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Endocrinology Vol. 144, No. 9 3783-3788
Copyright © 2003 by The Endocrine Society

Thyroid Hormone Administration to Hypothyroid Rats Restores the Mitochondrial Membrane Permeability Properties

Rosa A. Vacca, Loredana Moro, Giovanni Caraccio, Ferruccio Guerrieri1, Ersilia Marra and Margherita Greco

Institute of Biomembranes and Bioenergetics (R.A.V., L.M., E.M., M.G.), Consiglio Nazionale delle Ricerche, Bari, Italy; and Department of Medical Biochemistry and Biology (G.C., F.G.), University of Bari, I-70126 Bari, Italy

Address all correspondence and requests for reprints to: Dr. Margherita Greco, Institute of Biomembranes and Bioenergetics, Consiglio Nazionale delle Ricerche, Via Amendola 165/A, I-70126 Bari, Italy. E-mail: csmmmg14{at}area.area.ba.cnr.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the effect of thyroid hormone on the mitochondrial membrane permeability properties in a hypothyroid rat model. The role played by calcium in affecting these properties has been also examined. Cyclosporin A-sensitive mitochondrial calcium efflux, swelling, and external release of matrix proteins are events that occur normally during the permeability transition process induced by calcium loading of mitochondria. We demonstrate that these events are impaired in mitochondria isolated from the liver of hypothyroid rats, even in the presence of high calcium content. However, after thyroid hormone administration to hypothyroid rats, the mitochondrial permeability transition process in response to calcium loading is restored. Consequently, mitochondrial calcium efflux, swelling, and release of matrix proteins, like glutamate dehydrogenase, malate dehydrogenase, and aspartate aminotransferase occur. These effects are abrogated by the concomitant administration of cyclosporin A. The results of the present study suggest that hypothyroidism may be a potential source of adverse effects in patients receiving cyclosporin A.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CALCIUM-DEPENDENT PERMEABILITY transition pore (PTP), inhibited by cyclosporin A (CsA), regulates most of the permeability properties of the inner mitochondrial membrane (1, 2, 3). Such a putative pore, responsible for the mitochondrial permeability transition (MPT), plays a major role in many physiological and pathophysiological processes including intracellular signal transduction, ischemia reperfusion damage, liver regeneration, apoptosis, and mitochondria anoxic-reoxygenation damage (3, 4, 5, 6, 7, 8). Although the nature of the pore is still under investigation, it is commonly accepted that mitochondrial calcium level is involved in MPT and the mitochondrial adenine nucleotide translocase (ANT) participates in the formation of the transmembrane nonspecific pore, in a manner dependent on the presence of cardiolipin molecules, which can bind ANT in high amounts (9, 10, 11). Independent of the nature of this pore, its function depends on the appropriate localization in the inner membrane, which, in turns, depends on the composition of the lipid milieu of the phospholipid bilayer (11).

Because T3 induces changes in the fatty acid and phospholipid composition of the inner mitochondrial membrane, affecting its fluidity (12), and because hyperthyroidism results in the occurrence of MPT (13), it is likely that T3 deficiency can result in altered mitochondrial membrane permeability properties, thus contributing to the onset of mitochondrial dysfunctions (14, 15, 16, 17, 18, 19). In this paper, we have investigated, in a hypothyroid rat model, whether hypothyroidism causes MPT dysfunction and whether mitochondrial membrane permeability properties can be restored by T3 administration. The role played by calcium in inducing permeability transition (PT) in the mitochondria of hypothyroid rats and T3-treated hypothyroid rats was also examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
6-n-Propyl-2-thiouracil (PTU), T3, and Arsenazo III were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of high-purity grade. CsA was a gift from Sandoz Pharmaceutical Products (Milan, Italy).

Animals
Male Wistar rats (200–250 g) were housed at a temperature of 22 C with food and water ad libitum. Chemical hypothyroidism was induced in laboratory animals by administration of 0.1% wt/vol PTU in drinking water for 21 d as previously described (14). Hyperthyroidism was induced in PTU-treated rats by ip injection of 30 µg T3/100 g body weight for 3 d (14). Twenty-four hours after the final administration, the animals were anesthetized with an ether/oxygen mix, killed by decapitation, and the trunk blood was collected. The liver was excised and used for mitochondria preparation. Control animals received only the solvent, for the same period of time. All operations were carried out under sterile conditions. The animals received humane care, and the study was approved by the State Commission on Animal Experimentation.

Determination of T3
Blood, collected from animals, was quickly mixed with an equal volume of ice-cold 0.9% NaCl containing 0.24 mg EDTA x 100 ml-1. Plasma was separated by centrifugation in the cold and the samples stored at -70 C until assayed. Plasma T3 was determined using commercial T3 LIA kits (Diagnostic Products BYK-Gulden, Cormano-Milano, Italy). After incubation, the tubes were thoroughly decanted, and the luminescence was determined. Standard curves were constructed by plotting the amount of total luminescence against the hormone concentration.

Preparation of mitochondria
Rat liver mitochondria were prepared at 4 C according to Bustamante et al. (20) using a medium containing 0.25 M sucrose and 5 mM Tris/HCl (pH 7.4) as isolation buffer. In the preparation of mitochondria used for measurement of calcium content, 1.6 µM ruthenium red and 1 mM EGTA were added in the isolation buffer to restrict calcium movement during the subfractionation technique (21). Protein concentration was determined using a kit (Bio-Rad Laboratories Inc., Segrate-Milano, Italy) and albumin as standard.

Determination of mitochondrial calcium content
For determination of the endogenous mitochondrial calcium content, mitochondria (0.1 mg/ml) were suspended in isolation buffer [0.25 M sucrose, 5 mM Tris/HCl (pH 7.4)] in the presence of 40 µM Arsenazo III. The absorbance change at 675–685 nm was monitored by dual wavelength spectrophotometry, using a spectrophotometer (Lambda 3B, Perkin-Elmer, Norwalk, CT). After reading a baseline for 1 min, 0.2% Triton X-100 plus 3.3 µM sodium dodecyl sulfate were added to disrupt the mitochondrial membranes. The absorbance change was calibrated by adding standard aliquots of CaCl2 to the incubation medium. A standard curve was obtained from the pooled results of five independent series of determinations and used for analysis of the mitochondrial calcium content (21).

Determination of mitochondrial calcium influx and efflux
For determination of calcium influx, mitochondria (10 mg protein) were washed once with the isolation buffer, with exclusion of EGTA and ruthenium red, and incubated at 25 C in 1 ml swelling medium [0.2 M sucrose, 5 mM succinate/Tris, 10 mM 3[N-morpholine]propanesulfonic acid/Tris, 1 mM Pi/Tris, 2 µM rotenone, and 1 µg/ml oligomycin (pH 7.4)] containing CaCl2 (50 nmol/mg mitochondrial proteins). After 1 min, mitochondria were recovered by centrifugation at 7300 x g for 40 sec and washed twice with isolation buffer, and the calcium content was determined on 0.1 mg mitochondrial proteins, as described above.

Calcium efflux from mitochondria (10 mg protein/ml) either preincubated or not, for 1 min, with CaCl2 (50 nmol/mg mitochondrial proteins) was measured during a 15-min incubation time in the swelling medium above reported. Where indicated, CsA (1.7 nmol/mg mitochondrial protein) was added to the medium. At the indicated time, aliquots (0.1 ml) were taken and centrifuged at 7300 x g for 40 sec. The mitochondrial pellet was washed twice with the isolation buffer, and the calcium content was determined as described above.

Swelling assay
To analyze the mitochondrial swelling properties, mitochondria (0.35 mg protein/ml) were suspended in the swelling medium above reported and incubated at 25 C. During a 10-min incubation time, the absorbance change of the mitochondrial suspension, which is an index of change in mitochondrial membrane permeability (22), was followed at 540 nm, using a DU7400 spectrophotometer (Beckman, Palo Alto, CA) equipped with magnetic stirring and thermostatic control. Where indicated, 1 µM CsA and/or 15 µM CaCl2 were added to the medium.

Matrix protein release assay
For the assay of the in vitro release of mitochondrial matrix proteins, isolated mitochondria (10 mg protein/ml) were suspended in the swelling medium above reported and incubated at 25 C for 10 min. Where indicated, CsA (1.7 nmol/mg mitochondrial protein) and/or CaCl2 (50 nmol/mg mitochondrial protein) was added to the incubation medium. At the indicated times, aliquots (0.1 ml) were taken and the mitochondria were precipitated by centrifugation at 8000 x g for 40 sec. The supernatants were centrifuged for 2 min at 10,000 x g. Mitochondrial aspartate aminotransferase (AAT) (23), glutamate dehydrogenase (GDH) (24), and malate dehydrogenase (MDH) (25) activities were determined in the final supernatants. Mitochondrial AAT, GDH, MDH, adenylate kinase (ADK) (26) and monoamine oxidase (MAO) (27) activities were also determined in isolated mitochondria.

Statistical analysis
Data are reported as the mean ± SEM of five independent measurements on the samples obtained from five different animals for each experimental group (normal, hypothyroid, and T3-treated hypothyroid rats). The statistical significance of differences among groups was determined by the one-way ANOVA followed by a Student-Newman-Keuls test. Comparison between independent means was performed using the t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thyroid hormone status
Chemical hypothyroidism was induced in rats by administration of PTU, as previously described (14). PTU-treated rats exhibited a significant decrease in T3 serum levels when compared with the control group (86 ± 5 ng/dl vs. 182 ± 11 ng/dl, P < 0.0001). The switching from hypo- to hyperthyroidism was induced by administration of T3 (30 µg/100 g body weight) for 3 d to PTU-treated rats (14). After T3 administration, T3 serum levels in hypothyroid rats rose to values higher than 800 ng/dl.

MPT in hypothyroid and T3-treated hypothyroid rats: content, influx, and efflux of calcium
Because a large number of agents that induce PTP opening can stimulate the mitochondrial Na-independent calcium efflux through the opening of the calcium-activated nonspecific channel in the mitochondrial membrane (1), measurements of content, influx, and efflux of calcium were performed in liver mitochondria isolated from hypothyroid rats (H-RLM), compared with mitochondria isolated from normal rats (N-RLM), and in mitochondria isolated from T3-treated hypothyroid rats (T3-H-RLM).

N-RLM, H-RLM and T3-H-RLM suspended in a calcium-free medium were found to differ from each other with respect to their calcium content (P < 0.0001) (Fig. 1AGo, empty columns). This proved to be significantly elevated (18.7 ± 3 nmol/mg mitochondrial protein) in H-RLM compared with N-RLM (7 ± 1 nmol/mg mitochondrial protein) (P < 0.01). T3 treatment of hypothyroid rats resulted in a significant decrease of the mitochondrial calcium content (12 ± 2 nmol/mg mitochondrial protein), compared with H-RLM (P < 0.01); the calcium content proved higher compared with N-RLM (P < 0.01) (Fig. 1AGo, empty columns). When mitochondria were supplemented with calcium (50 nmol/mg protein), the mitochondrial calcium content was increased in N-, H-, and T3-H-RLM (15 ± 1, 27 ± 2, and 17 ± 3 nmol/mg mitochondrial protein, respectively; P< 0.01 vs. no calcium loaded mitochondria) (Fig. 1BGo, empty columns). These results indicate that even if H-RLM contain a high calcium content, they are still able to take up calcium from the external medium, as occurs in N- and T3-H-RLM.



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  		FIG. 1. Mitochondrial calcium content, influx, and efflux in N-RLM, H-RLM, and T3-H-RLM. N-RLM, H-RLM, and T3-H-RLM (5 mg mitochondrial protein), were incubated in 0.5 ml of a medium (swelling medium) containing 0.2 M sucrose, 5 mM succinate/Tris, 10 mM 3[N-morpholine]propanesulfonic acid/Tris, 1 mM Pi/Tris, 2 µM rotenone and 1 µg/ml oligomycin in the absence or presence of CsA (1.7 nmol/mg protein) and/or CaCl2 (50 nmol/mg protein). At the indicated time, 0.1-ml aliquots were taken, centrifuged at 7300 x g for 40 sec, and the calcium content was determined on the mitochondrial pellet, as described in Materials and Methods. A, Calcium-free mitochondria. B, Calcium-loaded mitochondria. The mean values ± SEM of five different measurements, on the samples obtained from five different animals for each experimental group, are reported.

 
Measurements of calcium efflux were performed either in the absence or presence of CaCl2 and CsA (1.7 nmol/mg mitochondrial protein), the specific inhibitor of mitochondrial PTP (2) (Fig. 1Go, A and B). In calcium-free medium (Fig. 1AGo), the mitochondrial calcium content remained rather constant in N- and H-RLM, whereas, as a result of T3 administration to hypothyroid rats, calcium release with a rate of 0.4 ± 0.05 nmol/mg protein min-1 was found to occur, with a consequent decrease in mitochondrial calcium content. The addition of CsA to the incubation medium resulted in preventing calcium efflux from T3-H-RLM.

In the same experiment, mitochondria were loaded with CaCl2 (50 nmol/mg mitochondrial protein) and calcium release was monitored as above (Fig. 1BGo). T3-H-RLM were found to release calcium, in a CsA-sensitive manner, with a rate (0.8 ± 0.03 nmol/mg protein min-1) higher with respect to that measured in calcium-free mitochondria (P < 0.01). As a control, calcium efflux from mitochondria was also evaluated in N-RLM; the rate of calcium efflux was found to be comparable with that of T3-H-RLM. On the contrary, no calcium efflux was found to occur from CaCl2-loaded H-RLM.

MPT in hypothyroid and T3-treated hypothyroid rats: swelling and matrix protein release
To investigate further the effect of thyroid status in modulating the mitochondrial membrane permeability properties, MPT was examined (as in Ref.7) by monitoring swelling of N-, H-, and T3-H-RLM, suspended in swelling medium, as the absorbance change of the mitochondrial suspension, either in the absence or presence of 1 µM CsA (Fig. 2AGo). H-RLM showed a resistance to swell during the 10 min incubation time (Fig. 2AGo, trace a), comparable with that of mitochondria isolated from normal rats (Fig. 2AGo, trace b) ({Delta}A after 10 min were 0.2 ± 0.05 and 0.18 ± 0.06, respectively); however, T3 administration to hypothyroid rats conferred on mitochondria the capability of slow swelling in a CsA-sensitive manner ({Delta}A after 10 min was 0.35 ± 0.05, in the absence of CsA) (Fig. 2AGo, traces c and c'). In a parallel experiment, CaCl2-loaded mitochondria were used. H-RLM already showed a resistance to swelling (Fig. 2BGo, traces a and a'), but, as expected, the calcium pulse induced swelling in N-RLM ({Delta}A after 10 min was 0.7 ± 0.1) (Fig. 2BGo, trace b) and in T3-H-RLM ({Delta}A after 10 min was 0.75 ± 0.05) (Fig. 2BGo, trace c) with respect to no-calcium loaded mitochondria. In the presence of CsA, a strong prevention of swelling was found (Fig. 2BGo, traces b' and c'). It has been reported that an increase in mitochondrial calcium content causes swelling of isolated mitochondria (22). Interestingly, our results show that in hypothyroid rats the mitochondrial swelling properties did not correlate with the mitochondrial calcium content. In fact, even if H-RLM contain a higher calcium content, compared with both N- and T3-H-RLM, they show a resistance to swelling. However, T3-H-RLM, which contain a higher calcium content, compared with N-RLM, show a swelling capability even without CaCl2 added to the incubation medium.



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  		FIG. 2. Analysis of mitochondrial permeabilization to sucrose in N-RLM, H-RLM, and T3-H-RLM. H-RLM (trace a), N-RLM (trace b) and T3-H-RLM (trace c) (0.35 mg mitochondrial protein/ml) were added with swelling medium and the absorbance change at 540 nm at 25 C was monitored as reported in Materials and Methods. Traces a', b', and c' show the same experiments, run in the presence of 1 µM CsA, added to the suspension medium before mitochondria. A, Calcium-free mitochondria. B, Calcium-loaded (50 nmol/mg protein) mitochondria.

 
As a further confirmation of the capability of T3 treatment to restore the PT, the occurrence of the CsA-sensitive release of certain mitochondrial proteins in the suspension medium was checked (as in Refs.6, 7). We first measured the intramitochondrial activity of the matrix proteins GDH, AAT, and MDH in N-, H-, and T3-H-RLM and statistically significant differences were found among the groups (P < 0.001) (Table 1Go). A significant increase in the activity of these mitochondrial enzymes was found in H-RLM, compared with N-RLM (P< 0.01). After administration of T3 to hypothyroid rats, the matrix enzyme activities were comparable with those observed in N-RLM (P > 0.05) (Table 1Go). To verify whether H-RLM accumulated only enzymes of the matrix compartment, we measured also the activities of ADK (a protein of the mitochondrial intermembrane space) and MAO (a protein of the outer mitochondrial membrane). We found that ADK enzyme activity was comparable among N-, H-, and T3-H-RLM (P > 0.05) (Table 1Go). On the contrary, MAO activity was found to differ among the groups (P < 0.001) (Table 1Go). In particular, no significant difference in MAO activity was found between N-RLM and H-RLM, but a decrease in activity was observed in T3-H-RLM, compared with N-RLM (P < 0.01) and H-RLM (P < 0.01). The decrease in MAO activity in the hyperthyroidism status is consistent with results obtained by others (28).


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  		TABLE 1. GDH, AAT, MDH, ADK, and MAO activities in isolated mitochondria

 
We then checked whether N-, H-, and T3-H-RLM, incubated in calcium-free swelling medium, either in the absence or presence of CsA (1.7 nmol/mg mitochondrial protein), differentially lost mitochondrial GDH, AAT, and MDH activity (Fig. 3Go). Both N- and H-RLM retained activity of matrix enzymes; on the contrary, T3-H-RLM slowly lost mitochondrial enzyme activity, as revealed by the appearance of GDH (Fig. 3AGo), AAT (Fig. 3BGo), and MDH (Fig. 3CGo) enzyme activities in the supernatants. The mitochondrial enzyme activity release was inhibited by externally added CsA. As a control, the release of enzymes from mitochondria was also evaluated by measuring the enzyme activities in the mitochondrial pellets during the incubation period. As expected, a decrease in enzyme activity was observed in T3-H-RLM during the incubation period (data not shown). The slow release of matrix enzymes from T3-H-RLM could be caused by the higher calcium content in T3-H-RLM, compared with N-RLM (Fig. 1Go).



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  		FIG. 3. Release of GDH (A), AAT (B), and MDH (C) in N-RLM, H-RLM, and T3-H-RLM. N-RLM ({blacksquare}), H-RLM ({bullet}), and T3-H-RLM ({blacktriangleup}) (10 mg mitochondrial protein/ml) were incubated for 10 min in swelling medium in the absence (—) or presence (----) of CsA (1.7 nmol/mg protein). At the indicated time, 0.1-ml aliquots were taken, the mitochondria were precipitated, and the enzyme activities were determined in the supernatants. The amount of enzyme activities released from mitochondria are shown as enzymatic units per milligram of incubated mitochondrial protein. The data reported are means (±SEM) of five different mitochondrial preparations.

 
After the calcium pulse (50 nmol/mg protein), a statistically significant difference was found among N-, H-, and T3-H-RLM with respect to GDH release (P < 0.0001) (Fig. 4Go). GDH release was observed in N-RLM (4 ± 0.24-fold increase vs. noncalcium-loaded mitochondria, P < 0.01). A further release of matrix enzyme from T3-H-RLM was also observed (1.8 ± 0.25-fold increase vs. no calcium-loaded mitochondria, P < 0.01). However, after a calcium pulse, no release of GDH in the incubation medium was observed for H-RLM. A calcium pulse induced release of AAT and MDH in N- and T3-H-RLM but not in H-RLM (data not shown).



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  		FIG. 4. Calcium-induced release of GDH in N-RLM and T3-H-RLM. N-RLM, H-RLM, and T3-H-RLM (10 mg mitochondrial protein/ml) were incubated for 10 min in the swelling medium containing CaCl2 (50 nmol/mg protein) either in the presence (dotted columns) or absence (dashed columns) of CsA (1.7 nmol/mg protein). Empty columns, Mitochondria incubated in the absence of CaCl2. The amount of enzyme activity released from mitochondria is shown as enzymatic units per milligram of incubated mitochondrial protein. The data reported are means (± SEM) of five different mitochondrial preparations.

 
Overall, these results show that CsA-sensitive mitochondrial calcium efflux, swelling, and release of matrix proteins are impaired in mitochondria isolated from the liver of hypothyroid rats, even if they have a high calcium content. T3 administration to hypothyroid rats facilitates the opening of CsA-sensitive PTP and responsiveness to calcium loading.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thyroid hormone is considered a major regulator of mitochondrial activity (for reviews see Refs.19, 29). Previous studies have shown that mitochondria from hypothyroid rats exhibit a decrease in oxygen consumption rate as a result of decreased activity of both respiratory chain and oxidative phosphorylation (14, 15), decrease in the transport of phosphate (16) and adenine nucleotide (17) across the inner mitochondrial membrane, and decrease in gene expression of ANT (18) and F0F1-ATP synthase (14). In addition, changes in the lipid composition and lipid-protein interaction (for review see Ref.30) and, in particular, a decrease in cardiolipin and an increase in cholesterol content have been shown to occur in the mitochondrial membrane of hypothyroid rats (16). Some of these adverse effects have shown to be reversed by T3 administration to hypothyroid rats (14, 17, 18).

In this article we have investigated whether and how thyroid hormone regulates the mitochondrial permeability properties by analyzing the occurrence of PT in mitochondria isolated from normal and hypothyroid rats and from hypothyroid rats after administration of T3.

Extensive evidence indicates that changes in the permeability properties of mitochondrial membranes are caused by changes in calcium content (1). Most of the mitochondrial calcium content is regulated by a voltage-dependent calcium uniporter, which catalyzes calcium uptake (31), and a Na-independent Ca2+/nH+ transport, which catalyzes calcium efflux with occurrence of the CsA-sensitive gated PTP opening (32). It has been reported that the properties of the mitochondrial protonmotrice force generators do not change in hypothyroid rats (33) and that the mitochondria isolated from hypothyroid rats have a membrane potential higher than mitochondria isolated from euthyroid controls (34). Consistently, we show here that H-RLM are still able to take up calcium from the external medium and that calcium is retained inside. Mitochondrial calcium accumulation should increase the probability of the CsA-sensitive PTP opening (1). Interestingly, we found that PTP is less likely to be opened in H-RLM, even if they contain a high amount of calcium. We show that switching from hypo- to hyperthyroidism induces a decrease in the mitochondrial calcium content and CsA-sensitive calcium efflux with opening of PTP. These results suggest a role for thyroid hormone in the regulation of mitochondrial calcium content in intact cells.

We demonstrate that, besides calcium, H-RLM accumulate matrix enzymes (GDH, MDH, AAT), but not proteins of the intermembrane space (ADK) or the outer membrane (MAO), compared with N- and T3-H-RLM. The accumulation of matrix enzymes in hypothyroidism could be partly explained by the alteration in mitochondrial membrane permeability, conceivable results in an increased retention of the matrix proteins into mitochondria, and/or changes in matrix enzyme turnover process.

The role played by T3 in regulating the membrane permeability has been confirmed by the occurrence of CsA-sensitive swelling and slow external release of matrix enzymes observed in T3-H-RLM but not in H-RLM. Calcium loading of both N- and T3-H-RLM causes CsA-sensitive swelling and release of matrix enzymes. In contrast, hypothyroidism does not make mitochondria prone to swelling or to releasing proteins, even in the presence of calcium overload. This might be explained in terms of changes in the mitochondrial membrane structure during hypothyroidism, such as alteration of the physical connection between the outer and the inner mitochondrial membrane (35, 36), and decrease in the phospholipids and ANT content in the inner membrane of rat liver mitochondria (16, 18). After T3 administration, an increase in expression of the ANT-2 isoform in rat liver, reported by Dümmler et al. (18), could be involved, at least in part, in the onset of MPT in T3-H-RLM. In fact, ANT is known to participate to the formation of the transmembrane nonspecific pore (9, 10). The MPT might induce mitochondria to reach their normal membrane permeability properties and might provide a way to eliminate excess calcium or other solutes and molecules that have been accumulated in mitochondria during the hypothyroidism condition. As proposed by Minamikawa et al. (37), the MPT could occur reversibly, without inducing long-lasting impairment of mitochondrial functions.

In conclusion, we show here that thyroid status is essential for MPT regulation. The identification of MPT as a target of both T3 (Ref.13 and our results) and CsA (Ref.2 and our results) could be of special clinical interest. It has been reported that CsA may induce nephrotoxicity in patients with hypothyroidism (38). Thus, the identification of MPT dysfunctions associated to the thyroid status might provide a potential help in designing improved clinical applications of CsA.


   Acknowledgments
 
We thank Professor S. Papa for stimulating discussions and suggestions.


   Footnotes
 
This work was supported in part by a grant from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (Piani di potenziamento della rete scientifica e Tecnologica-Cluster 03) (to E.M.).

Abbreviations: AAT, Aspartate aminotransferase; ADK, adenylate kinase; ANT, adenine nucleotide translocase; CsA, cyclosporin A; GDH, glutamate dehydrogenase; H-RLM, mitochondria isolated from hypothyroid rats; MAO, monoamine oxidase; MDH, malate dehydrogenase; MPT, mitochondrial permeability transition; N-RLM, mitochondria isolated from normal rats; PT, permeability transition; PTP, permeability transition pore; PTU, 6-n-propyl-2-thiouracil; T3-H-RLM, mitochondria isolated from T3-treated hypothyroid rats.

1 F.G. is deceased. Back

Received March 11, 2003.

Accepted for publication May 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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