Placenta
Volume 31, Issue 10 , Pages 860-866, October 2010

Contribution of Potassium in Human Placental Steroidogenesis

Department of Biochemistry, School of Medicine, National Autonomous University of Mexico, Mexico City, Mexico

Accepted 17 July 2010. published online 11 August 2010.

Article Outline

Abstract 

The role of K+ on steroidogenesis in isolated mitochondria from the human placenta was explored. Cholesterol uptake and progesterone synthesis were stimulated by K+, and by the further addition of ATP. In the presence of glibenclamide or quinine (inhibitors of the K+ channel mito-KATP), the synthesis of progesterone was improved, indicating that K+ acts outside the mitochondria. Valinomycin, a K+-ionophore, inhibited mitochondrial steroidogenesis only in the absence of K+. The mitochondrial K+ channel in human placental mitochondria is formed by the subunit Kir 6.1 which was detected by Western blot with polyclonal antibodies. These results suggest that K+ contributes placental mitochondrial steroidogenesis facilitating cholesterol uptake and intermembrane translocation through a mechanism non-dependent of the transport of K+ inside the mitochondria.

Keywords: Human placental mitochondria, Potassium, Cholesterol transport, Progesterone synthesis

 

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1. Introduction 

Monovalent cations contribute to maintain some physiological functions in cells such as the resting and action membrane potential, accumulation of glucose, amino acids and nucleotides, as well as keeping the cell volume and osmolarity [1]. While the K+ concentration in the cytoplasm is around 120mM [2], [3], [4] in mitochondria, it is between 140 and 180mM [5].

An increase of the matrix volume in rat liver mitochondria after a hormonal stimulus by glucagon was described to be associated to net transport of K+ [6] favoring contact sites. Similarly, an increase in extracellular [K+] associated with a reduction of mitochondrial pyridine nucleotides was observed in rat adrenal glomerulosa cells. An increase of [K+] as little as 0.5mM stimulated steroid hormone production [7]. Although the [K+] in cytoplasm is above 100mM, only small amounts of K+ transport are evidently needed to exert specific effects on the mitochondrial metabolism.

Steroidogenesis takes place in mitochondria and requires the movement of cholesterol between mitochondrial membranes. In the human placenta the transport of cholesterol between mitochondrial membranes is carried out by the protein MLN64 [8], [9], since the protein StAR is not expressed [10].

Mitochondrial contact sites participate in the transport of several metabolites [11] including cholesterol [12]. In the human placental mitochondria these contact sites were isolated and several proteins were identified [13], including a novel porin-like protein [14].

Cytochrome P450 side chain cleavage (P450scc) is associated to the inner mitochondrial membrane with adrenodoxin and adrenodoxin reductase. This electron transport chain is responsible to transform cholesterol into pregnenolone. In acute steroidogenic tissues like bovine adrenocortical mitochondria, in the absence of hormonal stimulus, cytochrome P450scc is associated to proteins unrelated to steroidogenesis, but when [K+] is increased the cytochrome P450scc is released favoring its interaction with adrenodoxin and adrenodoxin reductase, thus increasing pregnenolone synthesis. A reverse effect is observed when K+ is removed from the medium [15].

The positive effect of K+ on the association of steroidogenic proteins is also closely related to the formation of contact sites to transport cholesterol into mitochondria [16], [17]. Morphological changes were also observed in human placental mitochondria isolated in the presence of K+ as opposed to the ones isolated in sucrose showing close approximations between mitochondrial membranes [18].

It has been proposed that K+ uptake is performed by a mitochondrial ATP-regulated potassium channel (mito-KATP) modulated by nucleotides and composed of a Kir-SUR (sulfonylurea receptor) subunits [2], [3], [4], [5]. However, its presence apparently is not universal. Recently it was described a mitochondrial multi-protein complex reconstituted in liposomes containing the phosphate carrier, adenine nucleotide translocator, mitochondrial ATP-binding cassette protein 1, succinate dehydrogenase and ATP synthase (α-subunit) that transports K+ in a way similar to that of mito-KATP, which was inhibited by glibenclamide and open with malonate, an inhibitor of succinate dehydrogenase [19], [20].

As mentioned before, the influx of K+ increases the volume of the mitochondrial matrix producing contact sites stimulating oxidative phosphorylation in the rat liver [6]. However, the role of K+ in the metabolism of cholesterol during steroidogenesis in the human placenta has not been evaluated. In this study, we show that steroidogenesis increases in the presence of K+ suggesting its role in the endocrine functions of the human placenta.

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2. Materials and methods 

2.1. Isolation of mitochondria 

Human placentas were obtained according the Ethical Committee of the School of Medicine. Human placental mitochondria (HPM) from syncytiotrophoblast were isolated as reported by Martinez et al. [21]. Mitochondrial proteins were quantified as described by Bradford [22], using bovine serum albumin (BSA) as standard. Only mitochondria with a respiratory control upper than 4 were used assuring their integrity and intactness [23].

2.2. Cholesterol incorporation 

The incorporation of cholesterol into placental mitochondria was performed as described by Martinez et al. [24] by using a cholesterol-BSA-complex. In all the experiments the osmolarity was kept constant with sucrose. Progesterone synthesis was examined in the cholesterol-enriched-HPM with 100mMK+ in the presence or absence of ATP. Cholesterol incorporation was stopped by 5-fold dilution with cold 250mM sucrose, 1mM EDTA, pH 7.4, followed by centrifugation to remove free cholesterol. The mitochondrial pellet was washed three times with a fresh cold solution of 250mM sucrose, 1mM EDTA, pH 7.4. After protein determination, cholesterol concentration was quantified with a commercial kit (Spinreact, S.A. Ctra Santa Coloma, Spain), following the manufacturer’s instructions.

2.3. Progesterone synthesis 

Both intact HPM and cholesterol-enriched-HPM were incubated at 37°C in a medium with malate 10mM as oxidative substrate in the presence or absence of ATP or ADP. After incubation, the reactions were stopped with 1.5 volumes of cold methanol. In further experiments, HPM were incubated in a medium named progesterone synthesis medium, containing 5mM isocitrate, 10mM MOPS, 0.5mM EGTA, 120mM KCl and protease inhibitors, adjusted to pH 7.4 [25]. Progesterone determinations were made by radioimmunoassay using a commercial kit (Diagnostic Products, USA), according to the manufacturer’s specifications.

2.4. Protein phosphorylation by HPM 

Mitochondrial protein phosphorylation was performed by incubating the HMP in the presence of radioactive ATP (ADPγ32P) with a specific activity 3000Ci/mmol in the progesterone synthesis medium. At the times indicated in each figure, an aliquot was taken and 1.5 volumes of cold methanol were added to stop the reaction. Mitochondrial proteins (10μg per lane) were separated on a 10% polyacrylamide gel under denaturing conditions by SDS-PAGE according to Laemmli [26]. The gels were exposed to autoradiography film and developed according to the manufacturer’s instructions.

2.5. Membrane potential 

The contribution of K+ to the mitochondrial membrane potential was determined in the progesterone synthesis medium in a final volume of 2.5ml containing 1mg of protein/ml, measuring the Safranine O (2.5 μM) optical density changes at 511nm and using 533nm as an isosbestic point in a DW 2000 Aminco spectrophotometer, as described by Åkerman and Wikström [27]. As a control, the mitochondrial potential generated was depleted by adding 0.1mM valinomycin.

2.6. Western blots 

Whole mitochondrial proteins (10μg per lane) were separated on a 10% SDS-polyacrylamide gel using electrophoresis according to Laemmli [26] and transferred to polyvinylidene fluoride membrane (PVDF). After blocking with 10% fat free milk, the primary antibody against the KATP-receptor subunit Kir 6.1 was added at a dilution 1:500. Membranes were incubated at 4°C on ice during 12h and then incubated with horseradish peroxidase conjugated with the secondary antibody at a dilution 1:15000 [28]. Chemiluminescence was used to visualize the bands. Plasma membrane of rat heart was used as positive control.

2.7. Materials 

Sucrose, KCl, cholesterol, bovine serum albumin (BSA) and protease inhibitors were purchased from Sigma-Chemical, Mexico. The radioimmunoassay kit for progesterone was purchased from Diagnostic Products Corporation, Los Angeles, CA, USA. Radioactive ATP (ADPγ32P) was obtained from NEN, USA. The kit for cholesterol determination was purchased from Spinreact, S.A. Ctra Santa Coloma, Spain. Other reagents were analytical grade. Antibody anti Kir 6.1 was purchased from Santa Cruz and GR Healthcare Life Sciences.

2.8. Data analysis 

At least three independent experiments were performed by triplicate. Data are expressed as mean ± standard deviations (SD) and, according to the experimental characteristics; an appropriate statistical analysis was performed as indicated in each figure or table legend.

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3. Results 

3.1. Mitochondrial steroidogenesis 

Although HPM has a high content of cholesterol, their incubation with exogenous cholesterol increased from 35.6 to 61.1μg cholesterol/mg protein with 100mMK+ to 102.5μg cholesterol/mg protein when 2mM ATP plus K+ were added (Table 1) (p < 0.05). In the absence of K+, the incorporation was 48.2μg cholesterol/mg protein and 89.1μg cholesterol/mg protein in the presence of sucrose plus 2mM ATP (Table 1)(p < 0.05). HPM with an excess of cholesterol after the incorporation were named cholesterol-enriched-HPM.

Table 1. Cholesterol incorporation in human placental mitochondria and its further transformation into progesterone.
Cholesterol (μg/mg)ng P4/mg/min
Incubation condition + 2mM ATP
Untreated mitochondria35.6±8.4a10.74±1.15c16.61±1.94
Sucrose48.2±9.812.16±1.4724.78±1.15
KCl61.1±16.4b16.55±2.98d20.94±1.49

Sucrose+ATP89.1±9.5b15.72±0.87d19.9±2.81
KCl+ATP102.5±14.9b22.24±3.2d28.29±2.45

n=4n=4n=4

Human placental mitochondria were enriched with cholesterol as mentioned in the Materials and Methods section. In the medium the concentration was K+ 120mM or sucrose 240mM with 30μg cholesterol-BSA-complex. When ATP was present, it is concentration 2mM. The results are the mean±SD. The numbers of independent experiments made are indicated as n. The results present statistical difference in the cholesterol incorporation when was compared “a” vs “b” (p < 0.05). Fort the progesterone synthesis the statistical differences were between “c” vs “d” (p < 0.05). In the presence of 2mM ATP there was statistical difference for progesterone synthesis between untreated mitochondria vs all groups, which was analyzed by Tukey test (p < 0.05).

Steroidogenesis was performed in both HPM and cholesterol-enriched-HPM. The results showed that the synthesis of progesterone was consistently high in the presence of K+. When ATP was added to the incubation media, the amount of progesterone increased, being higher in mitochondria previously incubated with K+ plus ATP (Table 1) (p < 0.05).

The incubation of HPM with exogenous cholesterol in the presence of increasing concentrations of K+ plus a constant concentration of ATP or ADP showed a raise of cholesterol mitochondrial contents in a dose-response behavior (Fig. 1A), suggesting that this process was favored by K+ and required the presence of ATP or ADP. Although there was not statistical difference between the nucleotide used, the results strongly suggest that to incorporation of cholesterol into the HPM in the presence of ADP or ATP is required (Fig. 1A).

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  • Fig. 1 

    A. Incorporation of exogenous cholesterol in the presence of K+ and nucleotides. HPM were incubated for 10min at 37°C in the presence of increasing concentrations of K+ and 30μg cholesterol/mg BSA in 200μl of the medium containing 10mM malate, 0.2% BSA, 1mM EDTA, 1mM MgCl2, 10mM Tris–HCl, pH 7.4. The osmolarity was maintained at 250 mOSM with sucrose. ATP or ADP were 2mM. The results are the mean of independent experiments made in triplicate. By t student analysis there is not significant difference. The bars indicate the SD. B. Synthesis of progesterone in the presence of ATP or ADP and increasing K+ concentrations. Progesterone synthesis was determined at 37°C in the presence of increasing concentrations of K+ (n=5); plus 2mM ATP (n=10) or plus 2mM ADP (n=9). The results are the mean of at least three independent experiments made in triplicate. The one-factor ANOVA analysis shows statistical differences between control vs ATP (p < 0.05) and ATP vs ADP (p < 0.05). The bars indicate the SD.

Fig. 1B shows that the steroidogenic activity of HPM was 7.82±1.14 ng P4/mg/min in the absence of K+ and increased to 10.05±1.7 ng P4/mg/min in the presence of 100mMK+, following a dose-dependent curve. Similar results were observed when the incubation medium was supplemented with ATP, but the synthesis of progesterone increased to 14.93±5.13 ng P4/mg/min at 100mMK+. Despite the dispersion of the data, the results showed statistical differences between control and ATP (p < 0.05) at 100mMK+; and there is also statistical difference in progesterone synthesis with ATP or ADP (p < 0.05).

In order to further analyze the role of K+ on the transformation of cholesterol into progesterone, the effect of several inhibitors of mitochondrial K+ uptake were tested. Glibenclamide is a mito-KATP inhibitor that binds the SUR subunit, while tetraphenylphosphonium (TPP) associates to the Kir subunit; both close the channel and therefore stop the influx of K+ into the mitochondrial matrix. In the presence of glibenclamide or TPP the steroidogenesis of HPM was stimulated (p < 0.05), suggesting that the effect of K+ was outside the mitochondria (Table 2). Quinine is an inhibitor of the SUR subunit of the K+ channel; however progesterone synthesis did not show differences between control and inhibitor at 120mMK+. Interestingly, in the absence of K+ quinine increased 300% the progesterone production (Fig. 2A) and raising the concentration of K+ support the steroidogenesis reaching a plateau at 80mM (p < 0.05). It has been proposed that the mitochondrial K+ channel can be a multi-protein complex with the same sensibility to channel inhibitors and opened by malonate [19]. Then, the effect of malonate in progesterone synthesis was determined, and the results showed no differences with the control (Table 2).

Table 2. Progesterone synthesis by HPM in the presence of inhibitors of mitochondrial K+ uptake.
ControlaGlibenclamideb (50μM)Quinine (1mM)TPPb (100nM)Malonate (10mM)
11.72±3.216.29±2.2410.48±3.617.33±3.512.52±2.51
n157456

Effect of the inhibitors of the channel of K+ in the progesterone synthesis from HPM. The incubation medium contained: 5mM isocitrate, 0.5mM EGTA, 10mM MOPS, 120mM KCl, 1μg/mg leupeptine and 4μg/mg aprotinine. The results are expressed in ng progesterone- min-mg of HMP and are the mean from independent experiments±SD. The numbers of independent experiments made are indicated as n. The results were analyzed by Tukey test. Statistical differences between control (a) and glibenclamide (b) or TPP (b) was p < 0.05.

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  • Fig. 2 

    A. Quinine increases progesterone synthesis in HPM. The synthesis of progesterone was determined at 37°C in the medium of progesterone synthesis in the absence (●) or presence (○) of 1mM quinine. The results are the mean of four independent experiments. Bonferroni analysis showed statistical differences between 0 and 80mM KCl in both cases (p < 0.05). The bars indicate the SD. B. Mitochondrial membrane potential. The membrane potential of HPM was determined in the progesterone synthesis medium supplemented with Safranine O (2.5 μM) in a final volume of 2.5ml containing 1mg of protein/ml, measuring the optical density changes at 511nm and using 533nm as an isosbestic point. As a control, the mitochondrial potential generated was depleted by adding 0.1mM valinomycin and 5μM CCCP.

Additional evidence of the effect of K+ outside HPM was obtained with valinomycin, a natural ionophore for K+ which decreased progesterone synthesis (Table 3). Unexpectedly, in the presence of K+ plus quinine and valinomycin the steroidogenesis was not inhibited, but in the presence of 120mM or 160mMK+ the progesterone synthesis remains without any change between the control and any other experimental condition. In the presence of malonate as an opener of the channel progesterone synthesis remain the same as the control (Table 2).

Table 3. Progesterone synthesis in the presence of channel inhibitors and valinomycin.
A. Without valinomycinB. With valinomycin
KCl (mM)ControlQuinine (1mM)Malonate (10mM)ControlQuinine (1mM)Malonate (10mM)
02.87±0.58 b7.75±2.29a5.93±0.99c1.58±0.41d5.21±1.145.91±0.98
12012.72±1.317.07±0.9711.92±0.0813.18±3.613.92±1.2913.01±2.42
16013.11±1.0419.44±0.8513.02±0.8313.12±3.614.03±2.4314.47±0.86

Effect of valinomycin on the progesterone synthesis from HPM with different K+ concentrations and K+ channel inhibitors. Synthesis of progesterone with different K+ concentrations and 1mM quinine as potassium channel inhibitor or 10mM malonate as opener of the channel. Valinomycin (0.1mM) was added to the reaction medium. The results are expressed in ng of progesterone/min/mg of HMP and are the mean from independent experiments made in triplicate±SD. The date were analyzed by a Bonferroni test. Statistical difference were observed as compared “a” vs “b”, “c”, “d” (p < 0.05).

It is important to note that the mitochondrial membrane potential measured with Safranine O collapsed with valinomycin at the same concentration used for progesterone synthesis, whereas steroidogenesis remained unchanged (Fig. 2B).

3.2. Localization of the Kir 6.1 subunit 

The composition of the mitochondrial K+ channel remains unclear and at least two models to explain the K+ transport into mitochondria have been suggested. One model is the mito-KATP composed of the SUR and Kir 6.1 subunits [2], [3], [4], [5]. The other one is a multi-protein complex described by Marban et al. [19]. The results showed that Kir 6.1 is present in HPM and in the plasma membrane of rat heart (Fig. 3A), which was used as a positive control since the presence of the Kir 6.1 subunit has been described [5]. Then, our results support the assumption that mito-KATP is the mechanism through which K+ is transported in the HPM.

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  • Fig. 3 

    A. Western blot analysis of Kir 6.1 subunit. The proteins (20μg each) from the plasma cellular membrane of rat heart (lane 1) and HPM (lane 2) were processed by SDS-PAGE and electrotansferred to PVDF membrane or stained with Coomassie. Protein phosphorylation of HPM by radiolabel ATP incubated in the presence of sucrose (B) or K+ (C). HPM were incubated in the progesterone synthesis media in the presence of ADP-γ32P and sucrose or K+; at the times indicated the reaction was stopped with 1.5 volumes of cold methanol. After centrifugation, the pellet was processed to SDS-PAGE and the gels were exposed to autoradiography film as described in Materials and Methods. This is a representative experiment.

3.3. Phosphorylation of mitochondrial proteins 

During acute hormonally induced steroidogenesis in adrenal glands, StAR enhances its activity by phosphorylation mediated by a protein kinase A (PKA) [29]. Although the human placenta is not a hormonal induced-acute steroidogenesis tissue, the PKA activity is present and is related to steroidogenesis [30], [31]. The incubation of HPM with radioactive ATP showed protein phosphorylation (Fig. 3B). Although four proteins between 30 and 66kDa were detected, their intensity changed notably in the presence of K+, mainly in the first seconds of incubation (Fig. 3C).

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4. Discussion 

Potassium participates in several biological functions, including modulation of hormonal homeostasis [6] or enzyme activity [15], [32], [33]. Our results show that K+ participates in the steroidogenic functions of HPM as a cofactor and not just through a simple osmotic effect, since in all experiments the osmolarity was maintained constant and the effect of the osmolarity on the HPM has been previously evaluated [17].

In cells, variations of [K+] in the cytoplasm are related to metabolic changes. In vivo, [K+] did not change in magnitude as in our experimental assays, but they allowed us to understand how K+ modifies steroidogenesis. Even more, it has been reported that changes in [K+] as small as 0.1mM have an important physiological repercussion [7], i.e. it was reported that the rotational diffusion of mitochondrial cytochrome P450scc was significantly dependent on K+ concentration [15]. The effect of K+ on placental steroidogenesis could not be explained by a charge effect because in the presence of 120mM Na+ progesterone synthesis was 5.31±3.51 ng P4/mg/min, this value is near to that of progesterone synthesis in the absence of K+.

Although HPM have enough endogenous cholesterol to support progesterone synthesis, steroidogenesis occurs at a very low rate probably because no contact sites are formed [34]. Therefore, to initiate steroidogenesis, HPM require a stimulus, and in this sense, isolated HPM are a good model to study cholesterol transport and steroidogenesis.

In the presence of K+ and ATP both cholesterol transport and steroidogenesis increased, suggesting that K+ and/or ATP are related to placental steroidogenesis. This assumption is based first on the results from the incorporation of cholesterol into the mitochondria in the presence of K+, which was higher than in the presence of sucrose. Second, because in the presence of ATP the increase was two-fold, even when the mitochondria were incubated in the presence of sucrose (Table 1), suggesting that this transport requires energy, probably provided by ATP hydrolysis catalyzed by apyrase [35], similar to the GTPase observed in rat adrenal mitochondria [36].

HPM membranes incorporate cholesterol even in the absence of K+ or ATP in part due to the partition coefficient of cholesterol. Despite the high contents of endogenous cholesterol [37], in the presence of K+, ATP or both a significant increase in the transport of cholesterol into HPM was observed (Fig. 1A and Table 1). However, the electron transport chain of cytochrome P450scc is not the rate limiting step in placental steroidogenesis since progesterone synthesis could be stimulated in the presence of 22-hydroxycholesterol plus K+ reaching a value of 31.42±5.09 ng P4/mg/min that is 2.6 times more than only with K+ that was 11.72 ng/P4/mg/min. Then, like in other steroidogenic tissues, in HPM the transport of cholesterol between mitochondrial membranes is the rate limiting step of placental steroidogenesis.

Mitochondrial cholesterol transport requires contact sites and proteins such StAR [10], [36], [38], [39], [40], [41], [42]. It has been reported that mitochondrial K+ uptake is associated to water transport producing swelling of mitochondria [6], changing the volume of mitochondrial matrix and favoring contact sites formation for oxidative phosphorylation [11]. In HPM the isolation of contact sites with steroidogenic capacity has been previously reported [13]. Proteins from contact sites isolated in the presence of sucrose showed a different electrophoretic pattern when compared to the contact sites isolated in the presence of K+ (Fig. S1). Uniform contact sites were observed in the presence of K+, suggesting a homogeneous protein composition. Then, it is possible that proteins in these membrane structures increased their activity under the influence of K+ which in turn induces mitochondrial morphological changes [18].

Our results show that progesterone synthesis increased in the presence of K+ over the control containing sucrose. There are two ways to increase progesterone synthesis; one is by increasing cholesterol transport between mitochondrial membranes and the other is by supplying 22-hydroxycholesterol that freely cross mitochondrial membranes without requirements of a protein transport. The first one is the physiological and the second is the artificial way. Then, it is reasonable to suggest that K+ contributes to the transport of cholesterol from the outer to the inner mitochondrial membranes. Besides, although HPM have a high content of cholesterol in the outer membrane [34] the concentration of progesterone is low in the presence of sucrose at the same osmolarity as our results show.

Steroidogenesis and protein phosphorylation are linked in certain steroidogenic cells, with StAR being one of the key phosphoproteins [30]. Although the human placenta does not contain StAR [10], the phosphorylation of proteins in HPM by PKA activity could modulate steroidogenesis [31]. Our results show that the phosphorylation of a 46kDa protein increases in the presence of K+ (Fig. 3C) compared to sucrose (Fig. 3B). Thus kinase activity or specific protein phosphorylation induced by the presence of K+ could also be a contributor to the changes observed in steroid biosynthesis.

When mito-K+ATP was maintained open by the presence of malonate, progesterone synthesis remained as in the control (Table 2), and the influx of K+ was unchanged (Fig. S2).

The increase of steroidogenesis observed with the inhibitors of K+ uptake suggests that K+ has a relevant role outside the mitochondria to modulate the synthesis of progesterone. Under physiological conditions, K+ is moving dynamically between the cytoplasm and the mitochondria, but in the absence of K+ in the incubation media, mitochondria release K+ from matrix to cytoplasm. Quinine blocks the uptake of K+ which remains outside the mitochondria stimulating progesterone production (Fig. S3). Statistical differences were observed for progesterone synthesis in the presence of quinine at concentrations between 0 and 80mM of K+, where the stimulation by the presence of K+ and the inhibitor were in the same magnitude (Fig. 2A), which correlates with the results of Table 3 where there are statistical differences between the control without K+ and the presence of the inhibitors.

The Kir subunit from mito-KATP channel was identified in HPM by Western blot using an affinity purified goat polyclonal antibody against human C–terminal Kir 6.1 (Fig. 3A). In the presence of valinomycin, which equilibrates the [K+] across mitochondrial membranes, the synthesis of progesterone was reduced from 1.7±0.7 to 0.9±0.3 (Table 3). Interestingly, the presence of valinomycin did not swell the HPM as compared to yeast mitochondria (data not shown). Under these experimental conditions, in the presence of 120mMK+ the HPM membrane potential was lost, but not the synthesis of progesterone. It has been reported that the mitochondrial membrane potential is necessary for the processing of StAR to the active form of 30kDa, and therefore for the transport of cholesterol [43]. However, our data suggest that the membrane potential is not necessary for steroidogenesis in HPM. Even more, progesterone synthesis has been observed in isolated contact sites of HPM where there is no membrane potential [13]. Thus, the effects observed could be explained if it is proposed that K+ is a cofactor in the mechanism responsible for cholesterol transport between mitochondrial membranes.

The relevance of K+ in mitochondria has been described previously. It was reported an increase of respiration in rat liver mitochondria promoted by K+ and due to an increase in state 3 of respiration [32]. Martinez et al. [18] reported that in HPM the mitochondrial membrane potential was not modified by osmolarity, but morphological changes were induced when HPM were isolated in K+ probably because it induces the formation new contact sites [20].

An apyrase activity associated to progesterone synthesis in HPM has been reported [35]. Interestingly, K+-channel inhibitors glibenclamide, quinine or TPP did not modify the apyrase activity (data not shown). These inhibitors bind to the same place of ATP [4], [5]. Indeed, ATP is the physiological inhibitor of the channel [3]. Because the activity of apyrase is so high in HPM [44], these inhibitors associate to ATP-binding site producing a partial inhibition of ATP hydrolysis (data not shown). This data suggests that the basal activity of apyrase is enough for progesterone synthesis supporting the assumption that apyrase participates in placental steroidogenesis [35], similar to GTPase from rat adrenal mitochondria [36].

In conclusion, the data presented suggest that K+ contributes to placental steroidogenesis by modulating the formation of mitochondrial contact sites and therefore promoting the transport of cholesterol and its transformation into progesterone.

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Acknowledgements 

This work was partially supported by Grant IN217609 and IN203006 from Dirección General de Apoyo al Personal Académico de la UNAM, and 37263M from the National Council of Science and Technology (CONACyT). R. Milan is a student from the Programa de Doctorado en Ciencias Biomédicas from the Universidad Nacional Autónoma de México and this work is part of her thesis dissertation. The authors thank Dr. J.F. Strauss 3rd for his suggestions and the critical revision of the manuscript and Esther Urrutia M.Sc. for the statistical support and analysis; and also to Dr. José Luis Pérez-García for his in the reviewing the usage of Englsih in this text.

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Appendix A. Supplementary material 

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PII: S0143-4004(10)00277-8

doi:10.1016/j.placenta.2010.07.008

Placenta
Volume 31, Issue 10 , Pages 860-866, October 2010

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