Action of thyrotropin on thyroid energetic metabolism

Q 1967 by Academic Press Inc. Experimental

518

ACTION

OF THYROTROPIN

Cell Research 46, 518-532 (1967)

ON THYROID

ENERGETIC

METABOLISM VI.

REGULATION

F. M. LAMY,

OF MITOCHONDRIAL F. R. RODESCHI

RESPIRATION

and J. E.

_

DUMONT

Laboratory of Nuclear Medicine and Gynecology, School of Medicine, Urziversity of Brussels, and Biology Department, Euratom, Brussels, Belgium

Received November 7, 1966

ADMINISTRATION of thyrotropin,

in uiuo as in vitro, stimulates the uptake of oxygen by thyroid slices [12, 13, 16, 17, 211. As this stimulation is accompanied by an increased oxidation of exogenous and endogenous pyruvate, it has been ascribed to an enhanced mitochondrial respiration [12]. How TSH” Possible explanations are that stimulates this respiration is not known. TSH directly stimulates thyroid mitochondria, or that it induces the release from thyroglobulin of large amounts of thyroxin, which in turn could activate mitochondrial respiration. The experiments reported in this paper show that the increase in the respiration of thyroid slices is indeed mainly of a mitochondrial nature; they allow us to reject the two proposed hypotheses on the mechanism of this stimulation; they strongly support our suggestion that TSH activates mitochondrial respiration by increasing ADP formation, i.e. ATP consumption in the stimulated tissue. As a preliminary to this work, previous observations on the metabolic charac.teristics of thyroid mitochondria [9, 23, 331 have been extended and compared to data obtained on the well-known rat liver mitochondria. MATERIAL

AND

METHODS

The isolation of mitochondria was carried out by a method adapted from Schneider [39]. Sheep and veal thyroids were removed from dying animals at the slaughterhouse. They were immediately placed in chilled sucrose 0.25 M and brought back to the laboratory.

Rat liver was removed

from animals

exsanguinated

by decapitation.

This work was made possible thanks to grant F.R.F.C. 22 of the Fonds de la Recherche Fondamentale Collective. It was realized partially under contract Euratom/University of Brussels/ University of Pisa No. BIAC 026-63-h. 1 Aspirant at the Fonds National de la Recherche Scientifique. 2 Abbreoiclfions. BSA, bovine serum albumin; T,, thyroxine; TSH, thyroid stimulating hormone; DNP, 2,4 dinitrophenol, Na salt. Ezcperimenfal

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They were extensively washed in chilled sucrose 0.25 M. The tissues were dissected free of fat and connective tissue, then cut with scissors and homogenized in 10 vol of the isolation medium with a Potter Elvehjem homogenizer equipped with a teflon pestle. All the manipulations were performed at 2”. The isolation medium contained: 0.25 M sucrose, 2 mM 4TP, 1 mM EDTA, and 2.5 g/l BSA. The separation of cellular fractions was performed at 2” in a Sorvall RC2 centrifuge (Norwalk, U.S.A.) equipped with a GSA rotor. The homogenate was centrifuged at 2000 rpm for IO min. After the first centrifugation the pellet was discarded and the supernatant fluid was recentrifuged at 7000 rpm for 15 min. The mitochondria were washed at least once (8500 rpm) and finally taken up in a minimal volume of the isolation medium. All the experiments with mitochondria mere carried out at 20°C. Respiration was measured by polarography, with a vibrating platinum electrode (Gilson Medical Electronics Oxygraph, Model K, Middleton, U.S.A.). The cell contained: 150 mM sucrose, 20 mM KCl, 15 mM potassium phosphate buffer (pH = 7.2), 15 mM Tris/HCl buffer (pH x7.2), 5 rnfif MgCl,, 1 m&1 EDTA, 1 rnJ@ NAD+, 15 $W cytochrome c, and I g/l BSA in a total volume of 2 ml. Between 1 and 4 mg of mitochondrial protein were added to the cell. Phosphorylation efficiency (P/O ratio) and “Respiratory Control Index” were evaluated after Chance [6]. The fluorescence of reduced pyridine nucleotides in extracts and in whole mitochondria was measured with an Eppendorf fluorimeter (Netheler and Hinz, Hamburg, Germany) modified after Estabrook and MaTtra [19]. Oxidized and reduced pyridine nucleotides in the mitochondria were extracted at 100” in acid medium and in alkaline the extracts were immediately medium respectively [3]. After neutralization, assayed for NAD+, NADP+, NADH +H+, and for NADPH +H+ [15]. The concentration of oxidized and reduced pyridine nucleotides was measured in mitochondria brought to different metabolic states [31]. The incubation medium contained: 130 md# sucrose, 50 m&f pyruvate, 50 m&f malate, 7 mM KCI, 5 mM potassium phosphate buffer (pH =7.2), 5 m2CITris/HCl buffer (pH =7.2), 1 mM MgCl,, I mM EDTA and I g/l BSA. Mitochondria (10 to 40 mg protein) were added up to a total volume of 1 ml. For state 3 and state 5 evaluations, 20 mM ADP was added to the medium. For state 3 and state 4, the reaction was stopped a few seconds after the addition of mitochondria. For state 5 evaluation, the reaction was stopped 6 min after the addition of mitochondria. The reaction was stopped by pouring the boiling extraction medium in the incubation medium. For swelling experiments, sheep thyroid and rat liver mitochondria mere prepared as usual but for the fact that they were washed once and resuspended in 0.25 nl sucrose containing I m&f EDTA. Swellin g was measured after Lehninger et al. [34] in: 0.3 df sucrose, 0.02 11t Tris/HCl buffer (pH =7.4) and 0.15 bf KCI, 0.02 A1 Tris/HCI buffer (pH =7.4). Mitochondrial ATPase activity was measured by estimating the liberation of Pi from ATP after Fiske and Subbarow [20]. Mitochondria (1 to 2 mgr proteins) were incubated for IO minutes, with: 150 mM sucrose, 40 rnJJ KCl, 30 mil/l Tris/HCl buffer (pH ==7.2), 5 rnhf MgCl,, 5 mM ATP, 1 m&Z EDTA, I g/l BSA, in a total volume of 1.0 ml. The reaction was stopped with 1.0 ml of 5 per cent perchlorie acid. The method of Nielsen and Lehninger [36] was used to measure the rate of ATP--32Pi exchange reaction. The incubation medium contained: 150 mM sucrose, 40 rnnl KCI, 20 mM Tris/HCl buffer (pH =7.2), 10 m&1 32P-labeled potassium phosphate buffer Experimental

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F. M. Lamy, F. R. Rodesch and J. E. Dumonf

(pH =7.2) (0.05 Curie/mole), 5 mM MgCl,, 5 rnnr ATP, 1 m&f EDTA, 1 g/l BSA and mitochondria (1 to 2 mg proteins), total volume 1.0 ml. After 10 min of incubation the reaction was stopped with 1.0 ml 10 per cent trichloracetic acid. Protein was determined by the Biuret method [24] with bovine serum albumin as a standard. The suspensions were cleared by 0.2 ml 5 per cent sodium deoxycholate. Materials Thyroid slices have been prepared and incubated as previously described [13]. Nucleotide tripbosphates (ATP, GTP, ITP, UTP), nucleotide diphosphates (,4DP, CDP, GDP, IDP, UDP) and adenosine monophosphate, have been extracted and measured after Adam [I]. Means and standard deviations of the mean have been calculated on the logarithms of the data obtained on different thyroids [13]. Results are expressed as antilogarithms of the mean and of the mean minus or plus the standard deviation of the mean or as arithmetic means of at least two closely agreeing duplicates. Substrates have been obtained from Calbiochem (Los Angeles, U.S.A.), Fluka (Bucks, Switzerland) and Merck (Darmstadt, Germany); coenzymes from Boehringer (Mannheim, Germany); antimycin A and oligomycin from Sigma Chemical Co. (St. Louis, U.S.A.); thyrotropin from Armour Co. (Kankakee, U.S.A.) and rotenone from Penick Co. (New York, U.S.A.).

RESULTS

Metabolic properties of thyroid mitochondria Mitochondria with reasonable respiratory control indexes and phosphorylative activity can be prepared from sheep thyroids. Typical results obtained with one mitochondrial preparation are shown in Fig. 1. Thyroid mitochondria have been studied simultaneously by polarography and fluorimetry (Fig. 1). In the absence of substrate and ADP (state l), the respiratory rate was low; the addition of ,4DP (state 2) did not increase the respiratory rate or decrease the fluoresc.ence of pyridine nucleotides. When glutamate was added (state 3) a 6 fold increase in the respiratory rate and a slight reduction of mitochondrial pyridine nucleotides was observed: this was followed, after ADP exhaustion (state 4) by a decrease of the respiratory rate aud a further reduction of the pyridine nucleotides. After exhaustion of the dissolved oxygen a great reduction of the pyridine nucleotides was observed (state 5). Similar results were obtained with succinate as a substrate, though the reduction of mitochondrial pyridine nucleotides at state i was higher. In rat liver mitochondria, the pyridine nucleotides were markedly oxidized and the respiratory rate was stimulated when ADP was added in the absence of Experimental

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exogenous substrate (state 2). AMP and ADP stimulated similarly the rate of oxygen uptake by sheep thyroid mitochondria, but the exhaustion of AMP required twice as much oxygen as the exhaustion of ADP. Sheep thyroid mitochondria prepared in 0.25 Jf sucrose, 2mM ATP, 1 ml11 EDTA and 2.5 g/l BSA had a better respiratory control than mitochondria TABLE

I. Rates of respiration, oxidative phosphorylation and respiratory control index in liver and thyroid mitochondria.

Substrate

Oxygen uptake &atoms oxygen/g protein/min)

p/o

Sheep thyroid Succinate ru-Glutamate n-Ketoglutarate Pyruvate + nL malate on-Isocitrate cis-Aconitate on-Malate DL-B-Hydroxybutyrate n-cc-Glycerophosphate Fumarate Pgruvate Citrate Oxaloacetate No exogenous substrate

73 44 30 28 26 19 IT 13 13 8 8 8 7 6

-

T’eal thyroid Succinate L-a-Glycerophosphate No exogenous substrate

85 24 6

-

Rat iiaer Succinate L-a-Glycerophosphate No exogenous substrate

73 13 10

i.7

1.8 2.4 1.8 1.9 2.2 1.2 1.8

Respiratory control index

4.6 4.3 3.5 2.8 3.1 2.2 1.4

N

8 S 3 2 4 2 3 2 4 2 2 3 2 8

3.1

Incubation medium contained: 1 to 4 mg mitochondrial proteins, 150 mM sucrose, 20 mM KCl, 15 mM potassium phosphate buffer (pH = 7.2), 15 mdf Tris/HCl buffer (pH = 7.2), 5 mM MgCl,, 1 mM EDTA, 1 mM NAD+, 174 p& ADP, 15 ,LGMcytochrome c, 1 g/l BSA. Substrates concentrations were 10 m&I (20 mill for the racemic preparations). In the case of suecinate and cc-glycerophosphate: no NAD +. Total volume: 2.0 ml.; temperature: 20”. N, number of mitochondrial preparations which were investigated. For veal thyroid and rat liver mitochondria, values of a representative experiment are given. Ezperimentul

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F. M. Lamy, F. R. Rodesch and J. E. Dumont

prepared in 0.25 M sucrose and 1 ml\{ EDTA, in 0.88 M swrose and 1 mM EDTA, or in 0.15 M KCl, 2 mill ATP, 1 mdi EDTA, and 2.5 g/l BSA. hlitochondria prepared in sucrose 0.25 M had no respiratory control, which may perhaps be due to the high concentration of calcium in the thyroid [29]. The rate of oxidation of various substrates by sheep and veal thyroid mitochondria and by rat liver mitochondria has been studied (Table I). For sheep thyroid mitochondria, succinate was by far the best substrate, but these mitochondria also oxidized, in a decreasing order, glutamate, cc-ketoglutarate, pyruvate in the presence of catalytic amounts of malate, isocitrate, cisaconitate, malate, /I-hydroxybutyrate and cc-glycerophosphate. Satisfactory respiratory control indexes were only obtained with the best substrates. With these substrates, the P/O ratios were consistent with the known ratios. a-Glycerophosphate was oxidized at a higher rate in calf than in sheep mitochondria, but this oxidation was nevertheless low; it was not stimulated by ADP. The oxygen uptake of sheep thyroid mitochondria was not significantly increased in the presence of NADH or NADPH (10 mM). When cytochrome c (30 ,uuM) was added, a marked enhancement of the osygen uptake was observed; this enhancement was inhibited by cyanide (5 mM) but not by antimycin (5 mg/l) or oligomycin (2.5 mg/l). Sntimycin (0.5 mg/l and 5 mg/l) and cyanide (0.5 mM and 5 mM) abolished the respiration of sheep thyroid mitochondria incubated with various substrates. This inhibition was not reversed by dinitrophenol. Rotenone (0.1 pulli and 10 PM) inhibited this respiration in the presence of NAD linked substrates, but not in the presence of succinate. The inhibitory effects of rotenone and amytal were not reversed by dinitrophenol. The inhibition by oligomycin of mitochondria respiration was reversed by dinitrophenol. The respiration of sheep thyroid mitochondria in the presence of succinate (10 mM) was inhibited by oxaloacetate (10 mM). Glutamate (20 mM) abolished this effect. Malonate (10 mM) which inhibits competitively thyroid succinic dehydrogenase, nearly suppressed the oxidation of succinate and of glutamate by thyroid mitochondria. Although malate and fumarate (2 mM) did not by themselves stimulate the respiration of malonate inhibited mitochondria, these substrates restored the oxidation of glutamate by such mitochondria. These experiments evidence the existence in thyroid mitochondria of an active glutamic-aspartic cycle [26, 281. Oxidized and reduced pyridine nucleotides were measured in preparations of rat liver and sheep thyroid mitochondria brought to a definite metabolic state. The total NAD and NaDP contents of the mitochondria did not change Experimental

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VI

with the metabolic conditions. In sheep thyroid mitochondria, the NAD content was lower, and the NADP content was much lower than in rat liver mitochondria (Table II). Whatever the metabolic state, NADP was always almost completely reduced in both types of mitochondria. The proportion of reduced TABLE II. Pyridine

Total NAD Total NhDP Total NADP TotaI NXD I

nucleotides

in sheep thyroid

and rat liver mitochondria.

Rat liver

N

Sheep thyroid

3.77 ri: 0.84 4.29 + 1.63

4 4

2.36 10.44 0.47 * 0.04

6 6

1.10

4

0.20

6

Results are expressed as means plus or minus standard deviation: pyridine nucleotides content in pmoles/g protein. The ratio total NXDP/total N-ID is the logarithmic mean of the individual results [3J. N: number of experiments.

NAD was very low in state 3 (0.09 and 0.13), low in state 4 (0.20 and 0.21) and high in anaerobiosis (0.50 and 0.74) in both liver and thyroid mitochondria. Regulation.

of m.itochondrial

respiration

The respiration of sheep thyroid and rat liver mitochondria with succinate as a substrate has been measured in the presence of exc.ess ADP and of various phosphate concentrations and in the presence of excess phosphate and of various concentration of ADP. In one preparation of liver mitochondria half maximal acceleration of the oxygen uptake was obtained for a concentration of 0.5 mdl of phosphate; for ADP, maximal acceleration was observed at a concentration of 58 ,uM. These results are of the same order of magnitude as those obtained by Chance and Connelly [7] by a spectroscopic method on rat liver mitochondria. In two preparations of sheep thyroid mitochondria, the half maximal acceleration of respiration was obtained for a concentration of 0.5 mM of phosphate. The results of a representative experiment are shown in Fig. 2. In one preparation of sheep thyroid mitochondria, the half maximal acceleration of respiration was obtained for a concentration of 44 tlil1 of XDP (Fig. 3). The sensitivities to exogenous ADP and phosphate are therefore of the same order of magnitude in sheep thyroid mitochondria and in rat liver mitochondria. TSH did not modify the respiration, the phosphorylation efliciency, or the Experimental

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F. M. Lamy, F. R. Rodesch and J. E. Dumon f

respiratory control index of thyroid mitochondria with succinate (Fig. 4) or glutamate as a substrate: it did not stimulate the oxidation of external NADPH f H + in the presence of ,4DP. The addition of H,O, to thyroid and liver mitochondria determined a sudden increase in the concentration of dissolved oxygen in the medium. This fact is consistent with the strong catalase activity

Fig. l.-Respiratory control in sheep thyroid mitochondria with glutamate as a substrate. The cell contained: 3.1 mg mitochondrial proteins, 150 m&f sucrose, 20 mM KCl, 15 mM potassium phosphate buffer (pH =7.2), 15 mill Tris/HCl buffer (pH =7.2), 5 mM MgCl,, 1 m&f EDTA, 1 m&f NAD+, 15 ,U M cytochrome e, 1 g/l BSA. Total volume: 2.0 ml; temperature: 20°C. Successive additions are inclicated on the graph. The time scale increases from left to right. A, Polarographic recording. The figures along the traces indicate oxygen uptake in piatoms oxygen/g protein/min. B, Fluorimetric recording 366 m/l excitation, 400-3000 rnbl measurement. In ordinate, the fluorescence measures the reduction of the pyridine nucleotides in mitochondria. Mito, mitochondria. Fig. 2.-Stimulation of mitochondrial respiration by phosphate. The cell contained: 1.45 mg mitochondrial proteins, 150 miW sucrose, 30 m&Z KCl, 20 mM Tris/HCl buffer (pH = 7.2), 10 mM succinate, 5 mM MgCl,, 1.2 mhf ADP, 1 mM EDTA, 15 ,uM cytochrome c, i-g/l BSA: Total volume: 2.0 ml: temperature: 20°C. A 90, indicates the difference between respiration with and without phosphate, expressed in Alatorn; ixygen/g proteinlmin. [Pi] =potassi&n phosphate buffer (pH = 7.X) concentration in the cell. Fig. 3.-Stimulation of mitochondrial respiration by ADP. The oxygen uptake of a preparation of sheep thyroid mitochondria was measured in the presence of various concentrations of ADP. The cell contained: 1 mg mitochondrial proteins, 150 m&f sucrose, 20 mM KCl, 15 miW potassium phosphate buffer (pH = 7.2), 15 mM Tris/HCl buffer (pH = 7.2), 10 mM succinate, 5 mM MgCl,, 1 mM EDTA, 15 ,LLM cytochrome e, 1 g/l BSA. Total volume: 2.0 ml; temperature: 20°C. A QO, indicates the difference between respiration with and without ADP, expressed inpatoms oxygen/g protein/min. [ADP] = concentration of ADP in the cell. Fig. 4.-Influence of TSH on the respiration of sheep thyroid mitochondria. The figures along the traces indicate oxygen uptake in [latoms/g protein/minute. The incubation medium contained: 150 md sucrose, 20 mM KCl, 15 mM potassium phosphate buffer (pH = 7.2), 15 mM Tris/HCl buffer (pH = 7.21, 10 mill succinate, 5 mll MgCl~, 1 mM EDTA, 15 @f cytochrome c, 1 g/l BSA. Total volume: 2.0 ml; temperature: 20°C. Successive additions are indicated on the graph. The time scale increases from left to right. Fig. 5.-Effect of thyroxine on the respiration of sheep thyroid and rat liver mitochondria. Successive additions are indicated on the graph. The figures along the traces indicate oxygen nptake in htatoms/g protein/min. The incubation medium contained: 150 mM sucrose, 20 mill KCl, 15 mM potassium phosphate buffer (pH = 7.2), 15 mM Tris/HCl buffer (pH = 7.2), 10 mM succinate, 5 mM RIgCl,, 1 m&f EDTA, 15 FM cytochrome c, 1 g/l BSA. Tot’al volume: 2.0 ml; temperature: 20°C. A, Liver mitochondria; B, Thyroid mitochondria. Fig. 6.-Influence of thyroxine and dinitrophenol on ATPase activity of sheep thyroid and rat liver mitochondria. The cell contained: mitochondria 1 to 2 mg proteins, 150 m114 sucrose, 40 mM KCl, 30 m&f Tris/HCl buffer (pH =7.2), 5 mM MgCl,, 5 mM ATP, 1 mM EDTA, 1 g/l BSA. Total volume: 1 ml; temperature: 20°C. A, Rat liver. ATPase activity in the control mitochondria was: 0.01 ,umoles phosphate/l.45 mg proteinlmin. B, Sheep thyroid. ATPase activity in the control mitochondria was: 0.03 p,moles phosphate/l.82 mg proteinlmin. T,,Thyroxine dissolved in 99 per cent ethanol-O.1 N NaOH (24/l v/v). Fig. 7.-Swelling of rat liver and sheep thyroid mitochondria. The incubation medium was 0.3 M sucrose, 0.02 ill Tris/HCl buffer (pH = 7.4). Temperature: 20°C. 1, Spontaneous swelling. 2, Swelling induced by 30 @f T,. 3, Swelling in the presence of 5 m&f succinate. 4, Swelling in the presence of 5 mM succinate and 30 ,uM T,. A, Rat liver; B, Sheep thyroid. Experimental

Cell Research 46

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50 40 30.---_-.--- - 0.6mt4 Pi 20 11) 0k lo” 10’ 1r’ M[pi’

525

VI

-

44 pM AUP

2

‘ml”

[O&D

-----------

4

It

Abmbancy

520

“W A

0.8

Ezperimenful

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F. M. Lamy, F. R. Rode&

and J. E. Dumont

of mitochondria [35]. Five minutes after the addition of 1 md4 H,O,, the respiratory control was still normal in both types of mitochondria. Iodide (0.1 mM, 1 mM and 10 ml\!), perchlorate (1 mM), thiocyanate (1 mM) and methimazole (1 mM) did not modify the respiration, the P/O ratio or the respiratory control index of thyroid mitochondria.

Efects of thyroxine The effects of thyroxine and dinitrophenol on mitochondrial functions hare been compared in sheep thyroid mitochondria and in rat liver mitochondria. In state 4, the respiration of both types of mitochondria was stimulated in the presence of dinitrophenol; at the optimal concentration of dinitrophenol (0.04 mM to 0.1 mM), respiration was as high as in state 3 and respiratory c.ontrol was lost. The effec.ts of dinitrophenol were reversed when albumin (8.5 g/l) was added to the medium. The respiration of rat liver mitochondria incubated with succinate as a substrate, but without ADP (state 4) was inc.reased in the presence of thyroxine 40 ,uM or 0.1 md (Fig. 5). In similar conditions the respiration of thyroid mitochondria was not modified or slightly reduced. Whether mitochondrial respiration was enhanced (rat liver) or not (sheep thyroid) by thyroxine in state 4, its stimulation by ADP was diminished in the presence of T, 10 ~41 and abolished in the presence of T, 0.1 mM. The inhibitory effect of thyroxine on mitochondrial respiration could not be reversed by dinitrophenol while BSA partially restored both respiration and respiratory control. The ATPase activities in the presence of 5 mM ATP, expressed as pmoles of phosphate liberated per minute, were 0.01 pmoles/1.45 mg protein and 0.05 pmoles/2.05 mg protein for rat liver mitochondria and 0.02 pmoles/l.66 mg protein and 0.03 ,umoles/l.S mg protein for thyroid mitoc.hondria. ATPase activity was highly enhanced by dinitrophenol in liver mitochondria; it was increased to a lesser extent in thyroid mitochondria. Results of a representative experiment are outlined in Fig. 6. Thyroxine stimulated the ATPase activity by a factor of 2 in rat liver mitochondria, and by a factor of 1.5 in sheep thyroid mitochondria. For both compounds, and for both types of mitochondria, the optimal concentration varied from one experiment to another from 1O-4 M to 4.10-& M. Sheep thyroid mitoc.hondria, as rat liver mitochondria catalyzed an exchange reaction between inorganic phosphate and ATP in the absence of substrate. The average values of the exchange rates, expressed as /cmoles Experiment&

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phosphatefmg protein/hr were 1.8 for sheep thyroid mitochondria and 1.7 for rat liver. As in rat liver mitochondria [5] thyroxine (1 mM and more) and dinitrophenol (0.4 mM and more) inhibited the ATP3*Pi exchange reaction in sheep thyroid mitochondria. TABLE III.

Total nucleotides NDP NDP/NTP

Effect of thyrotropin

on the nucleotide phosphate thyroid slices.

content of sheep

Units

Controls

TSH (128 iU/l)

N

P

pMoles/g wet weight Per cent of total Per cent

0.282 (0.260-0.304) 14.4 (11.6-17.5) 17.0 (15.7-18.4)

0.290 (0.269-0.31-t) 17.4 (16.6-18.3) 20.1 (19.2-20.9)

15 15 15

0.4 0.01 0.01 -

Incubation 1 hr. Means, standard deviations of the mean and statistical significance of the results have been calcuIated on the logarithms of the data [13]. Results are expressed as antilogarithms of the mean and antilogarithms of the mean minus and plus the standard deviation of the mean. N, number of experiments. NTP: XTP, GTP, ITP, UTP. NDP: ADP, CDP, GDP, IDP, UDP. Total nucleotides: NTP, NDP, AMP.

As reported in the literature [18, 341, rat liver mitochondria incubated in sucrose-Tris or in KCl-Tris medium swelled spontaneously after a variable lag period. The rate of swelling was higher in El-Tris medium. In the presence of thyroxine (30 ,L&), in sucrose-Tris medium, the swelling began immediately and went on at a higher rate (Fig. 7). Incubated in the same conditions, for a similar period, sheep thyroid mitochondria, in the presence or in the absence of thyroxine (30 ,uM or 3 ,uM) showed only a slight swelling (Fig. i). When succinate was added to the medium, the swelling of rat liver mitochondria began after more than 60 min, and was completed after 150 min. Thyroxine induced an immediate swelling (Fig. 7). Swelling proceeded at a very fast rate when rat liver mitochondria were incubated with glutamate + NAD +. When incubated for several hours in the presence of succinate, in thyroid mitochondria did not the presence or in the absence of thyroxine, swell (Fig. 7). However, when glutamate and NADt- were added to the sucrose-Tris medium, swelling started after about 40 minutes incubation; this brought the absorbanq of the suspension to a stable value of about 65 per cent of the initial value. Experimental

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F. M. Lamy, F. R. Rodesch and J. E. Dumont Mitochondrial

respiration in thyroid slices

Thyrotropin (128 iU/l) stimulated the respiration of sheep thyroid slices (to 143 per c.ent of the controls). Antimycin A (5 mg/ml) inhibited equally the respiration of normal (to 10 per cent of the controls) and stimulated slices (to 7 per cent of the controls). Oligomycin (1 mg/l) also inhibited in the same proportion, the respiration of normal and stimulated slices, respectively to 32 per cent and 19 per cent of the controls. TSH (128 iU/l) markedly enhanced the uptake of oxygen by dog thyroid slices: to 141 per cent (125-159) of the controls-Oligomycin (1 mg/l) equally inhibited the respiration of normal and stimulated slices, respectively to 30 per cent (24-37) and 28 per c.ent (23-34) of the controls. Nucleotide phosphates have been measured in sheep thyroid slices incubated with and without TSH. The total concentration of the nucleotides is not modified in the stimulated slices (Table III). However, the proportion of nucleotide diphosphates, and more significantly the ratio of nucleotide diphosphates to nucleotide triphosphates is increased in the stimulated slices. DISCUSSION

The method of Schneider [39j has been adapted to sheep thyroid tissue in order to isolate thyroid mitochondria. The isolated particles presented the classical metabolic properties of mitochondria: respiration, respiratory control, well defined inhibition of phosphorylation by oligomycin and of respiration by oxaloacetate, malonate, rotenone, amytal, antimycin and cyanide, and loss of respiratory c,ontrol with dinitrophenol. These mitochondria were minimally damaged as evidenced by the following observations: (1) They exhibited a satisfactory respiratory control. (2) They did not significantly oxidize exogenous NADH or NADPH. (3) They are not swollen at the time of preparation, as swelling was elicited by incubation in a deficient medium. (4) Addition of cytochrome c and NAD+ to the incubation medium did not markedly increase the oxidation of Krebs cycle intermediates, which shows that these coenzymes had not been washed out during the preparation. (5) Their ATPase activity was low and was normally stimulated by dinitrophenol [a]. The thyroid mitochondria have been compared to rat liver mitochondria. Results obtained with the latter preparation generally agree with the results in the literature. With succinate as a substrate, the maximal uptakes of Experimental

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oxygen were similar in thyroid and in liver mitochondria. In the presence of substrates, the respirations of both types of organelles were mainly regulated by the concentration of ADP and of inorganic phosphate; the control of respiration by these compounds operated in the same range of concentrations. blitochondrial pyridine nucleotides were reduced by NAD linked substrates, and even more by succinate in both tissues extracts. ATPase activity and A4TP-32Pi exc.hange rate were similar in thyroid and in liver mitochondria. In both types of mitochondria, respiration and ATPase activity were stimulated, and dTP-32Pi exchange was inhibited, by the same concentrations of dinitrophenol. A glutamic-aspartic cycle is active in thyroid mitochondria as in liver mitochondria [26, 281. The ratio of NADP to NAD content of thyroidal mitochondria is of the same order as in other tissues, brain, heart, kidney [32], but much lower than in liver mitochondria. This suggests that in the thyroid as in the former tissues, the mitochondria have mainly an energetic and not a biosynthetic role. When compared to liver mitochondria, thyroid mitochondria are characterized by a low supply of endogenous substrates. Indeed, in the absence of exogenous substrates, the addition of ADP to the medium does not increase the respiration nor the oxidation of pyridine nucleotides in these mitochondria. Sheep thyroid mitochondria did not oxidize NADH via the antimycin sensitive phosphorylating pathway. As cytochrome c is probably exclusively localized in the mitochondria, the external antimycin insensitive non phosphorylating pathway which has been evidenced is generally not believed to be operative in the intact cell [4]. Contrary to the mitochondria of tissues in which an a-glycerophosphate cycle seems to operate [4, 81 thyroid mitochondria only poorly oxidized cr-glycerophosphate; ,&hydroxybutyrate whic.h can also catalyze a “shuttle system” [lo] was not a good substrate either. None of the mechanisms proposed in the literature for the oxidation of the extramitochondrial NdDH +H + seems therefore to be very active in the sheep thyroid cell. This is consistent with the fact that, in this tissue, a great proportion of the ghmose metabolized through the Embden Meyerhof pathway is reduced to lactate [17]. The effects of specific physiological aud pharrespiratory control and oxidative macological agents on the respiration, phosphorylation in sheep thyroid mitochondria have been studied. H,O r did not modify mitochondrial activity, which shows that this cell organelle at least, would not be harmed by retrodiffusion of H,O, from the iodinating sites. Thyroid mitochondria were not affected by KClO, or methimazole. These compounds barely modify the respiration of whole cells [12]; their inhibitory action on iodine metabolism can therefore not be ascribed to a Experimenfal

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530

direct inhibition of oxidative phosphorylation. KI did not modify the mitochondrial activity. The stimulatory effect of this anion on the respiration of thyroid slices [ll, 251 is therefore not due to a direct action on this organelle. When added in uitro to thyroid slices, KSCN stimulated mitochondrial activity and decreased the incorporation of inorganic phosphate in organic acid soluble compounds [30]. These effects could be explained by a direct or indirect uncoupling of oxidative phosphorylation, or by the stimulation of some thyroid ATPase. Our data show that KSCN has no direct uncoupling action on thyroid mitochondria. Stimulated thyroid cells are exposed to high levels of thyroxine. After an injection of TSH, the thyroid hormone concentration in the thyroid vein, i.e. in the extracellular medium, may be multiplied by a factor of 3 to 5 [38]. In this case thyroid cells are therefore exposed to concentrations which for peripheral cells would be in the thyrotoxic range. The direct effects of thyroxine on the function of thyroid mitochondria have therefore been studied. The well known effects of thyroxine on liver mitochondria were reproduced: (1) state (2) (3) (4)

Stimulation of respiration in state 4 and inhibition of respiration in 3, these effects being accompanied by a loss of respiratory control [37]. Inhibition of ATP-32Pi exchange [5]. Enhancement of ATPase activity [5]. Acceleration of mitochondrial swelling in deficient medium [18, 341,

Neither the relation between these effects [5], nor the in uivo physiological or pathological significance of these in vifro effects is well established [40, 411. However there is some support for the hypothesis that the swelling effect of thyroxine is linked to the physiological action of this hormone [40, 411. Likewise, the stimulation of mitochondrial respiration (state 4) coupled to a loss of respiratory control is similar to the metabolic pattern observed in mitochondria of animals intoxicated with thyroxine, and reminds very much the clinical features of thyrotoxicosis [27]. It seems therefore relevant that thyroxine caused neither a stimulation of respiration nor an acceleration of swelling in thyroid mitochondria. Full understanding of our data on the effects of thyroxine on thyroid mitochondria should await further clarification of the biochemical basis of physiological and pharmacological action of thyroxine in uiuo. Nevertheless, these data suggest that thyroid mitochondria are insensitive to these effects. Furthermore, as thyroxine does not stimulate the respiration of thyroid mitochondria, the hypothesis that the stimulation of thyroid mitochondrial respiration by TSH is secondary to the release of thyroxine in the thyroid cell may be rejected. Experimenfal

Cell Research 46

Regulation

of thyroid

respiration.

VI

531

The respiration of thyroid slices is almost completely antimycin and oligomycin sensitive, i.e. mitochondrial. The stimulation of this respiration by TSH is also abolished by antimycin and oligompcin. This fact supports the suggestion [12] that the increased respiration is mainly mitochondrial. The respiration of thyroid slices is not stimulated by glucose or by NAD linked substrates [l-1], but it is markedly enhanced by dinitrophenol [ 12, 19 !; it is therefore not limited by the availability of substrates. The inhibition by oligomycin and the stimulation by dinitrophenol of the mitochondrial respiration in thyroid slices show that this respiration is tightly coupled to phosphorylation in the intact cell as in our isolated mitochondria. This respiration is mainly regulated by the concentrations of ADP and Pi. TSH does not stimulate the mitochondria directly or affect their respiratory control. The stimulation of mitochondrial respiration in thyroid slices must therefore be ascribed to an increased production of ADP or Pi in the cell. The concentration of inorganic phosphate is increased in stimulated thyroid slices [22]. However, the concentration of Pi in unstimulated slices ( >2 mM) [22] is already sufficient to cause maximal mitochondrial stimulation. As cells constitute just part of the thyroid space, the true intracellular concentration of Pi is probably even higher. It seems therefore improbable that the stimulation of mitochondrial respiration in thyroid slices is due to an increase in the cell content of Pi. An enhanced formation of ADP, i.e. ATP utilization is therefore the most likely explanation for this stimulation. The slight, but significant shift in the ratio of nucleotide diphosphates to nucleotide triphosphates in the stimulated slices strongly supports this conclusion.

SUMMARY

Well preserved sheep thyroid mitoc.hondria have been isolated. When compared to liver mitochondria, these mitochondria are characterized by a specific pattern of substrate oxidation, a low supply of endogenous substrates, a low ratio of NADP to NAD, and the low activity of pathways for the oxidation of exogenous NADH. The respiration of thyroid mitochondria is not affected by hydrogen peroxide, perchlorate, methimazole, iodide or thiocyanate. Thyroxine stimulates their ATPase activity, inhibits their ATPm3” Pi exchange and suppress their respiratory control but does not, as in liver mitochondria, stimulate their respiration or their swelling. Thyrotropin stimulates the mitochondrial respiration in thyroid slices. This effect is not due to a direct effect of the hormone on the mitochondria, on the stimulation of mitochondrial respiration by thyroxine liberated in the stimuErperimental

Cell Research 46

F. Al. Lnmy, F. R. Rodesch and J. E. llumont

532

lated tissue. It is ascribed to an increased thyroid cell. The

authors

Borrey

and

would lvlr

like

J. Eloy

for

lo thank their

I>r technical

W.

A’I’P consumption

C. Hiilsmann

for

his

in the stimulated

advice

and

Miss

Ch.

assistance.

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Cell Research 46

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