Ca2+ in rat liver mitochondria. Contrary to these results, Moreno-Sanchez
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8 -log
[Co*+]
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8 --log
[Ca*+]
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5
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I 5
(Ml
(m)
Figure3.
Effect of increasing free Ca*+ concentrations on NADH-cytochrome c reductase and succinatecytochrome c reductase activities. The activity of NADH-qtochrome c reductase (a) or auccinate-cytokome c reductase (b) was started by the addition of 0.5 mg of cytochrome c. Results show the mean of three experiments. Bars indicate the standard deviation.
(1985b) found that, at higher Ca2+ COnCentMiOnS (5 X 1oe6 M), the ITSpiratOIy rate in the presence of succinate was inhibited, supporting the results presented in this paper. The increase of activity in individual electron transport components could be explained based on the experimental conditions used, since the activity of the respiratory chain is related to the rate-limiting step that controls the overall process (Tager et al, 1983); on the other hand, the use of specific inhibitors to explore only a section of the electron transport chain allows us to study the specific effect of Ca2+ on the activity of the segment (johnston and Brand, 1987; Moreno-Sanchez, 1985b). Furthermore, some of the respiratory chain segments and ATPase activities were stimulated significantly by Ca2’ concentrations in the range of 0.1-0.3 m. This indicates that under phys comiitims, Ca2+ could regulate the respiratory activity, as has been described (Can&&, $982; Hansford, 1985;Crompton, 1990).
Gardurio et al: Effect
ofCd 2+
in Placental .Mitochondria
o-o LL 9
541
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-log
[Ca’+]
(h-l)
Figure 4. ATPase activity in the presence of extramitochondrial free Ca2+ concentrations. ATPase activity was determined in Medium A, described under Material and Methods, in the presence of 2,4 dinitrophenol(50/.& and 1 mg of protein in a final volume of 1 ml at 30°C. Bars indicate the standard deviation, n = 3.
0 9
I
I
I
8
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-log
[Ca”]
I 5
(M)
Figure.% Effect of free Ca2+ on the biosynthesis of progesterone in mitochondria from human term placenta. Mitochondria were recuperated after that the oxygen consumption was stimulated with ADP. Mitochondria were treated with diethyl ether to determine the amount ofprogesterone, as described under Material and Methods. The results are the mean of three experiments. Bars indicate the standard deviation.
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Regarding inhibition of respiration, it has been reported that oxidative phosphorylation is inhibited in mitochondria with a high content of endogenous Ca2+, i.e., mitochondria from tumor cells (Thorne and Bygrave, 1974; Abou-Khalil, Abou-Khalil and Yunis, 1981; Villalobo and Lehninger, 1980), and it has been proposed that the potential factor, which allows tumor mitochondria to accumulate large amounts of Ca2’, is their elevated level of membrane cholesterol (Parlo and Coleman, 1984; Murphy and Fiskum, 1988). In steroidogenic mitochondria, such as adrenal (della-Cioppa et al, 1986) and placental (unpublished data), the concentration of cholesterol is around 3Opg/mg (compared to 5 pugof cholesterol/ mg in rat liver mitochondria; Daum, 198.5), suggesting that this membrane composition, which resembles that of tumor mitochondria, is responsible for the large accumulation of Ca’+. The phenomenon described in this paper may be of importance for the overall functions of placental mitochondria, particularly considering that Ca’+ increases progesterone synthesis. It is well known that mitochondria from steroidogenic tissues, such as placenta, have another electron transport chain coupled to cytochrome P-450,, (Mason and Boyd, 1971; Meigs and Ryan, 1968), which is responsible for the cleavage of the side chain of cholesterol to yield pregnenolone. In a second step, progesterone is formed through the A5-ene-3/3hydroxysteroid dehydrogenase-isomerase complex localized in the mitochondrial matrix (Rabe et al, 1985; Boguslawski, 1983). 0 ur results show that the synthesis of progesterone was stimulated by Ca2+ at concentrations that decreased respiratory activity. It is possible that Ca2+ could regulate the distribution of substrates between the respiratory chain and the electron transport chain coupled to cytochrome P450. It has been described that metabolites from the tricarboxylic cycle are involved in the production of NADH and NADPH (Klimek et al, 1976), which are the substrates for both electron transport chains. It is also well known that Ca2+ regulates the dehydrogenase activity of several enzymes of this cycle. In support of the role of Ca*+ in the biosynthesis of progesterone it has been shown that, in placental explants, extracellular Ca*+ is apparently necessary for progesterone production in fizstimulation (Kasugai et al, 1987). In bovine placenta, Ca*+ increases the biosynthesis of progesterone by a pathway different from the classical &IMP-induced pathway (Shemesh et al, 1988), suggesting that a similar mechanism could exist in human placental mitochondria.
ACKNOWLEDGEMENTS The authors thank Dr Armando Gomez-Puyou for his helpful criticisms of the manuscript, and Federico Martinez, Jr for the illustrations. This work was partially supported by Grant IN200189 IFC/UNAM from the Direction General de Asuntos de1 Personal Academic0 de la Universidad National Autonoma de Mexico.
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Gardurirr et al: J&t
q/‘Ca -‘+ in Platen tal Ilitoc.hondria
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