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

0013-7227/03/$15.00/0 Printed in U.S.A. Endocrinology 144(9):3783–3788 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0310 Thyroid Hormone Administration to Hypothyroid Rats Restores the Mitochondrial Membrane Permeability Properties ROSA A. VACCA, LOREDANA MORO, GIOVANNI CARACCIO, FERRUCCIO GUERRIERI,* ERSILIA MARRA, AND MARGHERITA GRECO 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 Asensitive 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. T HE CALCIUM-DEPENDENT PERMEABILITY transition pore (PTP), inhibited by cyclosporin A (CsA), regulates most of the permeability properties of the inner mitochondrial membrane (1–3). 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– 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 –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 defiAbbreviations: 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. * F.G. is deceased. 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. (Endocrinology 144: 3783–3788, 2003) ciency can result in altered mitochondrial membrane permeability properties, thus contributing to the onset of mitochondrial dysfunctions (14 –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 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 ␮g 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 ⫻ 100 ml⫺1. Plasma was separated by centrifugation in the cold and the samples 3783 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 3784 Endocrinology, September 2003, 144(9):3783–3788 Vacca et al. • T3 and Mitochondrial Permeability Transition natants were centrifuged for 2 min at 10,000 ⫻ 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. Preparation of mitochondria Statistical analysis 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 ␮m 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. 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-NewmanKeuls test. Comparison between independent means was performed using the t test. Results Determination of mitochondrial calcium content Thyroid hormone s (...truncated)