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Introduction: Protein phosphorylation plays an important role in the modulation of steroidogenesis and it depends on the activation of different signaling cascades. Previous data showed that PKA activity is related to steroidogenesis in mitochondria from syncytiotrophoblast of human placenta (HPM). PKA localization and contribution in progesterone synthesis and protein phosphorylation of HPM was assessed in this work., Methods: Placental mitochondria and submitochondrial fractions were used. Catalytic and regulatory PKA subunits were identified by Western blot. PKA activity was determined by the incorporation of (32)P into proteins in the presence or absence of specific inhibitors. The effect of PKA activators and inhibitors on steroidogenesis and protein phosphorylation in HPM was tested by radioimmunoassay and autoradiography., Results: The PKAα catalytic subunit was distributed in all the submitochondrial fractions whereas βII regulatory subunit was the main isoform observed in both the outer and inner membranes of HPM. PKA located in the inner membrane showed the highest activity. Progesterone synthesis and mitochondrial protein phosphorylation are modified by inhibitors of PKA catalytic subunit but are neither sensitive to inhibitors of the regulatory subunit nor to activators of the holoenzyme., Discussion: The lack of response in the presence of PKA activators and inhibitors of the regulatory subunit suggests that the activation of intramitochondrial PKA cannot be prevented or further activated., Conclusions: The phosphorylating activity of PKA inside HPM could be an important component of the steroidogenesis transduction cascade, probably exerting its effects by direct phosphorylation of its substrates or by modulating other kinases and phosphatases., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The transfer of cholesterol to mitochondria, which might involve the phosphorylation of proteins, is the rate-limiting step in human placental steroidogenesis. Protein kinase A (PKA) activity and its role in progesterone synthesis by human placental mitochondria were assessed in this study. The results showed that PKA and phosphotyrosine phosphatase D1 are associated with syncytiotrophoblast mitochondrial membrane by an anchoring kinase cAMP protein-121. The ³²P-labeled of four major proteins was analyzed. The specific inhibitor of PKA, H89, decreased progesterone synthesis in mitochondria while in mitochondrial steroidogenic contact sites protein-phosphorylation was diminished, suggesting that PKA plays a role in placental hormone synthesis. In isolated mitochondria, PKA activity was unaffected by the addition of cAMP suggesting a constant activity of this kinase in the syncytiotrophoblast. The presence of PKA and phosphotyrosine phosphatase D1 anchored to mitochondria by an anchoring kinase cAMP protein-121 indicated that syncytiotrophoblast mitochondria contain a full phosphorylation/dephosphorylation system., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The human placenta, which does not express the StAR protein, synthesizes large amounts of progesterone. The rate-limiting step for steroidogenesis is the transport of cholesterol which is divided into two steps: 1) cholesterol flux from cytoplasm to outer membrane mitochondria, and 2) cholesterol transport from outer to inner mitochondrial membrane. The proteins mediating placental cholesterol influx have not been clearly identified. We investigated the proteins involved in the transport of cholesterol in syncytiotrophoblast mitochondria from human placenta. Two proteins, one of 30 kDa, and another of 60 kDa, were identified using anti-MLN64 antibodies. The 30 kDa protein corresponds to a fragment of MLN64, and the 60 kDa protein was identified as a heat shock protein. During steroidogenesis, mitochondria released MLN64 protein to supernatant. When this supernatant was added to fresh isolated mitochondria, progesterone synthesis increased; a similar result was obtained with the addition of the recombinant MLN64-START protein. In the presence of flurescein-5-maleimide or N-ethyl-maleimide, the mitochondrial synthesis of progesterone was inhibited in a dose-dependent fashion without changes in mitochondrial respiration. 2D-electrophoretic pattern showed that flurescein-5-maleimide- fluorescence was associated with HSP60. Both MLN64 and HSP60 were identified in mitochondrial contact sites. The results suggest that HSP60 is involved in the steroidogenic metabolism of human placenta. A tight association between MLN64 and HSP60 is suggested for cholesterol transport in the human placenta., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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-K(ATP)), 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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).

Placental submitochondrial particles can synthesize progesterone. Both, progesterone synthesis and oxygen consumption were stimulated in the presence of succinate, NADH, or NADPH. NADPH reduced the cytochromes of the respiratory chain in a similar way as NADH. NADPH oxidation was not achieved by NADH-DH from the respiratory chain, and the presence of an NAD(P)H-DH is suggested. Submitochondrial particles contain at least two b-type cytochromes, one inhibited by antimycin when the electron donor is succinate, whereas the other is less sensitive to antimycin in the presence of pyridine nucleotides. The results suggest a connection between the respiratory chain and cytochrome P450, through an NAD(P)H-DH and a b-type cytochrome, both different from the respiratory chain.