Membrane potential regulates mitochondrial ATP-diphosphohydrolase activity but is not involved in progesterone biosynthesis in human syncytiotrophoblast cells

Abstract

ATP-diphosphohydrolase is associated with human syncytiotrophoblast mitochondria. The activity of this enzyme is implicated in the stimulation of oxygen uptake and progesterone synthesis. We reported previously that: (1) the detergent-solubilized ATP-diphosphohydrolase has low substrate specificity, and (2) purine and pyrimidine nucleosides, tri- or diphosphates, are fully dephosphorylated in the presence of calcium or magnesium (Flores-Herrera 1999, 2002). In this study we show that ATP-diphosphohydrolase hydrolyzes first the nucleoside triphosphate to nucleoside diphosphate, and then to nucleotide monophosphate, in the case of all tested nucleotides. The activation energies (Ea) for ATP, GTP, UTP, and CTP were 6.06, 4.10, 6.25, and 5.26 kcal/mol, respectively; for ADP, GDP, UDP, and CDP, they were 4.67, 5.42, 5.43, and 6.22 kcal/mol, respectively. The corresponding Arrhenius plots indicated a single rate-limiting step for each hydrolyzed nucleoside, either tri- or diphosphate. In intact mitochondria, the ADP produced by ATP-diphosphohydrolase activity depolarized the membrane potential (ΔΨm) and stimulated oxygen uptake. Mitochondrial respiration showed the state-3/state-4 transition when ATP was added, suggesting that ATP-diphosphohydrolase and the F1F0-ATP synthase work in conjunction to avoid a futile cycle. Substrate selectivity of the ATP-diphosphohydrolase was modified by ΔΨm (i.e. ATP was preferred over GTP when the inner mitochondrial membrane was energized). In contrast, dissipation of ΔΨm by CCCP produced a loss of substrate specificity and so the ATP-diphosphohydrolase was able to hydrolyze ATP and GTP at the same rate. In intact mitochondria, ATP hydrolysis increased progesterone synthesis as compared with GTP. Although dissipation of ΔΨm by CCCP decreased progesterone synthesis, NADPH production restores steroidogenesis. Overall, our results suggest a novel physiological role for ΔΨm in steroidogenesis.

Mitochondrial isolation, cryopreservation and preliminary biochemical characterisation from placental cytotrophoblast and syncytiotrophoblast

“…The increased mean fluorescent intensity associated greater membrane potential in cytotrophoblast mitochondria suggests a mitochondrial subpopulation more involved in ATP production, which is supported by the ATP results obtained in this study. The physiological ramification of this result reflects what has been observed in other studies that ATP production is not crucial for progesterone synthesis in syncytiotrophoblasts [31].…”

Placental mitochondria and reactive oxygen species in the physiology and pathophysiology of pregnancy

“…Placental mitochondria are critical to cell function, and mitochondrial damage is a feature of pregnancy complications ∆Ψm regulates ATP-diphosphohydrolase (an enzyme involved in cholesterol transport), and the majority of ATP produced is utilized for cholesterol transport and steroidogenesis. 18 As the syncytiotrophoblast is formed via the fusion of cytotrophoblasts, these mitochondrial differences represent an important shift in cellular function. Cytotrophoblast differentiation into the syncytiotrophoblast leads to decreased mitochondrial respiration and altered metabolism, including an increase in lactate production.…”

“…Therefore, lower dimeric ATP synthase levels may explain decreased ATP production in the syncytiotrophoblast compared to cytotrophoblasts . In the syncytiotrophoblast, most ∆Ψm regulates ATP‐diphosphohydrolase (an enzyme involved in cholesterol transport), and the majority of ATP produced is utilized for cholesterol transport and steroidogenesis …”

Tert-buthylhydroquinone pre-conditioning exerts dual effects in old female rats exposed to 3-nitropropionic acid

“…The pellets were resuspended again in the working solution at 4 °C. Protein concentration was determined using the commercial agent Bradford (Bio-Rad, Hercules, CA) [37].…”

“…Complex II was activated by pre-incubation of mitochondria in the presence of 5 mM PMS and DCPIP (6.3 mM). The decrease in absorbance was recorded for 5 min [37].…”