Contribution of Potassium in Human Placental Steroidogenesis

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.

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.

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.