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Department of Biology, University of Rome Tor Vergata, 00133 Rome; and the Department of Biology, University of Rome 3 (S.I.), 00146 Rome, Italy; and the Departamento de Bioquimica de la Nutricion, Instituto Superior de Investigaciones Biologicas, Universidad Nacional de Tucuman (R.N.F.), 4000 Tucuman, Argentina
Address all correspondence and requests for reprints to: Dr. Sandra Incerpi, Department of Biology, University of Rome Tor Vergata, via della Ricerca Scientifica, 00133 Rome, Italy. E-mail: incerpi{at}uniroma3.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A membrane effect of L-T3 has been hypothesized since 1977, when Farias and co-workers showed that L-T3 affects the cooperative behavior of membrane-bound enzymes (12, 13, 14, 15) and that the activation or inhibition of Ca2+-ATPase by thyroid hormones depends on membrane fatty acid composition (16). The same group has also shown that rat and human erythrocyte membranes possess high affinity L-T3-binding sites (17, 18).
The Na/H antiport is a plasma membrane protein exchanging extracellular Na+ with cytoplasmic H+ ions according to the concentration gradient; therefore, it does not require energy supply for activity but depends on the Na/K-ATPase, the main modulator of the Na+ gradient (19). Besides its housekeeping functions (i.e. modulation of intracellular pH and cell volume), the Na/H antiport has been shown to play a more regulative role in the cell, being activated by phosphorylation with involvement of kinase activities such as protein kinase C and tyrosine kinase. At least theoretically all hormones and growth factors acting through kinases can modulate the antiport, resulting in variation in the intracellular pH, which can represent the first response of the cell to a wide range of signals (19, 20, 21, 22).
Thyroid hormones have well known genomic actions on the activity of the Na/H antiport in renal brush border membranes in opossum kidney cells in culture and on the expression of different isoforms of the Na/H antiport in the renal tubule (23, 24, 25). In contrast, no information is available on a possible short term modulation of this ion transport system.
This prompted us to study nongenomic effects of thyroid hormones on the Na/H antiport in L-6 cells, a cell line from rat skeletal muscle in which the nuclear effects of L-T3 have been well documented (26, 27). To assess the specificity of L-T3 on the plasma membrane, we used analogs of thyroid hormones, some of which have been reported to mimic the biological effects of L-T3 on the plasma membrane (6, 8, 28).
Our data show that physiological concentrations of both L-T3 and L-T4 increase intracellular pH through activation of the Na/H antiport, both in experiments at pH equilibrium and after an acid load with ammonium chloride. For L-T4, the time course of activation was slower with respect to L-T3, and dependent on hormone concentration; this suggests a high molecular specificity of L-T3 with respect to L-T4. In fact, experiments carried out in the presence of the inhibitor of the 5'-deiodinase, 6-n-propyl-2-thiouracil (PTU), indicate that L-T4 acts via deiodination to L-T3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Determination of intracellular pH
For the fluorescence assays cells were grown in chamber slides
(Lab-Tek, Nunc, Naperville, IL) and used at confluence. Before the
experiment cells were rendered quiescent by serum deprivation for
5 h. Intracellular pH was measured by the fluorescent
intracellular pH indicator
2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein
tetraacetoxymethylester (BCECF/AM). To rule out the contribution of
HCO3--dependent transport mechanisms (21), all
experiments were carried out in bicarbonate-free buffer with the
following composition: 135 mM NaCl, 5 mM KCl, 1
mM CaCl2, 1 mM MgCl2,
10 mM glucose, and 20 mM HEPES, pH 7.3. This
buffer (henceforth called 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.
To carry out experiments with acid load, solutions containing NH3/NH4+ were prepared from the above 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.
Incubation with the fluorescent dye was carried out as follows: cells were washed twice with Na+ buffer and were thus considered bicarbonate free. Cells were then incubated in Na+ buffer with the fluorescent dye (1 mg/ml in dimethylsulfoxide) at the final concentration of 1 µg/ml for 20 min at 37 C in the dark. Then the medium containing the dye was eliminated, and the cells were washed twice with the Na+ buffer.
Routinely at the end of each experiment, calibration of fluorescence vs. pH was carried out by an established method (29), using the K-H ionophore nigericin (5 µM) added to cells suspended in a potassium solution with the same composition as sodium buffer, but with NaCl substituted by equimolar concentrations of KCl. Under these conditions, intracellular and extracellular pH are equilibrated (pHi = pHo). The extracellular pH was changed with 10-µl aliquots of 1 M 2-[N-morpholino]ethanesulfonic acid or 1 M Tris (hydroxymethyl)aminomethane and determined with a glass electrode inserted directly into the cuvette. Intracellular fluorescence was determined and plotted vs. extracellular pH. 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 LS-5 luminescence spectrometer equipped with a chart recorder model R 100A (Perkin-Elmer, Norwalk, CT), with excitation and emission wavelengths of 500 and 530 nm, using 5- and 10-nm slits, respectively, for the two light paths. Fluorescence was also routinely measured at 450 nm excitation (at this wavelength the fluorescence is proportional to intracellular dye concentration but is relatively pH insensitive), and the value did not change more than 10% during the experimental period.
Determination of intrinsic ßi
The total intracellular buffering capacity, ßt, is
defined as: ßt = ßCO2 + ßi.
In the nominally HCO3--free solutions used in
this study, ßCO2 was assumed to be negligible, and
ßt was therefore taken to be equal to ßi.
The NH4+ pulse technique was used to determine
ßi, as previously described (30, 31), according to the
formula: ßi =
[NH4+]i/pHi,
where [NH4+]i represents the
change in concentration of intracellular NH4+
after exposure to or removal of extracellular NH3, and
pHi represents the corresponding change in
pHi. The intracellular concentration of
NH4+ during the NH4Cl pulse was
calculated as previously reported (30) from the following equation:
[NH4+]i =
[NH3]i x 108.92 -
pHi, taking into account that NH3 equilibrates
across the cell membrane (i.e.
[NH3]i =
[NH3]o) and that the pKa of
NH4+ (8.92) is the same intra- and
extracellularly. [NH4Cl] in the absence of
NH4Cl was assumed to be zero. From the product of ß and
the rate of pHi recovery can be calculated J, the net
efflux of acid, expressed as x10-4
mM/sec.
Solutions
BCECF/AM (1 mg/ml), and
5-(N-ethyl-N-isopropyl)amiloride (EIPA; 10
mM) were dissolved in dimethylsulfoxide, which did not
affect the fluorescence signal. Nigericin (10 mM) was
dissolved in ethanol. Thyroid hormones and analogs were dissolved in
0.1 M NaOH. PTU (1 mM) was added as an aqueous
solution.
Materials
DMEM, antibiotics, and sterile plasticware for cell culture were
obtained from Flow Laboratory (Irvine, UK). FBS was obtained from
Life Technologies (Grand Island, NY), and BCECF/AM was
obtained from Molecular Probes, Inc. (Eugene, OR).
Nigericin, HEPES, 2-[N-morpholino]ethanesulfonic acid,
Tris(hydroxymethyl)aminomethane, 3,3',5-triiodo-L-thyronine
(sodium salt, L-T3),
(3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]-L-alanine
(sodium salt, L-T4), 3,3',5-triiodothyroacetic
acid (Triac), 3,3',5,5'-tetraiodothyroacetic acid (Tetrac), and
3,3',5'-triiodo-L-thyronine (rT3) were supplied
by Sigma Chemical Co. (St. Louis, MO). 3,3',5-Triiodo-
D-thyronine (free acid, D-T3) and
PTU were purchased from ICN Pharmaceuticals, Inc. (Costa
Mesa, CA). EIPA was obtained from Research Biochemicals International (Natick, MA). All other chemicals were of the
purest grade available from Merck (Darmstadt, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The
effect of L-T3 normally started approximately 2
min after its addition for all concentrations tested (from
10-6–10-11 M) and reached a
maximum in about 10 min, whereas L-T4 showed a
lag time varying from 5 min at 10-6 M to 11
min at 10-11 M and reached a maximum in about
15 min (Figs. 1 and 2). The effect of
L-T3 and L-T4 added
together was a rapid increase after about 2 min that could be
attributed to T3; after reaching an initial plateau,
a second slower increase in intracellular pH attributable to
T4 appeared (Fig. 1C). Figure 1 also shows also the
inhibition of hormone effect at 10-7 M by EIPA
(10 µM), a derivative of amiloride and a specific
inhibitor of the Na/H antiport. EIPA alone is not reported, but it did
not significantly affect the basal pH.
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. The inhibition of the
L-T4 effect was in the range of 70%, whereas
the L-T3-induced response was completely
unaffected. The cell viability after PTU treatment was assessed by
trypan blue exclusion and was around 90%.
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).
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. At short
incubation times (5 min) L-T3 was the
only thyroid hormone/analog showing an effect on intracellular pH. Even
though Triac, Tetrac, rT3, and D-T3
were ineffective at physiological concentrations, a small but
significant increase was found for Triac and Tetrac at
10-7 M.
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(2 min for
L-T3); it is not shown for
L-T4 so as not to overload the recorder
tracing, but in any case it did not affect the fluorescent signal.
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and 6A) due to the
rapid diffusion of NH3. During the exposure to
NH4Cl (
4 min), pHi tended to decrease toward
the baseline due to slow inward diffusion of
NH4+. Removal of NH4Cl from the
medium rapidly decreased the pHi by approximately 0.75
± 0.10 pH units (mean ± SD; n = 10), with
respect to the baseline, due to NH3 leaving the cell. The
readdition of sodium buffer lead to the rapid recovery of intracellular
pH, with a time course normally fitted by a single exponential.
Therefore, a plot of dpHi/dt (x10-4 pH/sec)
vs. pHi is a straight line, intersecting the
x-axis at the final steady state pHi (Figs. 5B and 6B).
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). For L-T4 we tested different times of
preincubation from 2–10 min, but only 10 min was effective in
increasing both the rate of recovery and the pHi set point,
resulting in an alkalinization similar to that caused by
L-T3 (compare Figs. 5B and 6B). Shorter times
of preincubation were not effective, and for the sake of brevity we
report only the results for 5 min, which are similar to the control
values (Fig. 6). Figures 5A and 6A also
show the effect of EIPA, which completely prevented the recovery of
pHi. The regression lines (Figs. 5B and 6B), calculated
from experiments similar to those shown as representative in panels A
of the same figures, were parallel to the control (the same in both
panels), but the set-point was higher for the hormone-treated cells
(7.14 for control and 7.35 for L-T3) and also
for L-T4 preincubated for 10 min, whereas the
same hormone preincubated for 5 min gave an intercept with the
x-axis of 7.16, which was not significantly different from
the control value (Figs. 5B and 6B).
The pHi decrease caused by the removal of
NH4+ can be used to evaluate the buffering
capacity of the cells (ß) when the acid-base transport mechanisms are
blocked. The thyroid hormones did not affect ß, and the values
measured are in good agreement with the literature values for the
skeletal muscle (30), but the rate of recovery and the net efflux of
acid J (i.e. acid extrusion rate) were significantly
increased (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The stimulation of the Na/H antiport by physiological concentrations (10-10 M) of thyroid hormones results in a dose-dependent increase in intracellular pH. The involvement of the antiport is proven unequivocally by the effect of the specific inhibitor EIPA, both at equilibrium and under acid load with ammonium chloride. For L-T3, the intracellular pH started to increase approximately 2 min after its addition, independent of the concentration, whereas for L-T4 the effect was delayed and was always dependent on the concentration. Thus, 10-6 M L-T4 gave rise to an increase in the intracellular pH after 5 min, whereas about twice that time was required for a physiological concentration (10-10 M) of this hormone.
The stimulation of the Na/H antiport by thyroid hormones requires a number of conditions. The higher efficiency of L-T3 compared with L-T4 as well as the different time courses suggest that the active hormone is L-T3. The L configuration appears to be an essential requirement, as D-T3 is completely inactive. The particular 3,3',5-triiodo substituent arrangement of the aromatic rings also seems to be necessary for the stimulating effect, as rT3 is ineffective even at high concentrations. This 3,3',5-triiodo pattern is also found in the Triac and Tetrac analogs, which show some activity at high concentrations; there are, of course, no distinctions between D and L configurations for these two compounds.
L-T3 is formed by L-T4 after activation of a peripheral 5'-deiodinase resulting in the formation of bioactive L-T3 (33). In fact, deiodination is quantitatively the most important metabolic pathway of L-T4 and is responsible for about 80% of active hormone production. The reaction is catalyzed by a microsomal 5'-deiodinase, resulting in a reduction of the iodothyronine. This pathway gives rise to a cascade of less iodinated thyronines, including the most potent active form of the thyroid hormone, L-T3 (33). Two main types of 5'- deiodinase have been reported: type I and type II. Their tissue distribution and physiological roles are quite different. The extent of monodeiodination of L-T4 to L-T3 in skeletal muscle is not well established, but apparently varies much among different muscle types (34, 35). An uptake mechanism of 3,3',5-triiodo-L-thyronine entering the skeletal muscle cells by a stereospecific, energy- and temperature- dependent mechanism has also been reported (36). In this tissue L-T3 appears to derive almost exclusively from circulating L-T3 (37), without a contribution from the 5'-deiodination process of T4, thus indicating that the plasma membrane might modulate the availability of the active thyroid hormone (38). Moreover, using a PCR-based technique, the complementary DNA for type II 5'-deiodinase was recently found to be expressed at low levels in cardiac and skeletal muscle (39). It is not clear whether the deiodination activity in L-6 cells is due to a PTU-sensitive type II or to an as yet unknown type I enzyme that might be present in myoblasts but not in differentiated muscle. It should be noted that even low levels of 5'-deiodinase activity in muscle tissue may be physiologically relevant in peripheral L-T3 production due to the large overall mass of skeletal muscle.
The L-T3 uptake in rat skeletal muscle is a Na+ dependent process, even though the mechanism involved is not clear at present (38). In any case the presence of a sodium-dependent process connected to specific L-T3 uptake appears to be of particular interest in skeletal muscle, as it emphasizes the possible role of the cell membrane in regulating the intracellular availability of the hormone (38).
Specific receptors for thyroid hormones have been identified in erythrocyte membranes since 1983 (17, 18). Stimulation of rabbit erythrocyte phospholipid-dependent protein kinase activity by T3 (6, 8) and the presence of a Na/H antiport hormone activatable in red blood cells have also been reported (40, 41). We are unable at present to indicate the transduction pathway followed by L-T3 for the activation of the Na/H antiport, even though preliminary evidence from different cell types suggests that a protein kinase C might be involved (our unpublished observations).
From a comparison of our data on the Na/H antiport in L-6 cells with data available to date on the other nongenomic effects of thyroid hormones, it appears that they are in quite good agreement with those on the increase in Ca2+ content in rat thymocytes (10), where the fast increase in cytosolic calcium is more evident with L-T3 than with L-T4. Also, the stimulation of rabbit erythrocyte phospholipid-dependent protein kinase activity is more efficient with L-T3 than with L-T4 (6); the same is true for the cooperative behavior of acethylcolinesterase activity of rat erythrocyte (12). At variance with our results, L-T4 was more potent than L-T3 in the stimulation of erythrocyte Ca2+ ATPase activity (3). The data reported to date point to a difference in the dose responses of the two hormones (10, 16), but none of the studies points to a difference in the time course of the effect.
In summary, our data shed light on several aspects of thyroid hormone action at the cellular level: 1) the nongenomic activation of the Na/H antiport, and 2) the striking specificity of L-T3 with respect to L-T4 indicated by the lag time of the effect. These results appear to be of great relevance for the physiological role of thyroid hormones, confirming L-T3 as the main biological product triggering thyroid hormones action, as L-T4 probably produces an effect only after deiodination to L-T3. The data also confirm a role of L-T4 as a prohormone, as first suggested 20 yr ago (12).
It is interesting to speculate on the significance of our findings in
the light of existing models of muscle physiology. In the skeletal
muscle, a short term stimulation of the Na/H antiport by thyroid
hormones could contribute to counteract the tendency to acidosis due to
metabolic activity of the muscular contraction, for which one of the
main recovery mechanisms is the Na/H antiport (30). In fact, the
buffering capacity (ß) does not appear to be significantly affected,
but the net acid efflux (J) is increased (see Table 2), confirming a
nongenomic role for thyroid hormones in ion homeostasis (8) and in the
recovery from acidosis.
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Footnotes |
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Received June 8, 1998.
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-induced antiviral state
requires PKA and PKC activities. Am J Physiol 27:C1256–C1261
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pHi/20 min over basal pH and are the
mean ± SD of 4–10 different experiments. The
reported differences were significant (P < 0.05 at
least with respect to baseline pHi for both
L-T3 and L-T4, as
assessed by Student’s t test) starting from
10-10 M.
