Nongenomic Effect of T3 on the Na, K-ATPase Activity of Syncytiotrophoblast from Human Term Placenta

Several ion transporting mechanisms, such as the plasma and sarcoplasmic membrane Ca-ATPase, the Na,K-ATPase, the Na/Hex changer and the Na/Pico transporter can be modulated by thyroid hormones, i.e.3,5,3′-triiodo-L-thyronine (T3) and 3,5,3′,5′-tetraiodothyronine (T4) [1]. Although these transporters are positively modulated by thyroid hormones through genomic mechanisms [2] some of them have been also found to be affected by non genomic mechanisms [2-5], with short time courses and probably mediated by specific receptors of the plasma membranes. Na,K-ATPase is an important primary ion transport system that is essential to keep the ion homeostasis and cell volume as well as the resting membrane potential. This enzyme is widely distributed in practically all animal tissues and it is regulated by different hormonal and non-hormonal factors. Among the hormonal factors, the thyroid hormones regulate the functionality of the enzyme by both genomic and non genomic mechanisms [6].


Introduction
Several ion transporting mechanisms, such as the plasma and sarcoplasmic membrane Ca-ATPase, the Na,K-ATPase, the Na/Hex changer and the Na/Pico transporter can be modulated by thyroid hormones, i.e.3,5,3′-triiodo-L-thyronine (T 3 ) and 3,5,3′,5′-tetraiodothyronine (T 4 ) [1]. Although these transporters are positively modulated by thyroid hormones through genomic mechanisms [2] some of them have been also found to be affected by non genomic mechanisms [2][3][4][5], with short time courses and probably mediated by specific receptors of the plasma membranes. Na,K-ATPase is an important primary ion transport system that is essential to keep the ion homeostasis and cell volume as well as the resting membrane potential. This enzyme is widely distributed in practically all animal tissues and it is regulated by different hormonal and non-hormonal factors.
Among the hormonal factors, the thyroid hormones regulate the functionality of the enzyme by both genomic and non genomic mechanisms [6].
The syncytiotrophoblast of the human placenta is a polarized cell layer with a maternal-facing microvillous membrane (MVM) and a fetal-facing basal membrane (BM). This cell layer interfaces the maternal and fetal circulations. The Na,K-ATPase of the syncytiotrophoblast has been shown to be present in both MVM and BM, playing a key role in intracellular ion homeostasis as well as constituting the force generator for the growth of the developing fetus [7]. In fact, the Na + gradient between the cell and its medium, maintained by the enzyme, activates Na + dependent amino acid transporters, driving toward the fetus amino acids and nutrients essentials for his normal growth.
Besides, the Na + gradient is essential for the intracellular pH control and it is involved in the net transport of Na + to the fetus [7][8][9]. Considering the fact that the Na, K-ATPase activity plays a key role in the growth and development of the human fetus, it was evaluated the non genomic effect of the thyroid hormones on the activity of this enzyme in MVM and BM from human term placenta.

Placenta collection
Term placentas obtained from uncomplicated normal women were collected immediately after delivery, from the Maternity Hospital "Concepción Palacios" in Caracas, and transported to the laboratory on ice. This study was carried-out in accordance with the ethical standards established by the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of the Maternity ''Concepción Palacios'' and the Bioethics Committee of IVIC; all women gave signed informed consent.

Explant preparation and culture
After removal of the chorionic plate and about 0.25cm of deciduas, explants were prepared using only tissue from the intermediate region of the placenta and following the procedure described elsewhere [10].

Preparation of syncytiotrophoblast plasma membranes
The membranes (MVM and BM) were isolated from placental explants following a method previously described [11,12].

Na, K-ATPase activity assay
The ATPase activity was determined by measuring the quantity of inorganic phosphate liberated from the hydrolysis of ATP, according to the method described elsewhere [13]. The ATPase activity is expressed as nmol Pi/mg protein. min. The protein concentration, in all the cases, was determined according to the micro assay method of Bio Rad [14]. In order to avoid presence of membrane vesicles, the membrane fractions were pretreated before the assays with SDS, as previously described [15].

Statistical analysis
Statistical analysis was performed by the Student's t-test. All results are expressed as means±standard error (S.E.) and n represents the number of experiments performed with different preparations. ATPase activities were calculated from paired data. A p-value <0.05 was accepted as statistically significant. Figure 1 shows the effect of different concentrations of T 3 and T 4 in the incubation medium, on the Na,K-ATPase activity of human placenta syncytiotrophoblast microvillous (MVM, panel A) and basal (BM, panel B) plasma membranes. Notice how T 3 , at concentrations over 2.5nM inhibits in a concentration dependent way the activity of the enzyme of MVM, but it does not inhibit the ATPase activity of BM, even at a concentration of 15nM. On the other hand, T 4 even at a concentration of 500nM does not produce any effect on the Na,K-ATPase activity of both membranes. Considering the fact that T 3 and T 4 are both present in the blood plasma, there is a possibility that T 4 could interfere with the effect of T 3 on the Na,K-ATPase activity of MVM. To test this possibility, we determined the enzyme activity in the presence of both 10nM T 3 and 350nM T 4 . The results of this experiment are shown in Figure 2. It can be seen that T 4 does not interfere with the inhibitory effect of T 3 on the Na,K-ATPase activity of MVM.  The presented results indicate a nongenomic effect of T 3 on the Na,K-ATPase activity of MVM. The possibility of T 3 binding to receptors in MVM in order to exert its effect was tested, incubating placental explants for six hours at 37 °C under normoxia conditions (8% O 2 , 87% N 2 , 5% CO 2 ), in the presence and absence of 10nMT 3 . The explants were then washed, homogenized and assayed for Na,K-ATPase activity. The results of this experiment are shown in Table 1. The Na,K-ATPase activity of explant homogenates preincubated in the presence of T 3 , shows an important inhibition, as compared to explants incubated under the same conditions but without T 3 . The Na,K-ATPase activity from MVM fractions isolated from the same homogenates does not show any inhibition (data not shown). This result may be taken as a clear indication that T 3 is washed away from the membranes during the MVM isolation procedure. The four groups were then assayed for Na,K-ATPase activity. The results of this experiment are shown in Figure 3. Condition A: Freshly prepared MVM fractions (2 mg protein/ml) were preincubated for 10 min at 37 °C in the presence of 10 nMT3. The membrane fraction was then diluted 10 fold (in order to dilute T3), and assayed for Na, K-ATPase activity.

Results
Condition B: MVM fractions were preincubated with T3, washed by centrifugation at 100,000xg for 1 hour and then assayed for Na, K-ATPase activity.
Condition C: MVM fractions were preincubated with T3, washed by centrifugation, preincubated again with T3 and then assayed for Na, K-ATPase activity. Values expressed as means± standard error, for n = 6. Second preincubation with 10nMT 3 of the already preincubated and then centrifuged membranes, produces again a significant reduction of their ATPase activity.

Discussion
In general, the thyroid hormones effects have been explained in terms of the expression of specific genes, i.e. genomic effects, or direct effects through interactions at the plasma membrane, in cytoplasm or at intracellular organelles, such as mitochondria, i.e. non genomic effects. The genomic mechanism has been explained as a primary interaction of T 3 with nuclear thyroid hormone receptor proteins. This primary interaction leads to the formation of intranuclear complexes formed by T 3 and its nuclear receptor. These intranuclear complexes modulate transcription through binding to the promoter regions of thyroid hormoneresponsive genes [16][17][18].
It has been suggested that the nongenomic effects of the thyroid hormones in intact cells may be initiated at the plasma membrane, in cytoplasm or at intracellular organelles. In the current study it was found an inhibitory effect of T 3 on the Na,K-ATPase activity of microvillous membranes of syncytiotrophoblast from human term placenta (Figure 1). This inhibitory effect is due to a direct binding of T 3 to the plasma membrane, as indicated by the fact that the Na,K-ATPase activity is diminished either in homogenates of placental explants preincubated with T 3 (Table 1) or in MVM fractions preincubated with T 3 (Figure 3). Even more, T 3 bound to MVM can be washed away by centrifugation of the plasma membranes at 100,000xg ( Figure 3).
The Na, K-ATPase activity inhibition of the placental MVM produced by T 3 could have important implications for the growing fetus. On this regard, different transport processes across the plasma membranes of eukaryotic cells are sodium coupled, and depend on the maintenance of a low intracellular Na + concentration. This condition is mainly achieved by the Na, K-ATPase, exchanging intracellular Na + ions for extracellular potassium K + ions. Consequently, any alteration in the transport capacity and/or expression of the Na,K-ATPase, would lead to a dysfunction in the maintenance of the Na + gradient across the cell plasma membrane, which will indirectly affect the driving forces of other membrane transport systems.
The differential effect of T 3 on the Na,K-ATPase activity of the syncytiotrophoblast plasma membranes, inhibiting the enzyme activity on the maternal side of the membranes (MVM), but not on their fetal side (BM), could result in an important disorder in the transepithelial Na + transport between the mother and the fetus. Consequently, pregnant mothers with hyperthyroidism might be facing this specific disorder. Even more, the resulting intracellular Na + accumulation, which is accompanied by water, will affect some of the normal cell parameters and mechanisms, such as cell volume, intracellular pH and Ca 2+ concentration, glucose and amino acid transepithelial transport from the maternal blood to the fetal blood [4,19]. The presented results may be taken as an indication that inhibition of the Na,K-ATPase of syncytiotrophoblast plasma membranes by T 3 could be contributing to the low weight gain of fetuses of pregnant women with hyperthyroidism.