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Short-Term Effects of Thyroid Hormones and 3,5-Diiodothyronine on Membrane Transport Systems in Chick Embryo Hepatocytes

Department of Biology (S.I.), University of Rome ‘Roma Tre,’ 00146 Rome, Italy; Department of Biology (P.D.V., P.L.), University of Rome ‘Tor Vergata,’ 00133 Rome, Italy; and Department of Cellular and Developmental Biology (S.S., S.L.), University of Rome ‘La Sapienza,’ 00185 Rome, Italy

Address all correspondence and requests for reprints to: Prof. Sandra Incerpi, Department of Biology, University of Rome ‘Roma Tre,’ Viale Marconi 446, 00146 Roma, Italy. E-mail: . incerpi{at}uniroma3.it

	Abstract

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Rapid nongenomic effects of thyroid hormones L-T₃ and L-T₄ on two plasma membrane transport systems were investigated in 14-d-old and 19-d-old chick embryo hepatocytes. The Na⁺/H⁺ exchanger activity was measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, whereas the amino acid transport was estimated by [1-¹⁴C]-2-aminoisobutyric acid uptake. System A amino acid transport activation was linear to hormone concentration, whereas the Na/H exchanger gave a bell-shaped dose-response curve, with a maximum at the physiological hormone concentration of 1 nM. The specificity of the effect was verified by the use of inhibitors and analogues. The thyroid hormone analog 3,5-diiodo-L-thyronine was able to mimic some of the hormone effects, but with a lower efficiency. The effect on the Na⁺/H⁺ exchanger was identified for 14-d-old and 19-d-old cells, whereas the amino acid transport could only be activated at the late stage of embryo development. Both transport systems were activated through a signal transduction pathway involving PKC, MAPK pathway, and PI3K, even though the differences in response behavior indicate a differential modulation of the two transport systems by L-T₃ and L-T₄. These results clearly demonstrate the existence of rapid nongenomic action of thyroid hormones also in avian cells, and show that activation of System A amino acid transport is not directly correlated to changes in intracellular pH. For the first time, evidence is presented which suggests that short-term effects of thyroid hormones may play a role during fetal development and cell differentiation.

	Introduction

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THE THYROID HORMONES L-T₃ and L-T₄ play important roles in growth, development, cell differentiation, and metabolism through their interaction with nuclear receptors (1, 2). Nongenomic actions of thyroid hormones are now also widely acknowledged, but the specific targets and mechanisms of action are quite difficult to recognize because these effects are found at the level of the plasma membrane, cytoskeleton, cytoplasm, and organelles of mammalian cells (3). Some of these short-term effects appear to involve the phosphatidylinositol pathway, PKC and the modulation of protein phosphorylation, and recent evidence suggests a role for MAPK (4, 5). Taken together, these findings indicate the existence of a receptor-activated signal transduction mechanism. Membrane receptors for thyroid hormones were identified years ago in rat erythrocytes by the group of Farias (6, 7). Thyroid hormone receptors have also been found in chick embryo synaptosomes, where they seem to modulate G protein functions (8).

In addition to L-T₃ and L-T₄, other iodothyronines, such as 3,5 diiodo-L-thyronine (3,5 L-T₂) and 3,3' diiodo-L-thyronine (3,3'L-T₂) are present in biological fluids, but at present it is not clear whether they are derived by peripheral deiodinase activity from thyroid hormones or they play a physiological role into the cell. In fact, these analogs have been reported to be able to mimic some effects of thyroid hormones in mammalian cells. 3,5 L-T₂ stimulates oxygen consumption in isolated perfused liver from hypothyroid rats and in mononuclear red blood cells (9). Both 3,5 L-T₂ and 3,3'L-T₂ stimulate the mitochondrial respiratory rate and cytochrome oxidase activity more rapidly than L-T₃, and liver mitochondria have specific binding sites for 3,5 L-T₂ (10). Nuclear receptors for these compounds have not been found so far.

We have recently reported the short-term nongenomic effects of thyroid hormones on the stimulation of the Na⁺/H⁺ exchanger, in L-6 myoblasts (11). Thyroid hormones also have well known genomic actions on the activity of this transport system in renal brush border membranes of mammalian kidney cells in culture and on the expression of different isoforms of the exchanger in the renal tubule (12, 13, 14). The Na⁺/H⁺ exchanger is a plasma membrane protein exchanging extracellular Na⁺ with cytoplasmic H⁺ ions according to the concentration gradient; it does not require an energy supply for activity but depends on the Na⁺/K⁺-ATPase, the main modulator of the Na⁺ gradient. Several isoforms of the protein have been reported and identified as NHE1 through NHE6. The NHE1 isoform is present in all mammalian cells and is considered to be the housekeeping isoform of the exchanger family: it is involved in the control of intracellular pH and cell volume (15, 16). The Na⁺/H⁺ exchanger has been shown to play a regulatory role in the cell, being activated by phosphorylation with involvement of kinase activities, such as PKC and tyrosine kinase. Therefore, an activation of the exchanger resulting in an increase of intracellular pH may represent the first response of the cell to a wide range of signals (17, 18, 19, 20, 21).

The activity of the Na⁺/H⁺ exchanger is supposed to be important for the neutral amino acid transport System A, which is controlled by hormones and growth factors in both adult and fetal cells. Many age-related differences in amino acid uptake regulation have been described in rat hepatocytes following different stimuli (22, 23). The A System carries preferentially alanine, an important gluconeogenetic amino acid in the liver. The alanine transport is Na⁺ and intracellular pH dependent and is modulated by hormones, growth factors, and substrate availability (24, 25, 26, 27, 28). Activation of System A transport may therefore be linked to modifications of the Na⁺/H⁺ exchanger activity (23, 28, 29).

In the present work, we have examined short-term effects of thyroid hormones on the Na⁺/H⁺ exchanger and the A System. As an experimental system, we have used chick embryo hepatocytes, isolated at two different stages of development. It is known that the liver is a pivotal target for the genomic effects of thyroid hormones, and about 5% of hepatic genes are under thyroid hormone control (30), but little information is available on the presence and the physiological role of short-term effects in this tissue. Moreover, as thyroid hormones have a relevant role in the prenatal development also in birds, it is important to know whether short-term effects are present during embryonal life and whether they are dependent on developmental stage. Because both the A System and the exchanger activities are activated by growth factors and related to cell proliferation, we have used hepatocytes isolated at 14 and 19 d of embryonal life, which have different proliferation rates (Leoni, S., unpublished observations).

Our results show that thyroid hormones L-T₃ and L-T₄ activate both transport systems by a nongenomic mechanism but in a differential way: amino acid System A transport is modulated only in the last period of chick embryo development, whereas the Na⁺/H⁺ exchanger is stimulated in both 14- and 19-d-old hepatocytes; furthermore, the dose-response of activation by thyroid hormone is different for the two transport systems. The 3,5 diiodothyronine is able to mimic the hormonal response only as far as the Na⁺/H⁺ exchanger is concerned, being totally ineffective on amino acid transport. The stimulation of these transport systems by thyroid hormones and 3,5 L-T₂ involves PI 3K, MAPK pathway, and PKC.

	Materials and Methods

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Hepatocyte preparation
White Leghorn (Gallus gallus) chick embryos (14 or 19 d old) were used to prepare hepatocytes, by immersion of small tissues pieces into a collagenase solution (31). The cell yield was about 4.0–5.0 x 10⁷ cells/g wet tissue and the average viability index, evaluated by Trypan blue exclusion, was 90%. Contamination of hepatocytes by nonparenchymal cells was about 5%. Chick embryo hepatocytes were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 200 mM L-glutamine, 5% (vol/vol) FCS, 2.5 ng/ml Amphotericin, 0.1 mg/ml gentamicin, 0.25 µM dexamethasone in a humidified atmosphere of 5% CO₂ at 37 C. For the experiments described below, cells were seeded either on 60-mm collagen-coated dishes at the concentration of 4.0 x 10⁶ cells/dish [for the [1-¹⁴C]2-aminoisobutyric acid (AIB) uptake] or on collagen-coated chamber slides (four-well Permanox slide) at the concentration of 2.0 x 10⁵ cells/well (for the intracellular pH measurement) and used after 48 h. These studies were conducted in accordance with the directives of the European Community (86/609/EEC) on the care and use of laboratory animals.

Amino acid transport
For the amino acid transport assay, AIB was used (specific activity 40–60 µCi/µmol). Chick embryo hepatocytes, kept at 37 C and oxygenated (5% CO₂ and 95% air), were incubated 10 min in Krebs-Henseleit solution with and without Na⁺, containing 0.1 µCi (10 µM final concentration) of the labeled amino acid in the presence of different concentrations of thyroid hormones. In the sodium-free medium, sodium chloride and bicarbonate were replaced by equimolar choline salts (24). For experiments in the presence of activators and inhibitors of PKC and PI3K, and of MAPK pathway, these were added 10 min before the addition of thyroid hormones or analogs. At the end of incubation, cells were rinsed twice with cold phosphate buffer and disrupted in 200 µl NaOH (0.2 M) and then kept at 100 C for 30 min. Aliquots were taken and the radioactivity was measured, after the addition of Optifluor, in a liquid scintillation spectrometer (Packard Instruments, Downers Grove, IL). The Na⁺-dependent component of AIB transport was determined by subtracting the AIB uptake in the absence of sodium from the uptake in its presence. The Na⁺-dependent uptake was linear up to 15 min. The protein content of each sample was evaluated by the method of Lowry (32).

Determination of intracellular pH
For the fluorescence assays, cells were seeded (about 2 x 10⁵ cells/well) in collagen-coated chamber slides (Lab-Tek, Nunc, Naperville, IL) and used at confluency, after 48 h from seeding.

Intracellular pH was measured by the fluorescent intracellular pH indicator 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM) as already reported (11). To rule out the contribution of HCO₃^--dependent transport mechanisms (33), all experiments were carried out in bicarbonate-free buffer with the following composition (mM): 135 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and HEPES (pH 7.3). This buffer (henceforth reported as Na⁺ buffer) was used for the incubation with the fluorescent probe and for the determination of intracellular pH, unless otherwise stated; the cells incubated in this buffer were considered virtually depleted of bicarbonate.

The experiments with acid load were carried out as already reported (11): solutions containing NH₃/NH₄⁺ were prepared from the above mentioned buffer where 20 mM NaCl was replaced with 20 mM NH₄Cl, and the remaining external Na⁺ was routinely replaced by equimolar choline chloride to keep the antiport quiescent.

Routinely at the end of each experiment, calibration of fluorescence vs. pH was carried out by the well established nigericin method (34). The calibration curve was linear in the pH range 6.5–7.8 (not shown).

Fluorescence was measured under continuous magnetic stirring at a controlled temperature (37 C) in a Perkin-Elmer Corp. (Shelton, CT) LS-5 luminescence spectrometer equipped with a chart recorder model R 100A, with excitation and emission wavelengths of 500 and 530 nm, using 5- and 10-nm slits, respectively, for the two light paths.

Determination of intrinsic ß_i
The total intracellular buffering capacity (ß_t) is defined as follows: . In the nominally HCO₃^--free solutions used in this study, ß_CO2 was assumed to be negligible and ß_t was therefore taken to be equal to ß_i, which was determined by the NH₄⁺ pulse technique, as previously described (Ref. 11 ; for a review see Ref. 35) according to the formula:

where [NH₄⁺]_i represents the change in concentration of intracellular NH₄⁺ after exposure to or removal of extracellular NH₃ and pH_i represents the corresponding change in pH_i.

Solutions
BCECF/AM (1 mg/ml), amiloride (10 mM), 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 10 mM), 2'amino-3'methoxyflavone (PD 98059), phorbol 12-myristate 13-acetate (PMA), wortmannin, bisindolylmaleimide IX methanesulfonate (RO 31-8220), and 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS) were dissolved in dimethyl sulfoxide, which did not affect the fluorescence signal and in any case its concentration in the presence of cells did never exceed 0.1%. Nigericin (10 mM) was dissolved in ethanol. Thyroid hormones and analogs were dissolved in 0.1 M NaOH. 6-n-Propyl-2-thiouracil (PTU, 1 mM) was an aqueous solution.

Materials
Collagenase (type D) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). [1-¹⁴C]2-Aminoisobutyric acid was from NEN Life Science Products (Boston, MA). Roswell Park Memorial Institute 1640 medium and FCS were from HyClone Laboratories, Inc. (Logan, UT). BCECF/AM was obtained from Molecular Probes, Inc. (Eugene, OR). Nigericin, HEPES, MES, Tris(hydroxymethyl) aminomethane, 3,3',5-triiodo-L-thyronine sodium salt (L-T₃), 3,3',5,5'-tetraiodo-L-thyronine (L-thyroxine or L-T₄), 3,5-diiodo-L-thyronine (3,5 L-T₂), D-thyroxine (D-T₄), PMA, wortmannin, RO 31-8220, amphotericin, gentamicin and collagen were supplied by Sigma (St. Louis, MO). EIPA was obtained from Research Biochemicals International (Natick, MA). 3,5,3'-Triiodo-D-thyronine (free acid, D-T₃), DIDS and PTU were from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). PD 98059 was from Alexis Biochemicals (Laufelfingen, Switzerland). All other chemicals were of purest grade available from Merck \|[amp ]\| Co., Inc. (Darmstadt, Germany).

	Results

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Effect of thyroid hormones on steady-state intracellular pH in chick embryo hepatocytes
The experiments were carried out using HCO₃^--free media to minimize the contribution of HCO₃^--dependent transport systems (33). In the standard HEPES-buffered, bicarbonate-free solution, the mean steady-state intracellular pH of chick embryo hepatocytes was 7.08 ± 0.09 and 7.05 ± 0.09 (mean ± SD; n = 6) for 14- and 19-d-old chick embryo hepatocytes, respectively: these data are in good agreement with values reported for other types of avian cells (35) and fetal rat hepatocytes (35, 36).

The dose-response of thyroid hormones (L-T₃ and L-T₄) and 3,5 L-T₂ on the activation of the Na⁺/H⁺ exchanger at the steady state is shown in Fig. 1. Both hormones stimulated the Na⁺/H⁺ exchanger in a wide concentration range with a maximum increase of intracellular pH at 1 nM for both 14- and 19-d-old cells, after 15 min, and a minor effect at higher and lower concentrations. L-T₄ and L-T₃ gave rise to an increase of intracellular pH of about 0.20 U pH/15 min over basal value for 19-d-old hepatocytes and about 0.15 U pH/15 min for the 14-d-old cells, well in agreement with the effect on the intracellular pH in L-6 myoblasts from rat skeletal muscle (11). The 19-d-old cells were more responsive to thyroid hormones than the 14-d-old cells, in particular, the difference in response to L-T₃ was significant between the two stages. Because the 3,5 L-T₂ has been reported to be able to mimic some nongenomic effects of thyroid hormones in liver mitochondria (10), we wanted to verify whether this analog of thyroid hormone might play a functional role also in chick embryo hepatocytes. The dose-response of 3,5 L-T₂ in a wide concentration range on the activation of the Na⁺/H⁺ exchanger showed a significant increase in intracellular pH (Fig. 1C), with a maximum at 1 nM of 0.11 ± 0.02 pH units/15 min (mean ± SD; n = 3) over the basal value for the 14 d, whereas the maximum increase for the 19 d was 0.12 ± 0.02 pH units/15 min (mean ± SD; n = 4). The effect of both hormones and 3,5 L-T₂ was inhibited by EIPA, an amiloride derivative and specific inhibitor of the Na⁺/H⁺ exchanger, confirming the specificity of thyroid hormone/analog action on the Na⁺/H⁺ exchanger (Fig. 2). Even though a HCO₃^--free buffer has been used throughout the experiments, we cannot rule out possible HCO₃^- production in metabolically active hepatocytes. However, experiments in the presence of DIDS (0.1 mM), an inhibitor of Na⁺dependent HCO₃^- transport systems indicate that the contribution of this transport system to the increase of intracellular pH by both thyroid hormones and 3,5 L-T₂ is negligible for the 19-d-old cells (Fig. 2). As to the 14-d-old cells, it was not possible to measure properly the activity of the exchanger in the presence of DIDS because this compound affected cell viability at concentrations as low as 15 µM.

View larger version (12K): [in this window] [in a new window]   Figure 1. Dose response of the effects of L-T3, L-T4, and 3,5 L-T2 on the steady-state intracellular pH of 14 ()- and 19 ()-d-old chick embryo hepatocytes. The results, derived from experiments carried at equilibrium, are given as pHi/15 min over basal value and are the mean ± SD of three to six different experiments. The reported differences for both L-T4 (A), L-T3 (B), and 3,5 L-T2 (C) were significant with respect to baseline pH (P < 0.05 at least, as from a t test) starting from 10-10 M for both 14- and 19-d-old hepatocytes. The asterisk (*) indicates that the difference between 19 d and 14 d was significant (P < 0.05, at least as from a t test) at the physiological concentration of 1 nM.

View larger version (21K): [in this window] [in a new window]   Figure 2. Inhibiton by EIPA and DIDS of the effects of L-T3, L-T4 and 3,5 L-T2 on the steady-state intracellular pH of 14- and 19-d-old chick embryo hepatocytes. Results of experiments carried out at equilibrium are shown as pHi/15 min over the basal value and are the mean ± SD of three to four different hepatocyte preparations. EIPA (10 µM; black box) was given to the cells together with the hormone, whereas DIDS (0.1 mM; hatched box) was given 10 min before hormone addition. EIPA and DIDS alone did not significantly affect the fluorescent signal. The hormone/analog (empty box) concentration was 1 nM. Fourteen-day-old cells did not overcome DIDS treatment over the low concentration of 15 µM.

L-T₄ is nowadays considered as a pro-hormone that is converted to the active form, L-T₃ by different deiodinase activities, reported to be present also in chick embryo hepatocytes (37). In particular, type 1 iodothyronine deiodinase (D1) is expressed in the liver and is considered responsible for the production of circulating L-T₃. The amount of active D1 enzyme increases 2- to 3-fold between d 14 and 18 of incubation, and remains stable afterwards (37). Therefore, chick embryo hepatocytes at different stages of development were pretreated 20 min at 37 C with the 5'-deiodinase inhibitor PTU (10 nM), then the effect of L-T₃ and L-T₄ on intracellular pH was tested (Fig. 3). As expected, PTU did not affect the functioning of L-T₃, whereas the effect of L-T₄ was partially suppressed (50–70%). The inhibition appeared to be less efficient in 19-d-old cells. Stronger inhibition was observed at high PTU concentrations, but under those conditions cell viability was affected; the results were no longer considered reliable and are not included.

View larger version (24K): [in this window] [in a new window]   Figure 3. Effect of PTU pretreatment on the hormone-mediated increase in intracellular pH of 14- and 19-d-old chick embryo hepatocytes. Chick embryo hepatocytes 14-d-old (empty box) and 19-d-old (hatched box) were incubated with BCECF/AM as reported in Materials and Methods and were treated 20 min with PTU (10 nM), then the hormones (1 nM) were added, and the change in fluorescence was followed for an additional 20 min. PTU at the concentrations employed did not affect the fluorescence signal. Results are given as the mean ± SD of at least three similar experiments and are reported as a percentage of the effect found in the presence of hormone alone (100%).

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of L-T3 on the pHi recovery from an acid load carried out with a NH3/NH4+ pulse, in 19-d-old chick embryo hepatocytes. Upper panel, After 2 min equilibration in Na+ buffer, cells were exposed to 20 mM NH4Cl for about 4 min in a choline chloride buffer containing 20 mM NH4Cl as reported in Materials and Methods. During the pulse pHi increased (because of the entry of NH3 into the cell) and then tended to decrease toward the baseline (because of slower influx of NH4+). Abrupt removal of NH4Cl gave rise to intracellular acidification, because of NH3 leaving the cell. The Na+ buffer was reintroduced allowing the recovery of the steady-state pH. L-T3 (1 nM) and EIPA (10 µM) were added at the time of recovery. A representative experiment is shown. Lower panel, dpHi/dt vs. pHi during recovery from a NH3/NH4+ pulse in control () and L-T3 (1 nM; )-treated 19-d-old chick embryo hepatocytes. Data are reported as regression lines calculated from the recoveries of experiments similar to those reported in the upper panel. Results are the mean of at least three different experiments.

Transduction pathways involved in the activation of the Na⁺/H⁺ exchanger by thyroid hormones
Some experiments were made to assess the involvement of kinase activities in the stimulation of the Na⁺/H⁺ exchanger in chick embryo hepatocytes, using inhibitors of PKC, MAPK, and PI3K. The treatment with the PKC inhibitor RO 31–8220 prevented the thyroid hormone stimulation of the Na⁺/H⁺ exchanger at both stages of development, even though the 14-d-old hepatocytes were less sensitive to the inhibitor treatment than 19-d-old cells. The inhibitors of PI3K, wortmannin, and of MAPK pathway, PD 98059, also gave rise to significant inhibition in all cases suggesting a role for these three kinases in the mediation of the stimulation of Na⁺/H⁺ exchanger in both 14-d-old and 19-d-old chick embryo hepatocytes (Fig. 5). From a careful inspection of Fig. 5, it appears that wortmannin was less effective in the inhibition of both L-T₃ and 3,5 L-T₂ with respect to L-T₄ (P < 0.05, at least as from a t test). In fact, about 25% of the effect of L-T₃ on intracellular pH was retained after wortmannin treatment.

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of PKC inhibitor RO 31-8220, PI3K inhibitor wortmannin and MAPK pathway inhibitor PD 98059 on the effect of L-T3, L-T4 and 3,5 L-T2 on the steady-state intracellular pH in 14- and 19-d-old chick embryo hepatocytes. Results are reported as percentage of increase of pHi/15 min, with respect to 100% variation of intracellular pH in the presence of hormone or its analog (1 nM), and are the mean ± SD of three to four separate experiments carried out on different hepatocyte preparations either 14-d-old cells (empty box) or 19-d-old cells (hatched box). All inhibitors were given to the cells 10 min before hormone addition, and none of them at the concentrations employed affected the fluorescent signal: RO 31-8220 and PD 98059 (10 µM), wortmannin (50 nM). *, P < 0.05, at least, with respect to L-T3 and 3,5 L-T2, as from a t test.

View larger version (13K): [in this window] [in a new window]   Figure 6. Effect of down-regulation of PKC, by overnight treatment with PMA on the L-T3 increase of the steady-state intracellular pH in 14- and 19-d-old chick embryo hepatocytes. Results are reported as pHi/15 min, with respect to basal value of intracellular pH and are the mean ± SD of three to four experiments carried out on different hepatocyte preparations. Hormone concentration was 1 nM (empty box), whereas PMA was 10 µM (black box).

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of PKC inhibitor RO 31-8220, of PI3K inhibitor wortmannin and of MAPK pathway inhibitor PD 98059 on L-T3, L-T4, and 3,5 L-T2 activation of the AIB uptake in 19-d-old chick embryo hepatocytes. Hormone/analog concentration was 1 nM (empty box). Inhibitors (black box) concentration was as reported in the legend to Fig. 5. Results are reported as percentage stimulation over basal value (100%) and are the mean ± SD of three experiments carried out on different hepatocyte preparations. *, P < 0.05 at least, with respect to cells incubated with the hormone and without inhibitor, as evaluated by a t test; **, P < 0.05, with respect to L-T3 plus wortmannin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The activation by a nongenomic mechanism of the Na⁺/H⁺ exchanger in L-6 rat myoblasts has previously been reported by some of us (11). In chick embryo hepatocytes it appears that both L-T₃ and L-T₄ are more effective at the late stage of embryonal development, when the embryo is close to term. The 3,5 L-T₂ which is able to mimic some nongenomic actions of thyroid hormones (9), is also able to activate the Na⁺/H⁺ exchanger, although with a minor efficiency with respect to L-T₃. The specificity of the effect was assessed by the use of inhibitors of the exchanger such as EIPA, and by the use of the most common thyroid hormone analogues. Our data with DIDS, an inhibitor of the Na⁺-dependent HCO₃^- transporter, indicate that this system is not significantly involved in the increase of pH_i elicited by thyroid hormones in chick embryo hepatocytes.

The higher sensitivity to thyroid hormones in the cells close to term is in agreement with the higher activity of 5'-deiodinase (type 1, D1) in the chick embryo at this stage, with respect to an earlier stage of development (37). However, the remaining 30–50% of the effect might actually be due to L-T₄, as described below. An appropriate level of thyroid hormone is essential for normal development in vertebrate species. This appears to be particularly relevant in humans, where deficiency of thyroid hormone in the central nervous system during fetal development or the neonatal period results in mental retardation and cretinism. Also the exposure of the embryo to excessive thyroid hormone levels is detrimental, and may likewise result in fetal malformation, growth and mental retardation (38). In this context, the bell-shaped dose-response curve of thyroid hormones on the Na⁺/H⁺ exchanger activation is particularly intriguing. In fact, the lower sensitivity of the transporter to high hormone concentration could be a protection mechanism against excessive exposure of these cells to high hormone levels, as already reported for other hormones (39, 40).

The acid load experiments in 19-d-old fetal hepatocytes indicate that L-T₃ increases the rate of recovery of the steady-state pH, adjusting the set point to a new value. As a consequence the net efflux acid (J) is increased, as in mammalian cells (11). At variance with these results the 14-d-old hepatocytes were completely insensitive to the acid load with ammonium chloride. It is interesting to speculate about the significance of these findings. First, chick embryo hepatocytes appear to be less sensitive to the acid load with respect to mammalian cells, showing a high buffering capacity of about 100 mM/pH, as already reported for other cell types from avian tissue (38). It seems that these cells have a higher capacity of preserving intracellular pH. In fact, alterations of intracellular pH is a treatment that disrupts embryo development in vitro (41), and therefore the maintenance of an appropriate intracellular pH may be a pivotal feature for embryo survival. Second, thyroid hormone increases the net acid efflux and this might contribute to the ability of the cell to maintain the intracellular pH or, since the exchanger mediates electroneutral exchange of 1 Na⁺ for 1 H⁺, it might also contribute to preserve intracellular sodium ions, a behavior which in land birds seems to be of great physiological relevance (42).

The lack of a significant difference in the stimulatory effectiveness of thyroid hormones on the exchanger at both stages of embryonal development, suggests that the activation of this transport system is not related to the proliferative activity of the hepatocytes, which is significantly different in the two stages of development (our unpublished observations).

At variance with the Na⁺/H⁺ exchanger, the activation of AIB transport by thyroid hormones appears related to the proliferative activity and to the stage of embryonal development, being highly significant at 19 d when the embryo is close to term and insensitive to thyroid hormones at the 14-d stage. L-T₃ and L-T₄ appear to be able to activate this transport system in the physiological range, whereas 3,5 L-T₂ shows little effect and only when added at very high concentrations (not shown). The dependence of the hormonal control of System A on development has previously been observed in these cells upon treatment with EGF; it is also found in fetal rat hepatocytes treated with EGF, insulin, glucagon, or epinephrine, and can be related to the different distribution of these cells in the phases of cell cycle (27, 28, 43).

The System A amino acid transport is believed to be dependent on intracellular pH (27, 29), and it is therefore very interesting that our results clearly demonstrate that System A activity is not directly correlated with pH_i. In fact, high levels of thyroid hormones do not lead to an increase in pH_i but still give the largest increase in AIB transport capacity. This observation suggests that System A is not controlled directly through pH_i, but rather through a receptor-based signal transduction pathway, which also activates the Na⁺/H⁺ exchanger.

The signal transduction pathways of the short-term effects of thyroid hormones are not known, but recent papers indicate an involvement of PKC, PI3K, and MAPK pathway (4, 5). Because both Na⁺/H⁺ exchanger and amino acid transporter may be modulated by PKC and by PI3K products (28, 44), we studied the involvement of these kinases by a pharmacological approach, and our data indicate the involvement of all three kinases in the mediation of nongenomic actions of thyroid hormones. Some authors have reported that the activity of NHE1 and NHE3 isoforms of the exchanger are regulated by PI3K and by its products, as it contains binding sites for PIP₂ (44). The PI3K is important for the translocation to the cell membrane from intracellular vesicles of some transporter systems, and the Na⁺/H⁺ exchanger may be one of these (44). In our cells, wortmannin, a potent inhibitor of this kinase, gives rise to a net decrease of the stimulation by thyroid hormones of both the Na⁺/H⁺ exchanger, reduced by about 75% with slight differences between L-T₃ and L-T₄, and the amino acid transport, suggesting that these hormones activate PI3K. But the different extent of inhibition for L-T₄ and L-T₃ in the two transport systems also suggests differences in the transduction pathways between the two hormones. This point deserves further investigation and it is now under study in our laboratory.

In conclusion, the Na⁺/H⁺ exchanger and the System A amino acid transport show different short-term modulation by thyroid hormones. First of all, the dose-response to thyroid hormones shows a bell-shaped curve for the exchanger, whereas it is linear for the amino acid transport; second, the response of the exchanger to the hormones already appears in 14-d-old cells, whereas the amino acid transport is activated only at 19 d of fetal development; third, 3,5 L-T₂ mimics the thyroid hormone effect on the Na⁺/H⁺ exchanger, but not on the amino acid transport. The nongenomic actions of thyroid hormones are present in avian hepatocytes during prenatal life and probably play an important role during development and terminal differentiation, with a rapid regulation of intracellular pH, Na⁺ flux and amino acid transport.

	Acknowledgments

 
We thank Prof. Jens Z. Pedersen for helpful comments and stimulating discussion.

	Footnotes

 
This work was supported by: Grant No. MM05A75274 ‘Cofinanziamento 2000' from the Italian Ministry of University and Scientific-Technological Research, a grant from the Department of Biology, University of Rome ‘Roma Tre’ ‘Sviluppo 2000,’ and by a grant from the Italian Ministry of the University and Scientific-Technological Research, formerly 60% funds.

Abbreviations: AIB, [1-¹⁴C]2-aminoisobutyric acid; BCECF/AM, 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein acetoxymethyl ester; DIDS, 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid; EIPA, 5-(N-ethyl-N-isopropyl) amiloride; PMA, phorbol 12-myristate 13-acetate; PTU, 6-n-propyl-2-thiouracil.

Received November 2, 2001.

Accepted for publication January 3, 2002.

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