The University of Buenos Aires and the Atomic Energy Commission, Nuclear Medicine Center, Hospital de Clínicas "José de San Martín", Buenos Aires
RECIPROCAL EFFECTS OF THYROXINE AND TRIIODOTHYRONINE ON THEIR DEIODINATION BY RAT TISSUES IN VITRO
By
Angel
A. Zaninovich
ABSTRACT
reciprocal effects of loading doses of thyroxine (T4) or triiodothyronine (T3) on the deiodination of their 125iodine-labelled isotopes by rat muscle and liver homogenates were studied. In 21 experiments muscle homogenates deiodinated a mean 45.0 % of a tracer dose of [125I]T4 and 18.0% of [125I]T3. On addition of graded amounts of nonradioactive T4 or T3 the percentual deiodination of both labelled hormones progressively declined. This effect was significantly greater in homogenates incubated with non-radioactive T4, thus reflecting a stronger affinity of this hormone for muscle deiodinating sites. This correlate with as revealed by the percentage of the greater displacement of [125I]T3 recovered labelled hormone. In 18 experiments liver homogenates deiodinated a mean 14.6% of a tracer amount of [125I]T4 and 8.5% of [125I]T3. The addition of a T4- or a T3-load was followed by a smaller decrease in percentual deiodination of both labelled hormones as compared to muscle homogenates. Unlike the effects observed in muscle, the breakdown of [125I]T4 and [125I]T3 by liver homogenates was equally affected by similar amounts of stable T4 or T3. It is concluded that in The
the present in vitro system *
Career
Investigator, Consejo
T4
and
T3
Nacional de
share cellular sites of deiodination
Investigaciones
Científices y Técnicas of
Argentina (CONICET).
Supported by grant
No. 3728 from the CONICET. Presented in part at the 9th Acta Endocrinologica
510
Congress, Oslo, Norway,
1973.
in rat muscle and liver and that, at least in muscle, which constitutes one-half of the rat body weight T4 appears to be preferentially deiodinated.
over
Studies in this
laboratory first reported data suggesting a secondary role of plasma binding in the physiological disposal of triiodothyronine (T3) in man (Zaninovich et al. 1966, 1969a,i5, 1971). Further kinetic studies of T3 by Oppenheimer et al. (1970) lent support to this contention by demonstrating that T3 is primarily an intracellular hormone, both in man and the rat. The findings on T3 peripheral metabolism led us to postulate that T4 and T3 might compete for tissue sites with a selective uptake and degradation of T4 by certain tissues (Zaninovich et al. 1969a,t5, 1971). With this framework in mind the present investigations have been made on the reciprocal effects of T4 and T3 on their deiodination by rat muscle and liver homogenates.
MATERIAL AND METHODS Two-month old rats of the Wistar strain weighing approximately 200 g were used. Before the study the animals were maintained on commercial laboratory chow and water ad libitum. [125I]T4 and [125I]T3 were obtained from a commercial source (Sorin, Italy) with a specific activity of approximately 80 mCi/mg. These labelled compounds were freed of contaminating iodide by dialysis, as described by Nicoloff Se
Dowling (1968).
Deiodination of [125I]T4 and [125I]T3 was studied in fresh homogenates of muscle and liver1). The animals were killed by a blow on the head and tissues were homo¬ genized in chilled teflon or glass grinders using 1 g of tissue in 5 ml of ice-cold Krebs-Ringer phosphate buffer pH 7.4 prepared in the manner described by Galton 8c Ingbar (1961). Two ml aliquots of homogenate were separated and tracer amounts of [125I]T4 or [125I]T3 (1 ,aCi and approximately 0.015 fig) were added. These homo¬ genates containing only labelled hormones will be referred to in the text as control samples. In some of the samples, in addition, a freshly prepared solution of nonradioactive T4 or T3 was added to the homogenate to give a final hormone concen¬ tration ranging from 25 to 800 ug per 100 ml. The mixture was incubated at 37°C for 70 min in room air in a continuously-shaking apparatus. Following incubation 1 ml of human plasma was added to each homogenate to stop further hormone de¬ iodination. Paper chromatography was performed by descending technique in butanoldioxane-ammonia (4:1:5) and in butanol-acetic acid-water (80:10:12) during 20 h. The results obtained in both solvent systems were comparable and thereafter, studies were carried out in the alkaline system. Appropriate reference standards were used and their positions were located by staining with palladium chloride in the case of iodide and ninhydrin for the iodothyronines. In each experiment chromatography of ') Initial studies in which 300 mg-tissue slices were incubated with T4 or T3 and further homogenized and chromatographed, exhibited similar results to those ob¬ tained with tissue homogenates.
511
dialyzed
solution of both labelled hormones was similarly performed for assess¬ purity. The areas of radioactivity were located in a stripscanner equipped with an electronic integrator (Actigraph III, Nuclear, Chicago). A tissue-free system containing 2 ml of buffer and a tracer dose of labelled T4 or T3 was similarly incubated and chromatographed. All experiments were performed in duplicate. The major 125iodide-labelled products formed were inorganic iodide and origin material, as observed previously (Gallon 8c Ingbar 1961). These products were added together for assessing the percentile deiodination of the two radioactive hor¬ the
ment of radio-chemical
mones.
The absolute amount of hormone degraded may be determined from the data on the fractional deiodination of its labelled isotope. However, this does not provide information on the absolute deiodination of the other iodothyronine. Since this study was concerned with the reciprocal effects of T4 and T3 on their degradation to iodide, the results are expressed as a fraction of the radioactive hormone that have under¬ gone deiodination.
Reciprocal effects by rat
Table 1. of thyroxine (T4) and triiodothyronine (T3) on their deiodination muscle homogenates. Mean values from 21 experiments.
5I]T3
5I]T4 Hormone load
Concentration
/(g/100
ml
125iodide released*
% of control
'«iodide released*
%.**
% of control
45.0 ± 2.31§
100.0
18.0 ± 1.76
100.0
25.0 25.0
34.6 ± 1.92 37.3 ± 1.98a
77.0 83.0
9.0 ± 1.02 13.0 ± 1.32b
50.0 72.0
2.0
100.0 100.0
22.6 ± 1.48 30.1 ± 1.70b
50.2 67.0
6.0 ± 0.65 9.6 ± 0.84c
33.0 53.3
8.0
400.0 400.0
13.0 ± 0.84 20.1 ± 1.42d
28.8 44.7
2.1 ± 0.16 5.0 ± 0.23d
11.7 27.7
800.0 800.0
6.2 ± 0.56 12.5 ± 0.90d
13.8 27.7
0.5 ± 0.05 2.0 ± 0.14d
2.8 11.1
0.015t
Nonet 0.5
16.0
T,
T4
To
Free 125I plus 125I deposited in the origin material. Per cent of added radioactivity. It represents the percentage of labelled hormone that underwent deiodination. t Control sample containing approximately 0.015 fig of substrate carried by the tracer labelled hormone. § Mean ± sem. Probability value obtained using Student's ¿-test: a: NS, b: < 0.05, c: < 0.02 and d: < 0.001. It indicates the significance of the difference between the effects of an equal amount of T4 or T3 on the deiodination of [125I]T4 and [125I]T3.
**
512
RESULTS
Muscle
homogenates (Table 1)
Deiodination of [12äI]T4, which carried approximately 0.015 fig of substrate, reached 45.0 °/o of the added dose during the 90 min control incubation. Sub¬ sequent aliquots incubated in addition with graded amounts of non-radioactive T4 or T3 caused a gradual decline in percentual deiodination of labelled T4. The decline was greater in homogenates to which non-radioactive T4 was added. The difference between the effects of an equal amount of T4 or T3 on the deiodination of [125I]T4 was not significant when the smaller dose of stable hormones was used (0.5 fig) but it became statistically significant with higher doses. Deiodination of [125I]T3 by muscle was markedly slower and reached 18.0% in control homogenates with a substrate of approximately 0.015 fig. Sub¬ sequent addition of non-radioactive T4 or T3 to individual homogenates re¬ sulted in a substantial reduction in the percentual breakdown of [123I]T3. Thus, after the addition of 0.5 fig of stable T4, deiodination of [I25I]T3 de-
Reciprocal
Table 2. effects of thyroxine (T4) and triiodothyronine (T3) on their deiodination by rat liver homogenates. Mean values from 18 experiments.
5I] T4 Hormone load Mg
Concentration /rg/100 ml
Nonet
0.015t
125iodide released* %**
5I]T3 % of control
l^iodide released* %**
% of control
14.6 ± 1.08§
100.0
8.5 ± 0.68
100.0
0.5
25.0 25.0
11.2 ± 1.06 12.0 ± 1.12
76.7 82.2
6.1 ± 0.58 5.8 + 0.58
71.7 68.2
2.0
100.0 100.0
9.6 ± 0.90 10.1 ± 0.90
65.7 69.2
5.3 ± 0.54 5.0 ± 0.52
62.3 58.8
400.0 400.0
S.2 ± 0.86 8.4 ± 0.92
56.1 57.5
4.3 ± 0.33 3.8 ± 0.38
50.5 44.7
800.0 800.0
5.9 ± 0.63 5.4 ± 0.65
40.4 37.0
3.9 ± 0.41 3.4 ± 0.38
45.9 40.0
16.0
To
t § For explanation of symbols see legend to Table 1. At all levels of hormone concentration the difference between the effects of equal amounts of T4 or T3 on the deiodination of either labelled hormone was not statis¬ * **
tically significant.
513 Acta endocr. 82, 3
creased to 50 % of the control sample, and with the maximal amount added (16 fig of stable T4) the breakdown of [125I]T3 was only 0.5%, which re¬ presented 2.8 % of the control homogenate. Stable T3 similarly caused a decline in [123I]T3 deiodination although to a lesser degree and the difference with the effects induced by stable T4 was statistically significant at all levels of substrate concentration.
Liver homogenates (Table 2) The breakdown of both labelled hormones by liver homogenates proceeded at a slower rate than in muscle. Control samples deiodinated a mean 14.6% of [123I]T4 and 0.5% of [123I]T3. Incubation with non-radioactive T4 or T3 reduced the fraction of both labelled hormones which were degraded to iodide. The degree of decline was less than that observed in muscle. In the case of [123I]T4, deiodination was reduced to 40.4 % of the control sample following incubation with 16 fig of stable T4, and to 37.0 % after the addition of an equal amount of stable T3. Comparable effects were observed with [12äI]T3. It is noteworthy that the pattern of deiodination differed from that observed in muscle, in that the breakdown of [125I]T4 and [125I]T3 were equally affected by similar amounts of stable T4 or T3.
DISCUSSION
After leaving the circulation T4 and T3 are distributed in tissue pools for ultimate degradation. Different cellular components seem to be associated with binding and deiodination of both hormones (Tata et al. 1962; Stanbury et al. I960; Oppenheimer et al. 1970). It has not been established unequi¬ vocally whether both iodothyronines are bound and degraded by the same cell site, although recent studies seem to indicate that T4 and T3 share cellular bonds (Samuel 8c Tsai 1974; Oppenheimer et al. 1974; DeGroot 8c Torresani
1975). The displacement of a labelled iodothyronine from its cellular site by its stable isotope has been evaluated in the rat by means of in vivo and in vitro techniques (Tata 1957; Tata et al. 1957; Oppenheimer et al. 1972). The present experiments were primarily designed to assess to what extent de¬ iodination of one thyroid hormone in whole tissue, is affected by the presence of the other iodothyronine. The studies in muscle tissue which constitutes over one-half of the rat body weight and therefore may account for a sizeable portion of the utilization of the thyroid hormones, revealed that over a wide range of substrate concentration, the decline in deiodination of both labelled 514
hormones was consistently more pronounced in homogenates incubated with T(. These hormones displayed different degrees of deiodination; hence, a more reliable assessment of the effects exerted by a hormone load can be drawn from a comparison with the percentile iodide generated by the control homogenate, which was associated with physiological quantities of hormone. The pattern thus obtained shows that on addition to muscle homogenates of equal quantities of non-radioactive T4 or T3 a greater recovery of intact labelled T3 is found as compound to labelled T4, this reflecting a more labile binding and easier displacement or labelled T3 from tissue sites. Even in the set of experiments using each radioactive hormone there occurred once again a consistently greater effect of a T4 load in diminishing the proportion of either labelled thyronine degraded to iodide. However, in liver homo¬ genates the decline in deiodination of both labelled hormones following the addition of either stable isotope was comparable. These results correlate with the data of Oppenheimer et al. (1970) indicating that the strength of binding of T4 and T3 by rat liver is approximately the same. The present in vitro experiments provide data consonant with the view that there is a cross-reactivity of T4 and T3 at muscle and liver cell sites. Oppenheimer et al. (1972) reported the presence of relatively specific binding sites for T3 in the nuclei of rat liver. However, further studies by these in¬ vestigators (Oppenheimer et al. 1974) led to the conclusion that liver nuclear sites also interact with T4. Other workers have similarly demonstrated a cross-reactivity of both thyroid hormones for cell sites, albeit of a dissimilar strength (Samuels 8c Tsai 1974; DeGroot 8c Torresani 1975). The interconversion of T4 to T3 should be equated in the analysis of in vivo studies. In the present in vitro experiments T4 conversion has not been ob¬ served in the qualitative analysis of radioiodinated metabolites. The interpretation of data derived from in vitro experiments in individual tissues can not be extrapolated to explain phenomena taking place in the whole body. This is emphasized by the markedly faster metabolism of T3 in vivo in relation to T4 (Rail et al. 1964). Whereas in vitro the opposite seems to occur in most tissues (Tata et al. 1957; Galton 8c Ingbar 1962; Broverman 8c Ingbar 1962); this is also shown in the present study. Never¬ theless, preliminary data from this laboratory performed in a similar fashion in the present experiments revealed that the in vivo deiodination of as [125I]T4 in the rat, as measured by urinary radioactivity, is significantly diminished by the administration of a loading dose of T4, whereas a similar T3 load was without effect during the same period of study (Zaninovich
1975). It is concluded that in the present in vitro system T4 and T3 appear to share sites of deiodination in rat muscle and liver and that, at least in muscle, T4 is preferentially deiodinated. 515 33 *
REFERENCES Braverman L. E. Se Ingbar S. H.: Endocrinology 79 (1962) 391. DeGroot L. J. 8e Torresani J.: Endocrinology 96 (1975) 357. Galton V. A. Se Ingbar S. H.: Endocrinology 68 (1961) 435. Galton V. A. Se Ingbar S. H.: Endocrinology 70 (1962) 622. Nicoloff J. T. 8e Dowling J. T.: J. clin. Invest. 47 (1968) 26. Oppenheimer J. H., Koerner D., Schwartz H. L. Se Surks M. L:
J. clin. Endocr. 35 (1972) 330. Oppenheimer J. H., Schwartz H. L., Koerner D. Se Surks M. L: J. clin. Invest. 53 (1974) 768. Oppenheimer J. H., Schwartz H. L., Shapiro H. C, Bernstein J. Se Surks M. J.: J. clin. Invest. 49 (1970) 1016. Rail J. E., Robins J. Se Lewallen C. G. In: Pincus G, Thimann K. V. and Astwood E. B., Eds. The Hormones, Vol. 4. Academic Press Inc., New York (1964) 159. Samuels H. H. Se Tsai J. S.: J. clin. Invest. 53 (1974) 656. Stanbury J. B., Morris M. L., Corrigan H. J. Se Lassiter W. E.: Endocrinology 67 (1960) 353. Tata J. R.: Proc. Soc. exp. Biol. (N. Y.) 95 (1957) 362. Tata J. R., Ernster L. Se Suranyi E. M.: Biochim. biophys. Acta (Amst.) 60 (1962) 480. Tata J. R., Rail J. E. Se Rawson R. W.: Endocrinology 60 (1957) 83. Zaninovich A. A.: Acta endocr. (Kbh.) Suppl. 799 (1975) 310. Zaninovich A. A., Dégrossi O. Se Pecorini V.: J. clin. Endocr. 32 (1971) 509. Zaninovich A. A., Farach H., Ezrin C. Se Volpé H.: J. clin. Invest. 45 (1966) 1290. Zaninovich A. A., Volpé R. Se Ezrin C: J. clin. Endocr. 29 (1969a) 1601. Zaninovich A. A.. Volpé R.. Soto R. J. Se Ezrin C: Acta endocr. (Kbh.) 60 (19695) 412.
Received
on
June 13th,
1975.
516
RECIPROCAL EFFECTS OF THYROXINE AND TRIIODOTHYRONINE ON THEIR DEIODINATION BY RAT TISSUES IN VITRO
By
Angel
A. Zaninovich
ABSTRACT
reciprocal effects of loading doses of thyroxine (T4) or triiodothyronine (T3) on the deiodination of their 125iodine-labelled isotopes by rat muscle and liver homogenates were studied. In 21 experiments muscle homogenates deiodinated a mean 45.0 % of a tracer dose of [125I]T4 and 18.0% of [125I]T3. On addition of graded amounts of nonradioactive T4 or T3 the percentual deiodination of both labelled hormones progressively declined. This effect was significantly greater in homogenates incubated with non-radioactive T4, thus reflecting a stronger affinity of this hormone for muscle deiodinating sites. This correlate with as revealed by the percentage of the greater displacement of [125I]T3 recovered labelled hormone. In 18 experiments liver homogenates deiodinated a mean 14.6% of a tracer amount of [125I]T4 and 8.5% of [125I]T3. The addition of a T4- or a T3-load was followed by a smaller decrease in percentual deiodination of both labelled hormones as compared to muscle homogenates. Unlike the effects observed in muscle, the breakdown of [125I]T4 and [125I]T3 by liver homogenates was equally affected by similar amounts of stable T4 or T3. It is concluded that in The
the present in vitro system *
Career
Investigator, Consejo
T4
and
T3
Nacional de
share cellular sites of deiodination
Investigaciones
Científices y Técnicas of
Argentina (CONICET).
Supported by grant
No. 3728 from the CONICET. Presented in part at the 9th Acta Endocrinologica
510
Congress, Oslo, Norway,
1973.
in rat muscle and liver and that, at least in muscle, which constitutes one-half of the rat body weight T4 appears to be preferentially deiodinated.
over
Studies in this
laboratory first reported data suggesting a secondary role of plasma binding in the physiological disposal of triiodothyronine (T3) in man (Zaninovich et al. 1966, 1969a,i5, 1971). Further kinetic studies of T3 by Oppenheimer et al. (1970) lent support to this contention by demonstrating that T3 is primarily an intracellular hormone, both in man and the rat. The findings on T3 peripheral metabolism led us to postulate that T4 and T3 might compete for tissue sites with a selective uptake and degradation of T4 by certain tissues (Zaninovich et al. 1969a,t5, 1971). With this framework in mind the present investigations have been made on the reciprocal effects of T4 and T3 on their deiodination by rat muscle and liver homogenates.
MATERIAL AND METHODS Two-month old rats of the Wistar strain weighing approximately 200 g were used. Before the study the animals were maintained on commercial laboratory chow and water ad libitum. [125I]T4 and [125I]T3 were obtained from a commercial source (Sorin, Italy) with a specific activity of approximately 80 mCi/mg. These labelled compounds were freed of contaminating iodide by dialysis, as described by Nicoloff Se
Dowling (1968).
Deiodination of [125I]T4 and [125I]T3 was studied in fresh homogenates of muscle and liver1). The animals were killed by a blow on the head and tissues were homo¬ genized in chilled teflon or glass grinders using 1 g of tissue in 5 ml of ice-cold Krebs-Ringer phosphate buffer pH 7.4 prepared in the manner described by Galton 8c Ingbar (1961). Two ml aliquots of homogenate were separated and tracer amounts of [125I]T4 or [125I]T3 (1 ,aCi and approximately 0.015 fig) were added. These homo¬ genates containing only labelled hormones will be referred to in the text as control samples. In some of the samples, in addition, a freshly prepared solution of nonradioactive T4 or T3 was added to the homogenate to give a final hormone concen¬ tration ranging from 25 to 800 ug per 100 ml. The mixture was incubated at 37°C for 70 min in room air in a continuously-shaking apparatus. Following incubation 1 ml of human plasma was added to each homogenate to stop further hormone de¬ iodination. Paper chromatography was performed by descending technique in butanoldioxane-ammonia (4:1:5) and in butanol-acetic acid-water (80:10:12) during 20 h. The results obtained in both solvent systems were comparable and thereafter, studies were carried out in the alkaline system. Appropriate reference standards were used and their positions were located by staining with palladium chloride in the case of iodide and ninhydrin for the iodothyronines. In each experiment chromatography of ') Initial studies in which 300 mg-tissue slices were incubated with T4 or T3 and further homogenized and chromatographed, exhibited similar results to those ob¬ tained with tissue homogenates.
511
dialyzed
solution of both labelled hormones was similarly performed for assess¬ purity. The areas of radioactivity were located in a stripscanner equipped with an electronic integrator (Actigraph III, Nuclear, Chicago). A tissue-free system containing 2 ml of buffer and a tracer dose of labelled T4 or T3 was similarly incubated and chromatographed. All experiments were performed in duplicate. The major 125iodide-labelled products formed were inorganic iodide and origin material, as observed previously (Gallon 8c Ingbar 1961). These products were added together for assessing the percentile deiodination of the two radioactive hor¬ the
ment of radio-chemical
mones.
The absolute amount of hormone degraded may be determined from the data on the fractional deiodination of its labelled isotope. However, this does not provide information on the absolute deiodination of the other iodothyronine. Since this study was concerned with the reciprocal effects of T4 and T3 on their degradation to iodide, the results are expressed as a fraction of the radioactive hormone that have under¬ gone deiodination.
Reciprocal effects by rat
Table 1. of thyroxine (T4) and triiodothyronine (T3) on their deiodination muscle homogenates. Mean values from 21 experiments.
5I]T3
5I]T4 Hormone load
Concentration
/(g/100
ml
125iodide released*
% of control
'«iodide released*
%.**
% of control
45.0 ± 2.31§
100.0
18.0 ± 1.76
100.0
25.0 25.0
34.6 ± 1.92 37.3 ± 1.98a
77.0 83.0
9.0 ± 1.02 13.0 ± 1.32b
50.0 72.0
2.0
100.0 100.0
22.6 ± 1.48 30.1 ± 1.70b
50.2 67.0
6.0 ± 0.65 9.6 ± 0.84c
33.0 53.3
8.0
400.0 400.0
13.0 ± 0.84 20.1 ± 1.42d
28.8 44.7
2.1 ± 0.16 5.0 ± 0.23d
11.7 27.7
800.0 800.0
6.2 ± 0.56 12.5 ± 0.90d
13.8 27.7
0.5 ± 0.05 2.0 ± 0.14d
2.8 11.1
0.015t
Nonet 0.5
16.0
T,
T4
To
Free 125I plus 125I deposited in the origin material. Per cent of added radioactivity. It represents the percentage of labelled hormone that underwent deiodination. t Control sample containing approximately 0.015 fig of substrate carried by the tracer labelled hormone. § Mean ± sem. Probability value obtained using Student's ¿-test: a: NS, b: < 0.05, c: < 0.02 and d: < 0.001. It indicates the significance of the difference between the effects of an equal amount of T4 or T3 on the deiodination of [125I]T4 and [125I]T3.
**
512
RESULTS
Muscle
homogenates (Table 1)
Deiodination of [12äI]T4, which carried approximately 0.015 fig of substrate, reached 45.0 °/o of the added dose during the 90 min control incubation. Sub¬ sequent aliquots incubated in addition with graded amounts of non-radioactive T4 or T3 caused a gradual decline in percentual deiodination of labelled T4. The decline was greater in homogenates to which non-radioactive T4 was added. The difference between the effects of an equal amount of T4 or T3 on the deiodination of [125I]T4 was not significant when the smaller dose of stable hormones was used (0.5 fig) but it became statistically significant with higher doses. Deiodination of [125I]T3 by muscle was markedly slower and reached 18.0% in control homogenates with a substrate of approximately 0.015 fig. Sub¬ sequent addition of non-radioactive T4 or T3 to individual homogenates re¬ sulted in a substantial reduction in the percentual breakdown of [123I]T3. Thus, after the addition of 0.5 fig of stable T4, deiodination of [I25I]T3 de-
Reciprocal
Table 2. effects of thyroxine (T4) and triiodothyronine (T3) on their deiodination by rat liver homogenates. Mean values from 18 experiments.
5I] T4 Hormone load Mg
Concentration /rg/100 ml
Nonet
0.015t
125iodide released* %**
5I]T3 % of control
l^iodide released* %**
% of control
14.6 ± 1.08§
100.0
8.5 ± 0.68
100.0
0.5
25.0 25.0
11.2 ± 1.06 12.0 ± 1.12
76.7 82.2
6.1 ± 0.58 5.8 + 0.58
71.7 68.2
2.0
100.0 100.0
9.6 ± 0.90 10.1 ± 0.90
65.7 69.2
5.3 ± 0.54 5.0 ± 0.52
62.3 58.8
400.0 400.0
S.2 ± 0.86 8.4 ± 0.92
56.1 57.5
4.3 ± 0.33 3.8 ± 0.38
50.5 44.7
800.0 800.0
5.9 ± 0.63 5.4 ± 0.65
40.4 37.0
3.9 ± 0.41 3.4 ± 0.38
45.9 40.0
16.0
To
t § For explanation of symbols see legend to Table 1. At all levels of hormone concentration the difference between the effects of equal amounts of T4 or T3 on the deiodination of either labelled hormone was not statis¬ * **
tically significant.
513 Acta endocr. 82, 3
creased to 50 % of the control sample, and with the maximal amount added (16 fig of stable T4) the breakdown of [125I]T3 was only 0.5%, which re¬ presented 2.8 % of the control homogenate. Stable T3 similarly caused a decline in [123I]T3 deiodination although to a lesser degree and the difference with the effects induced by stable T4 was statistically significant at all levels of substrate concentration.
Liver homogenates (Table 2) The breakdown of both labelled hormones by liver homogenates proceeded at a slower rate than in muscle. Control samples deiodinated a mean 14.6% of [123I]T4 and 0.5% of [123I]T3. Incubation with non-radioactive T4 or T3 reduced the fraction of both labelled hormones which were degraded to iodide. The degree of decline was less than that observed in muscle. In the case of [123I]T4, deiodination was reduced to 40.4 % of the control sample following incubation with 16 fig of stable T4, and to 37.0 % after the addition of an equal amount of stable T3. Comparable effects were observed with [12äI]T3. It is noteworthy that the pattern of deiodination differed from that observed in muscle, in that the breakdown of [125I]T4 and [125I]T3 were equally affected by similar amounts of stable T4 or T3.
DISCUSSION
After leaving the circulation T4 and T3 are distributed in tissue pools for ultimate degradation. Different cellular components seem to be associated with binding and deiodination of both hormones (Tata et al. 1962; Stanbury et al. I960; Oppenheimer et al. 1970). It has not been established unequi¬ vocally whether both iodothyronines are bound and degraded by the same cell site, although recent studies seem to indicate that T4 and T3 share cellular bonds (Samuel 8c Tsai 1974; Oppenheimer et al. 1974; DeGroot 8c Torresani
1975). The displacement of a labelled iodothyronine from its cellular site by its stable isotope has been evaluated in the rat by means of in vivo and in vitro techniques (Tata 1957; Tata et al. 1957; Oppenheimer et al. 1972). The present experiments were primarily designed to assess to what extent de¬ iodination of one thyroid hormone in whole tissue, is affected by the presence of the other iodothyronine. The studies in muscle tissue which constitutes over one-half of the rat body weight and therefore may account for a sizeable portion of the utilization of the thyroid hormones, revealed that over a wide range of substrate concentration, the decline in deiodination of both labelled 514
hormones was consistently more pronounced in homogenates incubated with T(. These hormones displayed different degrees of deiodination; hence, a more reliable assessment of the effects exerted by a hormone load can be drawn from a comparison with the percentile iodide generated by the control homogenate, which was associated with physiological quantities of hormone. The pattern thus obtained shows that on addition to muscle homogenates of equal quantities of non-radioactive T4 or T3 a greater recovery of intact labelled T3 is found as compound to labelled T4, this reflecting a more labile binding and easier displacement or labelled T3 from tissue sites. Even in the set of experiments using each radioactive hormone there occurred once again a consistently greater effect of a T4 load in diminishing the proportion of either labelled thyronine degraded to iodide. However, in liver homo¬ genates the decline in deiodination of both labelled hormones following the addition of either stable isotope was comparable. These results correlate with the data of Oppenheimer et al. (1970) indicating that the strength of binding of T4 and T3 by rat liver is approximately the same. The present in vitro experiments provide data consonant with the view that there is a cross-reactivity of T4 and T3 at muscle and liver cell sites. Oppenheimer et al. (1972) reported the presence of relatively specific binding sites for T3 in the nuclei of rat liver. However, further studies by these in¬ vestigators (Oppenheimer et al. 1974) led to the conclusion that liver nuclear sites also interact with T4. Other workers have similarly demonstrated a cross-reactivity of both thyroid hormones for cell sites, albeit of a dissimilar strength (Samuels 8c Tsai 1974; DeGroot 8c Torresani 1975). The interconversion of T4 to T3 should be equated in the analysis of in vivo studies. In the present in vitro experiments T4 conversion has not been ob¬ served in the qualitative analysis of radioiodinated metabolites. The interpretation of data derived from in vitro experiments in individual tissues can not be extrapolated to explain phenomena taking place in the whole body. This is emphasized by the markedly faster metabolism of T3 in vivo in relation to T4 (Rail et al. 1964). Whereas in vitro the opposite seems to occur in most tissues (Tata et al. 1957; Galton 8c Ingbar 1962; Broverman 8c Ingbar 1962); this is also shown in the present study. Never¬ theless, preliminary data from this laboratory performed in a similar fashion in the present experiments revealed that the in vivo deiodination of as [125I]T4 in the rat, as measured by urinary radioactivity, is significantly diminished by the administration of a loading dose of T4, whereas a similar T3 load was without effect during the same period of study (Zaninovich
1975). It is concluded that in the present in vitro system T4 and T3 appear to share sites of deiodination in rat muscle and liver and that, at least in muscle, T4 is preferentially deiodinated. 515 33 *
REFERENCES Braverman L. E. Se Ingbar S. H.: Endocrinology 79 (1962) 391. DeGroot L. J. 8e Torresani J.: Endocrinology 96 (1975) 357. Galton V. A. Se Ingbar S. H.: Endocrinology 68 (1961) 435. Galton V. A. Se Ingbar S. H.: Endocrinology 70 (1962) 622. Nicoloff J. T. 8e Dowling J. T.: J. clin. Invest. 47 (1968) 26. Oppenheimer J. H., Koerner D., Schwartz H. L. Se Surks M. L:
J. clin. Endocr. 35 (1972) 330. Oppenheimer J. H., Schwartz H. L., Koerner D. Se Surks M. L: J. clin. Invest. 53 (1974) 768. Oppenheimer J. H., Schwartz H. L., Shapiro H. C, Bernstein J. Se Surks M. J.: J. clin. Invest. 49 (1970) 1016. Rail J. E., Robins J. Se Lewallen C. G. In: Pincus G, Thimann K. V. and Astwood E. B., Eds. The Hormones, Vol. 4. Academic Press Inc., New York (1964) 159. Samuels H. H. Se Tsai J. S.: J. clin. Invest. 53 (1974) 656. Stanbury J. B., Morris M. L., Corrigan H. J. Se Lassiter W. E.: Endocrinology 67 (1960) 353. Tata J. R.: Proc. Soc. exp. Biol. (N. Y.) 95 (1957) 362. Tata J. R., Ernster L. Se Suranyi E. M.: Biochim. biophys. Acta (Amst.) 60 (1962) 480. Tata J. R., Rail J. E. Se Rawson R. W.: Endocrinology 60 (1957) 83. Zaninovich A. A.: Acta endocr. (Kbh.) Suppl. 799 (1975) 310. Zaninovich A. A., Dégrossi O. Se Pecorini V.: J. clin. Endocr. 32 (1971) 509. Zaninovich A. A., Farach H., Ezrin C. Se Volpé H.: J. clin. Invest. 45 (1966) 1290. Zaninovich A. A., Volpé R. Se Ezrin C: J. clin. Endocr. 29 (1969a) 1601. Zaninovich A. A.. Volpé R.. Soto R. J. Se Ezrin C: Acta endocr. (Kbh.) 60 (19695) 412.
Received
on
June 13th,
1975.
516
