- Email: [email protected]
[20] Methods for assessing thyroid stimulating hormone effects on thyroidal iodide transport
256
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[20]
upon the ADH-induced stimulation of sodium transport? ~ Since the water flow response to exogenous cyclic AMP is not affected by high concentrations of these ions it seems likely that two adenylate cyelases are present in the tissue. One of these is unaffected by high calcium or magnesium and is responsible for the stimulation of sodium transport by ADH. The other is responsible for the stimulation of water flow and is inhibited by high concentrations of these divalent cations. A similar conclusion may be drawn from the fact that prostaglandin E, is capable of stimulating sodium transport by stimulation of an adenylate cyclase, while simultaneously it can inhibit the water flow response to ADH by inhibiting an adenylate cyclase. 5 Other agents known to influence the water flow response to ADH include phosphodiesterase inhibitors such as theophylline, adrenocortical steroids, and prostaglandin inhibitors, all of which enhance the response. Inhibitory agents in addition to calcium, magnesium, and prostaglandins include agents which interact with microtubules such as vinblastine.
[20] M e t h o d s for A s s e s s i n g T h y r o i d S t i m u l a t i n g H o r m o n e Effects on Thyroidal Iodide Transport By WINTON TONG The active transport process that functions in the thyroidal uptake and accumulation of ionic iodide, and the regulation of this process by the pituitary hormone, thyrotropin (TSH), comprise one of the few examples of a membrane transport mechanism for which a hormonal control system has been characterized in detail. The stimulation of glucose transport by insulin and of sodium transport by aldosterone have been studied more extensively, but the current understanding of how these hormones act at the cellular level is no more advanced than is the case for the actions of TSH. Current notions regarding the nature of the thyroidal iodide concentrating mechanism, and the experimental documentation supporting these notions have been reviewed in detail? Kinetic models formulating thyroidal iodide transport in terms of its component pump and leak fluxes have also been described in the review article cited above. Depending on the level of the antecedent iodide supply and TSH stimulation, intrathyroidal inorganic iodide concentration may achieve 1j. Wolff,Physiol. Rev. 44, 45 (1964).
[20]
TSH E F F E C T S ON THYROID IODIDE T R A N S P O R T
257
values of ten- to a thousand-fold above the concentration of iodide in the blood or in the media in which thyroid tissue slices or isolated thyroid cells are incubated. Operationally the ratio of intrathyroidal to extracellular fluid iodide concentrations is generally expressed in terms of T / S (I), T / M (I), or C / M (I) values as follows: T / S (I) -- ~g iodide per g thyroid gland ~g iodide per ml serum ~g iodide per g thyroid slice T / M (I) -ug iodide per ml incubation medium ug iodide per ml thyroid cells C / M (I) = ug iodide per ml incubation medium T / S (I) ratios would be observed in experiments using whole animals, while T / M (I) and C / M (I) ratios would be obtained from thyroid tissue slices and isolated thyroid cells incubated in vitro. The measurements are usually made with the aid of iodide labeled with the radioactive isotopes,lalI o r 12~I, and in order to insure that all the radioactivity recovered in the thyroid tissues will be in the form of the transported ion, the further utilization of the iodide for the iodination of thyroglobulin is blocked with the antithyroid drugs 6-propylthiouracit (PTU) or 1-methyl, 2-mereaptoimidazole ( M M I ) . Thus, the thyroid specimens may be assayed directly for radioactivity without prior separation into iodide and iodoprotein fractions, and the T / S (I), T / M (I), and C / M (I) may be computed from the ratios of counts per minute of radioactivity in lieu of micrograms of iodide. In the text that follows, experimental approaches will be described for studying the actions of T S H under "in vivo and in vitro conditions.
In Vivo Studies: The Actions of Injected TSH on Thyroidal Iodide
Transport In Rats As Hahni and his colleagues have shown, '-','~ the T / S (I) of rats fell from normal values of at)out 25 to values as low as 6 within 10 days after hypophysectomy. A single subcutaneous injection of 5 mg of T S H given to these rats returned the T / S (I) to 20 within 10 hours, and to " N. S. Halmi, B. N. Spirtos, E. M. Bogdanove, and H. J. Lipner, E~docrinology 52, 19 (1953). 3N. S. Halmi, D. K. Granner, D. J. Doughman, B. H. Peters, and G. Muller, Endocrinology 67, 70 (1960).
258
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[20]
a maximum of about 30 at 48 hours. In subsequent studies, it was found that the response to TSH was really biphasic in nature: The initial response was a peculiar, transient depression of the T / S (I), followed by the increase in the T / S (I) that developed slowly thereafter. Further studies into this biphasic action of TSH on isolated thyroid cells will be described below. In Halmi's experiments, T / S (I) values were determined by a modification of the method of VanderLaan and Greer 4 as follows: Rats were injected subcutaneously with 12 mg of PT U dissolved in dilute NaOH; 45 minutes later, 10 ~Ci of 131I were injected subcutaneously; finally, the rats were anesthetized with ether, blood samples were taken from the aorta, and thyroid glands were excised. The thyroid glands were weighed quickly on an analytical balance and then assayed for 13II in a well-type crystal gamma scintillation spectrometer. Aliquots of blood sera were similarly assayed for 131I. Then the T / S (I) values were computed from the radioassay data.
I n Vitro Studies: The Actions of T S H on Iodide Transport in Isolated Thyroid Cells The cells isolated from thyroid tissues dispersed by gentle digestion with trypsin and collagenase retain the capacity to transport and concentrate iodide. Moreover, such isolated thyroid cells respond to added TSH with biphasic changes in iodide transport that appear to be entirely analogous to those observed in the thyroid glands of rats following the injection of TSH2 Indeed, the cell suspension system is the only in vitro thyroid preparation that has been successfully employed to demonstrate the actions of TSH on iodide transport. Procedures for the preparation of dispersed thyroid cells have been reported elsewhere2 Techniques for observing the effects of TSH and of cyclic AMP derivatives on C / M (I) and on the component unidirectional pump and leak iodide fluxes are described below.
The Biphasic Effects of TSH and Dibutyryl cyclic A M P on C / M (I) Iodide concentrating activity, expressed in terms of steady state C / M (I) values, may be observed by incubating isolated thyroid cells in Eagle's minimum essential medium 6 supplemented with 1 toM iodide 4W. P. VanderLaan and M. A. Greer, Endocrinology 47, 36 (1950). 5W. Tong and M. R. Sherman, Vol. 36 [14]. GH. Eagle, Science 122, 510 (1955).
[20]
TSH EFFECTS ON THYROID IODIDE TRANSPORT
259
labeled with 13lI at a concentration of 0.1 ~Ci/ml of medium. Also, MMI is added to the medium at 2 mM concentration to block incorporation of the iodide into organically bound products, and penicillin (105 units/liter) and streptomycin sulfate (0.1 g/liter) may be added to inhibit bacterial growth during long-term incubations. For most experiments, 2 ml of cells are dispersed in 200 ml of medium and 5 ml aliquots of the resulting suspension are incubated under 95% 02, 5% CO~ in 25 ml Erlenmeyer flasks at 37 ° with shaking at a rate of 80-100 per minute. Steady state distribution of tile [l:~lI]iodide between the cells and the medium is usually reached within 30 minutes. Then the incubated cell suspensions are transferred to conical tubes, and the cells are sedimented by eentrifugation for 5 minutes at 150 g. The supernatant media are withdrawn, and 1 ml aliquots assayed for 131I. The pellet of packed cells is mixed by stirring, and carefully measured aliquots are taken using 25 ~1 disposable, glass micropipettes. These cell aliquots are also assayed directly for 131I. Finally, the C/M (I) is computed from the ratio of '3~I cpm/ml of cells to 1~I epm/nfl of media. The C/M (I) of healthy lamb or steer thyroid cells range between 8 and 20. Cell preparations giving C/M (I) of less than 8 should be rejected. For reasons that are not clear, the cells isolated from cow and pork thyroid glands give nmeh higher C/M (I) values ranging from 40 to 60. When TSH or the N~-O~'-dibutyryl derivative of cyclic AMP (DBC) are added to steer thyroid cells that had been previously equilibrated with I~'~II]iodid~~, the C/M (I) undergoes a peculiar biphasic alteration which seems to be the counterpart of the phenomenon occurring in rats injected with TSH as described by Halmi et al. 3 Following the addition of the TSH or DBC, the C/M (I) first declines rapidly to 30% reduced values after 30 minutes, and then gradually increases to about 100% increased values after about 6 hours. These alterations occur maximally in response to TSH concentrations as low as 0.5 mU/ml and DBC at 3 raM. The effects of TSH and DBC seem to take place through two different mechanisms. The rapid decline in C/M (I) is evidently a direct effect of TSH to augment the iodide leak flux; the effect is abolished and the C/M (Ii restored to control values when the added TSH is withdrawn and the cells treated with 0.06~, crystalline trypsin to inactivate TSH adherent to the cells. The slowly developed increase in C/M (1) seems to involve the induction of an enzyme that is rate limiting for the pump process. This part of the response to TSH and DBC can be blocked by 5 ~g/ml ,~ctinomycin D or by 0.1 mM cycloheximideJ Its initiation by 7j. Knopp, V. Stole, and W. Tong, J. Biol. Chem. 245, 4403 (1970).
260
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[20]
TSH or DBC is completed after about 2 hours. Thereafter, the TSH or DBC may be removed by the tryptic washout procedure and the C/M (I) will continue its rise to fully stimulated levels.
The E~ects of TSH and DBC on the Saturation Kinetics of the Iodide Pump The steady state C / M (I) seems to be the resultant of opposing pump and leak iodide fluxes. When the C/M (I) is increased by treatment with TSH or DBC, the primary change seems to be an increase in the pump flux. As a secondary consequence, the leak flux is also increased, but a new steady state is achieved with intracellular iodide concentration established at a higher level. Evidence for this has been obtained by applying the Lineweaver-Burk analysis to compare the saturation kinetics of unidirectional iodide influx in control and TSH- or DBC-stimulated steer thyroid cells. For such experiments, the cells are suspended in 10 volumes of Eagle's minimum essential medium supplemented with 2 triM MMI, penicillin, and streptomycin, and incubated under 95% O~, 5% CO~ at 37 ° for 6 hours. To obtain fully stimulated cells, half of the cell suspension is incubated with added TSH (50 mU/ml) or DBC (3 raM). Following this preincubation, the cells are recovered by centrifugation for 5 minutes at 150 g, washed, and treated with 0.06% crystalline trypsin in Eagle's medium for 10 minutes at 37 ° to remove adherent TSH and DBC. To determine unidirectional iodide influx, the cells are washed again and resuspended in 100 volumes of fresh incubation medium, and 5 ml aliquots are dispensed into 25 ml Erlenmeyer flasks. These specimens are then placed in the incubator shaker and brought to 37% Finally, at zero time, 100 ~1 of [131I]iodide solution containing 1 t~Ci of 131I is added, and after 10, 20, and 30 second incubation periods, specimens are transferred to centrifuge tubes, the cells sedimented by centrifugation for 30 seconds at 2000 g, and the 13lI concentration of the cells determined. The plot of cellular 131I concentration against time is a straight line whose slope is the rate of 13'I influx into the cells, which, divided by the specific radioactivity of the iodide will give the iodide influx in microgram per minute per milliliter of cells. By conducting these influx measurements in media containing 10, 13, 20, 40, and 60 ~M iodide, the saturation kinetics of the influx process will be observed. The Lineweaver-Burk plot of the reciprocal of iodide influx vs. the reciprocal of extracellular iodide concentration gives a straight line with the ordinate intercept equal to V..... having values of about 20 ~g/minute/ml of cells. The abscissal intercept gives a K,, of
[20]
261
TSH EFFECTS ON THYROID IODIDE TRANSPORT r-
~
~
- -
-'
- -
7
Vmax S.3xlO-SM 20ttglMINImlCELLS 57 ,, 37 ,, 5.7 37 ,, Km
-~u" 0'St oCONTROL • TSH O.IUlml o 3mM DBC
I i
.
_J 1
04 E z: ~
0.3
i-t
..> 02
~
01
-20
I
I
I
I
L
I
[
20
40
60
80
I00
120
140
I/S (mM -I) FIG. 1. Steer thyroid cells were preincubated for 6 hours without or with either 0.1 u n i t / m l TSH or 3 m M DBC. Then the cells were equilibrated with media containing 100, 150, 300, or 1000 t~g % iodide. Finally, ~31I was added, and from the observed rates of ~3~I uptake at 10, 20, and 30 seconds thereafter, unidirectional iodide influx rates were computed for each iodide concentration (from K n o p p et ~1.7).
about 55 uM. In cells fully stimulated by incubation with T S H or DBC, the Vm~ is increased to about 35 ug/minute/ml of cells, while the Km remains unchanged (Fig. 1).
The E#ects o] TSH and DBC on the Kinetics o] the [odide Leak Although the primary effect of T S H and DBC may be the increase in the iodide influx mediated by the iodide pump, augmentation of the opposing iodide leak also occurs at the same time. This aspect of the response to TSH and DBC seems to reflect direct actions of these agents on the thyroid cell membrane to increase its permeability toward the passive outward diffusion of iodide. The best quantitative indication of this permeability change is the rate constant for unidirectional iodide efflux which is most readily determined by observing the rate of discharge of radioactivity from cells previously loaded with [13'I]iodide. To load with '3'I, thyroid cells are incubated for 1 hour at 37 ° in 20 volumes of Eagle's minimum essential medium supplemented with 3 m M M M I and 10 ~M iodide labeled with 13'I at a concentration of
262
EVALUATION OF BIOLOGICAL E F F E C T S OF HORMONES
[21]
1 tLC/ml. The resulting suspension of 131I-labeled cells is then distributed in 2 ml aliquots (each containing 0.1 ml of cells) into a series of test tubes, the cells are sedimented by centrifugation for 5 minutes at 150 g, the media discarded, and the inside surfaces of each tube wiped with tissue paper to remove adherent 131I. To observe the rate of lalI discharge, 2 ml of fresh Eagle's medium containing 3 mM MMI, but no [13q]iodide, are added to each ~a~I-labeled cell aliquot along with a miniature Teflon-coated magnetic stirring bar; 131I discharge is allowed to occur at 37 ° with mixing for 30 seconds, following which the cells are sedimented for 30 seconds at 2000 g, and the supernatant fluid is decanted and assayed for radioactivity. Then another 2 inl portion of fresh medium is added to the cell pellet, and lalI discharge is allowed to proceed for another 30 second period. This procedure is repeated about 20 times. Finally, the cell pellet itself is assayed for lalI. The total ~'I originally present in the cell pellet (I0) is computed by adding together all of the 13'I discharged plus the 131I remaining in the final cell pellet. The '31I remaining in the cell pellet at each time interval (It) is calculated by subtracting from Io all of the ~31I discharged into the media samples collected previously. The rate of [~3~I]iodide discharge is invariably an exponential function so that In It/Io = --kot where/co is the rate constant of iodide efflux. Thus, In It~I,, plotted against time yields a straight line whose slope is equal to --ko. The /Co observed for control steer thyroid cells has a value of about 0.25 min-k When the ~3~I efftux determinations are performed on cells preincubated for 30 minutes with TSH at concentrations of 10 m units/ml or higher, the ko is increased to values approximately 0.35 rain -1.
[21] M e t h o d s for A s s e s s i n g H o r m o n e ( T S H ) E f f e c t s on Glucose Oxidation and Phospholipid Synthesis B y JAMES B. FIELD
Thyroid stimulating hormone (TSH) stimulates a variety of metabolic and morphologic parameters in thyroid cells. 1 TSH initially attaches to specific receptors on the cell membrane and subsequently activates the adenylate cyclase-cyclic AMP system. Most of the morphologic and metabolic effects of TSH including stimulation of glucose oxidation are probably mediated by cyclic AMP, but this is less well established for 1j. B. Field, Metab., Clin. Exp. 17, 226 (1968).
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[20]
upon the ADH-induced stimulation of sodium transport? ~ Since the water flow response to exogenous cyclic AMP is not affected by high concentrations of these ions it seems likely that two adenylate cyelases are present in the tissue. One of these is unaffected by high calcium or magnesium and is responsible for the stimulation of sodium transport by ADH. The other is responsible for the stimulation of water flow and is inhibited by high concentrations of these divalent cations. A similar conclusion may be drawn from the fact that prostaglandin E, is capable of stimulating sodium transport by stimulation of an adenylate cyclase, while simultaneously it can inhibit the water flow response to ADH by inhibiting an adenylate cyclase. 5 Other agents known to influence the water flow response to ADH include phosphodiesterase inhibitors such as theophylline, adrenocortical steroids, and prostaglandin inhibitors, all of which enhance the response. Inhibitory agents in addition to calcium, magnesium, and prostaglandins include agents which interact with microtubules such as vinblastine.
[20] M e t h o d s for A s s e s s i n g T h y r o i d S t i m u l a t i n g H o r m o n e Effects on Thyroidal Iodide Transport By WINTON TONG The active transport process that functions in the thyroidal uptake and accumulation of ionic iodide, and the regulation of this process by the pituitary hormone, thyrotropin (TSH), comprise one of the few examples of a membrane transport mechanism for which a hormonal control system has been characterized in detail. The stimulation of glucose transport by insulin and of sodium transport by aldosterone have been studied more extensively, but the current understanding of how these hormones act at the cellular level is no more advanced than is the case for the actions of TSH. Current notions regarding the nature of the thyroidal iodide concentrating mechanism, and the experimental documentation supporting these notions have been reviewed in detail? Kinetic models formulating thyroidal iodide transport in terms of its component pump and leak fluxes have also been described in the review article cited above. Depending on the level of the antecedent iodide supply and TSH stimulation, intrathyroidal inorganic iodide concentration may achieve 1j. Wolff,Physiol. Rev. 44, 45 (1964).
[20]
TSH E F F E C T S ON THYROID IODIDE T R A N S P O R T
257
values of ten- to a thousand-fold above the concentration of iodide in the blood or in the media in which thyroid tissue slices or isolated thyroid cells are incubated. Operationally the ratio of intrathyroidal to extracellular fluid iodide concentrations is generally expressed in terms of T / S (I), T / M (I), or C / M (I) values as follows: T / S (I) -- ~g iodide per g thyroid gland ~g iodide per ml serum ~g iodide per g thyroid slice T / M (I) -ug iodide per ml incubation medium ug iodide per ml thyroid cells C / M (I) = ug iodide per ml incubation medium T / S (I) ratios would be observed in experiments using whole animals, while T / M (I) and C / M (I) ratios would be obtained from thyroid tissue slices and isolated thyroid cells incubated in vitro. The measurements are usually made with the aid of iodide labeled with the radioactive isotopes,lalI o r 12~I, and in order to insure that all the radioactivity recovered in the thyroid tissues will be in the form of the transported ion, the further utilization of the iodide for the iodination of thyroglobulin is blocked with the antithyroid drugs 6-propylthiouracit (PTU) or 1-methyl, 2-mereaptoimidazole ( M M I ) . Thus, the thyroid specimens may be assayed directly for radioactivity without prior separation into iodide and iodoprotein fractions, and the T / S (I), T / M (I), and C / M (I) may be computed from the ratios of counts per minute of radioactivity in lieu of micrograms of iodide. In the text that follows, experimental approaches will be described for studying the actions of T S H under "in vivo and in vitro conditions.
In Vivo Studies: The Actions of Injected TSH on Thyroidal Iodide
Transport In Rats As Hahni and his colleagues have shown, '-','~ the T / S (I) of rats fell from normal values of at)out 25 to values as low as 6 within 10 days after hypophysectomy. A single subcutaneous injection of 5 mg of T S H given to these rats returned the T / S (I) to 20 within 10 hours, and to " N. S. Halmi, B. N. Spirtos, E. M. Bogdanove, and H. J. Lipner, E~docrinology 52, 19 (1953). 3N. S. Halmi, D. K. Granner, D. J. Doughman, B. H. Peters, and G. Muller, Endocrinology 67, 70 (1960).
258
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[20]
a maximum of about 30 at 48 hours. In subsequent studies, it was found that the response to TSH was really biphasic in nature: The initial response was a peculiar, transient depression of the T / S (I), followed by the increase in the T / S (I) that developed slowly thereafter. Further studies into this biphasic action of TSH on isolated thyroid cells will be described below. In Halmi's experiments, T / S (I) values were determined by a modification of the method of VanderLaan and Greer 4 as follows: Rats were injected subcutaneously with 12 mg of PT U dissolved in dilute NaOH; 45 minutes later, 10 ~Ci of 131I were injected subcutaneously; finally, the rats were anesthetized with ether, blood samples were taken from the aorta, and thyroid glands were excised. The thyroid glands were weighed quickly on an analytical balance and then assayed for 13II in a well-type crystal gamma scintillation spectrometer. Aliquots of blood sera were similarly assayed for 131I. Then the T / S (I) values were computed from the radioassay data.
I n Vitro Studies: The Actions of T S H on Iodide Transport in Isolated Thyroid Cells The cells isolated from thyroid tissues dispersed by gentle digestion with trypsin and collagenase retain the capacity to transport and concentrate iodide. Moreover, such isolated thyroid cells respond to added TSH with biphasic changes in iodide transport that appear to be entirely analogous to those observed in the thyroid glands of rats following the injection of TSH2 Indeed, the cell suspension system is the only in vitro thyroid preparation that has been successfully employed to demonstrate the actions of TSH on iodide transport. Procedures for the preparation of dispersed thyroid cells have been reported elsewhere2 Techniques for observing the effects of TSH and of cyclic AMP derivatives on C / M (I) and on the component unidirectional pump and leak iodide fluxes are described below.
The Biphasic Effects of TSH and Dibutyryl cyclic A M P on C / M (I) Iodide concentrating activity, expressed in terms of steady state C / M (I) values, may be observed by incubating isolated thyroid cells in Eagle's minimum essential medium 6 supplemented with 1 toM iodide 4W. P. VanderLaan and M. A. Greer, Endocrinology 47, 36 (1950). 5W. Tong and M. R. Sherman, Vol. 36 [14]. GH. Eagle, Science 122, 510 (1955).
[20]
TSH EFFECTS ON THYROID IODIDE TRANSPORT
259
labeled with 13lI at a concentration of 0.1 ~Ci/ml of medium. Also, MMI is added to the medium at 2 mM concentration to block incorporation of the iodide into organically bound products, and penicillin (105 units/liter) and streptomycin sulfate (0.1 g/liter) may be added to inhibit bacterial growth during long-term incubations. For most experiments, 2 ml of cells are dispersed in 200 ml of medium and 5 ml aliquots of the resulting suspension are incubated under 95% 02, 5% CO~ in 25 ml Erlenmeyer flasks at 37 ° with shaking at a rate of 80-100 per minute. Steady state distribution of tile [l:~lI]iodide between the cells and the medium is usually reached within 30 minutes. Then the incubated cell suspensions are transferred to conical tubes, and the cells are sedimented by eentrifugation for 5 minutes at 150 g. The supernatant media are withdrawn, and 1 ml aliquots assayed for 131I. The pellet of packed cells is mixed by stirring, and carefully measured aliquots are taken using 25 ~1 disposable, glass micropipettes. These cell aliquots are also assayed directly for 131I. Finally, the C/M (I) is computed from the ratio of '3~I cpm/ml of cells to 1~I epm/nfl of media. The C/M (I) of healthy lamb or steer thyroid cells range between 8 and 20. Cell preparations giving C/M (I) of less than 8 should be rejected. For reasons that are not clear, the cells isolated from cow and pork thyroid glands give nmeh higher C/M (I) values ranging from 40 to 60. When TSH or the N~-O~'-dibutyryl derivative of cyclic AMP (DBC) are added to steer thyroid cells that had been previously equilibrated with I~'~II]iodid~~, the C/M (I) undergoes a peculiar biphasic alteration which seems to be the counterpart of the phenomenon occurring in rats injected with TSH as described by Halmi et al. 3 Following the addition of the TSH or DBC, the C/M (I) first declines rapidly to 30% reduced values after 30 minutes, and then gradually increases to about 100% increased values after about 6 hours. These alterations occur maximally in response to TSH concentrations as low as 0.5 mU/ml and DBC at 3 raM. The effects of TSH and DBC seem to take place through two different mechanisms. The rapid decline in C/M (I) is evidently a direct effect of TSH to augment the iodide leak flux; the effect is abolished and the C/M (Ii restored to control values when the added TSH is withdrawn and the cells treated with 0.06~, crystalline trypsin to inactivate TSH adherent to the cells. The slowly developed increase in C/M (1) seems to involve the induction of an enzyme that is rate limiting for the pump process. This part of the response to TSH and DBC can be blocked by 5 ~g/ml ,~ctinomycin D or by 0.1 mM cycloheximideJ Its initiation by 7j. Knopp, V. Stole, and W. Tong, J. Biol. Chem. 245, 4403 (1970).
260
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[20]
TSH or DBC is completed after about 2 hours. Thereafter, the TSH or DBC may be removed by the tryptic washout procedure and the C/M (I) will continue its rise to fully stimulated levels.
The E~ects of TSH and DBC on the Saturation Kinetics of the Iodide Pump The steady state C / M (I) seems to be the resultant of opposing pump and leak iodide fluxes. When the C/M (I) is increased by treatment with TSH or DBC, the primary change seems to be an increase in the pump flux. As a secondary consequence, the leak flux is also increased, but a new steady state is achieved with intracellular iodide concentration established at a higher level. Evidence for this has been obtained by applying the Lineweaver-Burk analysis to compare the saturation kinetics of unidirectional iodide influx in control and TSH- or DBC-stimulated steer thyroid cells. For such experiments, the cells are suspended in 10 volumes of Eagle's minimum essential medium supplemented with 2 triM MMI, penicillin, and streptomycin, and incubated under 95% O~, 5% CO~ at 37 ° for 6 hours. To obtain fully stimulated cells, half of the cell suspension is incubated with added TSH (50 mU/ml) or DBC (3 raM). Following this preincubation, the cells are recovered by centrifugation for 5 minutes at 150 g, washed, and treated with 0.06% crystalline trypsin in Eagle's medium for 10 minutes at 37 ° to remove adherent TSH and DBC. To determine unidirectional iodide influx, the cells are washed again and resuspended in 100 volumes of fresh incubation medium, and 5 ml aliquots are dispensed into 25 ml Erlenmeyer flasks. These specimens are then placed in the incubator shaker and brought to 37% Finally, at zero time, 100 ~1 of [131I]iodide solution containing 1 t~Ci of 131I is added, and after 10, 20, and 30 second incubation periods, specimens are transferred to centrifuge tubes, the cells sedimented by centrifugation for 30 seconds at 2000 g, and the 13lI concentration of the cells determined. The plot of cellular 131I concentration against time is a straight line whose slope is the rate of 13'I influx into the cells, which, divided by the specific radioactivity of the iodide will give the iodide influx in microgram per minute per milliliter of cells. By conducting these influx measurements in media containing 10, 13, 20, 40, and 60 ~M iodide, the saturation kinetics of the influx process will be observed. The Lineweaver-Burk plot of the reciprocal of iodide influx vs. the reciprocal of extracellular iodide concentration gives a straight line with the ordinate intercept equal to V..... having values of about 20 ~g/minute/ml of cells. The abscissal intercept gives a K,, of
[20]
261
TSH EFFECTS ON THYROID IODIDE TRANSPORT r-
~
~
- -
-'
- -
7
Vmax S.3xlO-SM 20ttglMINImlCELLS 57 ,, 37 ,, 5.7 37 ,, Km
-~u" 0'St oCONTROL • TSH O.IUlml o 3mM DBC
I i
.
_J 1
04 E z: ~
0.3
i-t
..> 02
~
01
-20
I
I
I
I
L
I
[
20
40
60
80
I00
120
140
I/S (mM -I) FIG. 1. Steer thyroid cells were preincubated for 6 hours without or with either 0.1 u n i t / m l TSH or 3 m M DBC. Then the cells were equilibrated with media containing 100, 150, 300, or 1000 t~g % iodide. Finally, ~31I was added, and from the observed rates of ~3~I uptake at 10, 20, and 30 seconds thereafter, unidirectional iodide influx rates were computed for each iodide concentration (from K n o p p et ~1.7).
about 55 uM. In cells fully stimulated by incubation with T S H or DBC, the Vm~ is increased to about 35 ug/minute/ml of cells, while the Km remains unchanged (Fig. 1).
The E#ects o] TSH and DBC on the Kinetics o] the [odide Leak Although the primary effect of T S H and DBC may be the increase in the iodide influx mediated by the iodide pump, augmentation of the opposing iodide leak also occurs at the same time. This aspect of the response to TSH and DBC seems to reflect direct actions of these agents on the thyroid cell membrane to increase its permeability toward the passive outward diffusion of iodide. The best quantitative indication of this permeability change is the rate constant for unidirectional iodide efflux which is most readily determined by observing the rate of discharge of radioactivity from cells previously loaded with [13'I]iodide. To load with '3'I, thyroid cells are incubated for 1 hour at 37 ° in 20 volumes of Eagle's minimum essential medium supplemented with 3 m M M M I and 10 ~M iodide labeled with 13'I at a concentration of
262
EVALUATION OF BIOLOGICAL E F F E C T S OF HORMONES
[21]
1 tLC/ml. The resulting suspension of 131I-labeled cells is then distributed in 2 ml aliquots (each containing 0.1 ml of cells) into a series of test tubes, the cells are sedimented by centrifugation for 5 minutes at 150 g, the media discarded, and the inside surfaces of each tube wiped with tissue paper to remove adherent 131I. To observe the rate of lalI discharge, 2 ml of fresh Eagle's medium containing 3 mM MMI, but no [13q]iodide, are added to each ~a~I-labeled cell aliquot along with a miniature Teflon-coated magnetic stirring bar; 131I discharge is allowed to occur at 37 ° with mixing for 30 seconds, following which the cells are sedimented for 30 seconds at 2000 g, and the supernatant fluid is decanted and assayed for radioactivity. Then another 2 inl portion of fresh medium is added to the cell pellet, and lalI discharge is allowed to proceed for another 30 second period. This procedure is repeated about 20 times. Finally, the cell pellet itself is assayed for lalI. The total ~'I originally present in the cell pellet (I0) is computed by adding together all of the 13'I discharged plus the 131I remaining in the final cell pellet. The '31I remaining in the cell pellet at each time interval (It) is calculated by subtracting from Io all of the ~31I discharged into the media samples collected previously. The rate of [~3~I]iodide discharge is invariably an exponential function so that In It/Io = --kot where/co is the rate constant of iodide efflux. Thus, In It~I,, plotted against time yields a straight line whose slope is equal to --ko. The /Co observed for control steer thyroid cells has a value of about 0.25 min-k When the ~3~I efftux determinations are performed on cells preincubated for 30 minutes with TSH at concentrations of 10 m units/ml or higher, the ko is increased to values approximately 0.35 rain -1.
[21] M e t h o d s for A s s e s s i n g H o r m o n e ( T S H ) E f f e c t s on Glucose Oxidation and Phospholipid Synthesis B y JAMES B. FIELD
Thyroid stimulating hormone (TSH) stimulates a variety of metabolic and morphologic parameters in thyroid cells. 1 TSH initially attaches to specific receptors on the cell membrane and subsequently activates the adenylate cyclase-cyclic AMP system. Most of the morphologic and metabolic effects of TSH including stimulation of glucose oxidation are probably mediated by cyclic AMP, but this is less well established for 1j. B. Field, Metab., Clin. Exp. 17, 226 (1968).