exposure thyroid to thyroid-stimulating hormone induces cyclic...

Exposure of Thyroid Slices to Thyroid-Stimulating

Hormone Induces Refractoriness of the Cyclic AMP

System to Subsequent Hormone Stimulation

SANDRAJ. SHUMAN,URIEL ZOR, RUEBENCHAYOTH,and JAMESB. FIELD

From the Clinical Research Unit and the Department of Medicine, Universityof Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

A B S T R A C T These studies evaluated the influence ofan initial exposure of thyroid slices to thyroid-stimu-lating hormone (TSH) on the subsequent responsive-ness to the hormone. Bovine thyroid slices were incu-bated with or without 50 mU/ml TSH for varying pe-riods and then incubated in hormone-free medium forvarying periods. Subsequently, slices were incubatedfor 20 min with 10 mMtheophylline and with or with-out TSH. Cyclic AMPwas measured after the thirdincubation. Phosphodiesterase and adenylate cylase wereassayed in homogenates prepared from slices after thesecond incubation. In some experiments prostaglandinE1, puromycin, thyroxine, and triiodothyronine and pro-pylthiouracil were included in the media. In other ex-periments, low doses of TSH (1 and 10 mU/ml) wereused instead of 50 mU/ml.

Slices previously exposed to TSH have decreased re-sponsiveness of the adenylate cyclase-cyclic AMP sys-tem. Such refractoriness is hormone specific since ini-tial exposure to prostaglandin E1 decreases the subse-quent response to this substance but not to TSH. Re-fractoriness to TSH develops only when the first incu-bation is at least 30 min. It is not reversed by 5 h of in-cubation without hormone. Incubation of thyroid sliceswith puromycin does not eliminate refractoriness. Thedecreased response to TSH cannot be explained by re-lease of thyroxine, triiodothyronine, or iodide from theslices. Phosphodiesterase activity is not increased dur-ing the refractory period. The decreased cyclic AMPresponse to TSH is associated with diminished responseof adenylate cyclase activity to the hormone. Guanosinetriphosphate (1 mM) increased adenylate cyclase ac-

Sandra J. Shuman, a third year medical student, was sup-ported by a summer research fellowship from the MedicalAlumni Association of the University of Pittsburgh.

Received for publication 31 July 1975 and in revised form15 January 1976.

tivity in both control and TSH treated tissue, but theeffect was significantly less in the latter. Although withguanosine triphosphate, TSH increased adenylate cy-clase activity in TSH treated tissue, the enzyme activitywas still less than that present in control tissue incu-bated with guanosine triphosphate and TSH. NaFcaused an equivalent stimulation of adenylate cyclase inboth control and TSH treated tissue. These results sug-gest that the refractoriness represents an alteration inhormone binding or the coupling of the bound hormoneto the adenylate cyclase activity rather than any modi-fication of the catalytic site of the enzyme.

INTRODUCTION

The effects of thyroid-stimulating hormone (TSH)' onthyroid metabolism are probably mediated by activationof the adenylate cyclase-cyclic AMP system (1). Al-though this system can initiate the diverse metaboliceffects of TSH, less is known about the mechanisms bywhich the actions of TSH are terminated. Since TSHacutely does not increase cyclic AMPphosphodiesteraseactivity (1), this mechanism cannot account for theacute regulation of the hormone's effects on the thyroid.The development of refractoriness to hormonal stimula-tion after prior exposure to the hormone could providea regulatory mechanism for control of hormone action.The existence of such refractoriness to hormonal stimu-lation has been reported in adipose tissue (2-4), cere-bellum (5), ovary (6), liver (7), cerebral cortex (8),and lung fibroblasts (9). The present studies were doneto determine whether similar refractoriness of theadenylate cyclase-cyclic AMPsystem developed in the

'Abbreziations used in this paper: GTP, guanosine tri-phosphate; PGE1, prostaglandin E1; Ts, triiodothyronine;T4, thyroxine; TSH, thyroid-stimulating hormone.

The Journal of Clinical Investigation Volume 57 May 1976- 1132-11411132

NEVER EXPOSEDTO TSH

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FIGURE 1 Effect of length of the first incubation on the subsequent cyclic AMP response toTSH. The second incubation was 120 min and the third incubation was 20 min. A single beefthyroid was used for the entire experiment. The values are the mean±SEMof triplicate slices.The TSH concentration was 50 mU/ml.* P <0.01 when compared to slices exposed to TSH in the third incubation only.t P <0.05 when compared to slices exposed to TSH in the third incubation only.t P <0.02 when compared to slices exposed to TSH in the third incubation only.

thyroid after exposure to TSH. Additional experimentswere performed to elucidate the mechanism of suchrefractoriness.

METHODSExperiments were performed primarily on beef thyroids;however, dog, lamb, and human glands were also studied.The results presented in the figures and tables were allobtained using beef thyroid glands. Beef and lamb thyroidswere obtained from local abattoirs, transported to the labor-atory in iced saline and used within 2 h of the animal'sdeath. Human thyroids were obtained at the time of surgeryfor nonfunctioning nodules. Surrounding normal thyroid tis-sue was utilized. Dog thyroids were obtained from animalskilled by exsanguination. Thyroid slices were made witha Stadie-Riggs microtome. Slices were initially incubated ina Dubnoff metabolic shaker at 370C in 2 ml Krebs-Ringerbicarbonate buffer containing 1 mg/ml glucose, 1 mg/mlbovine albumin with or without TSH (50 mU/ml), orprostaglandin E1 (PGE1) (25 /hg/ml) where appropriate.The gas phase was 95% 02 and 5% C0O; The time of the

first incubation ranged from 5 to 120 min. After this firstincubation, the slices were washed in an excess of 0.85%saline for 30 s, and a second incubation (30-300 min) wasperformed at 370C in 2 ml Krebs-Ringer bicarbonate bufferwith 1 mg/ml glucose. Slices were washed in 0.85% saline,blotted on filter paper, and weighed. They were transferredto fresh buffer containing 1 mg/ml glucose and albumin and10 mMtheophylline, and incubated at 370C for 20 min.Half of the slices that had been incubated without TSH inthe first incubation were again incubated in buffer with-out TSH, while the other half were exposed to TSH forthe first time. Likewise, half of the slices incubated withTSH during the first incubation were now incubated with-out TSH while the other half were reexposed to TSH.After the third incubation the tissue was extracted in 0.4ml of hot 0.05 M sodium acetate. After centrifugation, thesupernate was assayed for cyclic AMP by a modificationof the protein binding method (10). Adenylate cyclase andphosphodiesterase activities (1) were measured in homoge-nates prepared from slices after the second incubation. TSHstimulation of adenylate cyclase was examined by adding 1or 10 mU of the hormone to the homogenate. To evaluate

Thyroid Refractoriness to TSH Stimulation 1133

phosphodiesterase activities with a low and a high Km forcyclic AMP (11), the assay was done using both 1 and100 j"M.

Puromycin and propylthiouracil were used, respectively, toinvestigate the effects of protein synthesis and of iodiderelease into the medium on the development of refractoriness.Appropriate thyroid slices were incubated in Krebs-Ringerbicarbonate buffer with or without puromycin (150 Ag/ml)or propylthiouracil (3 mM) for 15 min before commencingthe first incubation. During the subsequent three incubationsas outlined above, puromycin or propylthiouracil was againpresent. Experiments were also done with triiodothyronine(T.) or thyroxine (T4) (0.1 ,uM and 1 nM) in the mediumduring all three incubations to determine if this would in-duce refractoriness to TSH during the final incubation.

TSH (NIH-B6) was kindly provided by the NationalInstitute of Arthritis, Metabolic, and Digestive Diseases.The sources of other materials has been reported (1).

RESULTS

Initial exposure of beef thyroid slices to 50 mU/mlTSH for at least 30 min significantly diminished the

rise in cyclic AMPinduced by reexposure to the sameamount of hormone (Fig. 1). Despite an intervening in-cubation in the absence of TSH, slices exposed to TSHonly during the first incubation had cyclic AMPvaluessignificantly higher than those in slices that had neverbeen exposed to TSH. The higher cyclic AMPvaluesreflected the interaction of TSH still present on themembrane from the first incubation and theophyllinepresent in the third incubation (12). Nonetheless, com-pared to thyroid slices not incubated with TSH in thefirst incubation, the increase and absolute levels of cyclicAMP were significantly diminished in thyroid slicesupon reexposure to TSH in the third incubation. Simi-lar refractoriness was also found in dog, pig, lamb, andhuman thyroids (data not shown). The effect of vary-ing the length of the second incubation on the develop-ment and persistence of the refractoriness to TSH wasinvestigated (Fig. 2). The degree of unresponsivenessto TSH was not diminished even after a 5-h second in-

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TIME OF SECONDINCUBATIONFIGUR.E 2 Effect of length of the second incubation on the subsequent cyclic AMP responseto TSH. The first incubation was 120 min and the second incubation varied as indicated. Thethird incubation was 20 min. Different beef thyroid glands were used for each time. The valuesare the mean±SEM of triplicate slices. The TSH concentration was 50 mU/ml.* P < 0.02 when compared to slices exposed to TSH in the third incubation only.t P <0.01 when compared to slices exposed to TSH in the third incubation only.

1134 S. J. Shuman, U. Zor, R. Chayoth, and J. B. Field

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INCUBATION CONDITIONFIGURE 3 Failure of puromycin to modify refractoriness to TSH. Beef thyroid slices were

incubated with or without puromycin (150 gg/ml) for 15 min before commencing the firstincubation which was 120 min. All subsequent incubation media for slices initially exposed topuromycin also contained this substance. The second incubation was for 60 min and the thirdwas for 20 min. The values represent the mean±SEM of triplicate slices. The TSH concen-

tration was 50 mU/ml.* P <0.01 when compared to slices exposed to TSH in the third incubation only.

cubation in a hormone-free medium. Although puro-

mycin decreased basal- and TSH-stimulated cyclicAMP concentrations somewhat, the refractoriness was

not affected by incubation of thyroid slices with this ma-

terial (Fig. 3). The reason for this reduction is not ob-vious, but it may represent a toxic effect of puromycin.In these experiments puromycin inhibited ["4C] leucineincorporation into protein by over 95% (data notshown). Since it has been reported that iodide (13, 14)and thyroid hormones (15-18) can inhibit the TSH ac-

tivation of the adenylate cyclase-cyclic AMPsystem andsince incubation of thyroid slices with TSH releasesthese substances into the medium (19), studies were doneto determine whether they could be responsible for the

refractoriness. The inhibition by iodide of the stimula-tion of the adenylate cyclase-cyclic AMP system byTSH is abolished by propylthiouracil or Tapazole which

prevents organification of iodide (13, 14). These results

of Fig. 4 indicate that the TSH-induced refractorinessis not due to release of iodide and subsequent organi-


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FIGURE 4 Failure of propylthiouracil (PTU) to abolish TSH refractoriness. Beef thyroidslices were incubated with or without (3 mM) PTU for 15 min before commencing the firstincubation which was for 120 min. All subsequent incubation media for slices initially exposedto PTU also contained this substance. The second incubation was for 180 min and third for20 min. The values represent the mean±SEMof triplicate slices. The TSH concentration was

50 mU/ml.* P < 0.02 when compared to slices exposed to TSH in the third incubation only.t P < 0.01 when compared to slices exposed to TSH in the third incubation only.

fication since incubation of slices with propylthiouracildid not modify hormone-induced unresponsiveness. In-cubation of thyroid slices with T4 or T3 had minimaleffects on the cyclic AMPresponse to TSH added dur-ing the third incubation compared to slices incubated ina similar fashion but without thyroid hormones (Fig.5). In these experiments the thyroid hormones were

present in the appropriate flasks during all three incu-bations. The refractoriness induced by TSH was notinfluenced by the presence of either T4 or T3 during allthree incubations (data not shown).

The effect of submaximal doses of TSH on develop-ment of refractoriness was also examined (Table I).Incubation of thyroid slices with 1 and 10 mU/ml of


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FIGURE 5 Failure of T4 and T, to induce refractoriness to TSH. Beef thyroid slices were

incubated with or without 0.1 usM or 1 nM To or Ta during all three incubations. The first andsecond incubations were for 120 min each while the third incubation was for 20 min. Thevalues are the mean±SEM of triplicate slices. The TSH concentration was 50 mU/ml. Inthe presence and absence of 0.1 uM T8, the effect of TSH added in the third incubation onlywas significantly less (P < 0.05) in the former slices.* P < 0.01 when compared to slices exposed to TSH in the third incubation only.

TSH during the first incubation induced some unre-

sponsiveness of the tissue to subsequent exposure to thesame dose of hormone. However, the refractoriness in-duced by 1, but not 10 mU/ml of TSH in the first in-cubation could be overcome by utilizing 50 mU/ml TSHduring the third incubation.

The specificity of the TSH-induced refractoriness was

investigated using PGE1, a substance that mimics many

of the effects of TSH on the thyroid (1). Initial incu-bation of thyroid slices with PGE1 (25 /g/ml) signifi-cantly inhibited the increase in cyclic AMPupon re-

exposure to PGE1 but had no effect on the subsequentstimulation with TSH (Table II). The refractorinessinduced by PGE1 to subsequent stimulation by that sub-stance could be reversed when the second incubation was

prolonged from 2 to 5 h. In fact in the latter experi-ments, the response to PGE1 appeared to be augmentedby the prior incubation of thyroid slices with the sub-

stance in the first incubation. The reason for this in-creased responsiveness to PGE1 is not apparent. Theresults suggest that initial exposure of thyroid slicesto TSH did not induce refractoriness to PGE1 addedduring the third incubation. Two factors may influencethis conclusion. The stimulation induced by TSH greatlyexceeds that due to PGEL. In addition the persistentbinding of TSH from the first incubation (12) in-creases the cyclic AMPconcentration in the slices in-cubated in buffer and theophylline alone in the thirdincubation. This changing base line makes comparisondifficult, but even so the increase caused by PGE1 addedonly during the third incubation was as great in the thy-roid slices initially incubated with TSH as that in slicesincubated with buffer only. An attempt to overcome theeffects of persistent TSH binding by increasing the sec-ond incubation from 2 to 5 h was only partially success-ful. Again, however, the increment of cyclic AMP in-


r

TABLE I

Effect of Submaximal Doses of TSHon Refractoriness

Third incubation

TSH, mU/mlFirst

incubation Control 1 10 50

cA MPpmoi/gControl 235±32 1,76014203 2,196l197 1,922±326TSH, 1 mU/ml 265437 1,153432* 2,021±80 1,5874232TSH, 1OmU/ml 552±20 858±241 1,291±78§ 94343411TSH, 50 mU/ml 715 ±97 - - 84043311

Beef thyroid slices were first incubated for 120 min in buffer alone or with1, 10, or 50 mU/ml TSH. The second incubation, in buffer alone, was30 min. The third incubation of 20 min was performed as follows: slicesfrom each of the four groups in the first incubation were incubated in bufferalone or with 1, 10, or 50 mU/ml TSH. The values represent the mean±SEMof triplicate slices.* P < 0.05 when compared to slices first exposed to 1 mU/ml of TSH inthe third incubation.$ P < 0.02 when compared to slices first exposed to 1 mU/mIl of TSH inthe third incubation.§ P < 0.02 when compared to slices first exposed to 10 mU/ml of TSH inthe third incubation.11 P < 0.05 when compared to slices first exposed to 50 mU/ml of TSH inthe third incubation.

duced by PGE1 in the third incubation was certainly asgreat in the slices initially exposed to TSH as in thecontrol slices.

The diminished cyclic AMP concentrations duringreexposure of the thyroid slices to TSH could reflectaugmented phosphodiesterase activity. D'Armiento et al.reported that increased cyclic AMPlevels in fibroblastswere associated with augmentation of phosphodiesteraseactivity (20). Although TSH did not increase phos-

TABLE I IFailure of Prostaglandin El (PGE1) to Induce

Refractoriness to TSH

Third incubationFirst Second

incubation incubation Control TSH PGEi

h cAMPpmol/gControl 584±i52 6,978±+99 1,963±1-138TSH 2 2,310±4198 4,385 ±164t 4,157 ±104PGE1 671 ±48 6,893±4 186 1,364 ± 100*

Control 518±67 6,344±349 1,2194139TSH 5 1,053±i63 3,604 ± 108$ 2,428±4 182PGE1 60047 7,717+212 1,813+113*

Beef thyroid slices were first incubated in buffer alone or with TSH(50 mU/ml/) or PGE1 (25 jg/ml) for 120 min. They were then washed insaline and incubated for either 120 or 300 min in buffer. Subsequentlyslices from each of the three initial groups were incubated in buffer alone,TSH (50 mU/ml) or PGE1 (25 jsg/ml). A single beef thyroid gland wasused for the entire experiment. Each value represents the mean±SEMoftriplicate slices.* P < 0.05 when compared to slices first exposed to PGEI during the thirdincubation.* P < 0.01 when compared to slices first exposed to TSH during the thirdincubation.

TABLE IIIPhosphodiesterase Activities during TSHRefractoriness

First incubation

Control TSHPhosphodiesterase Activity cAMP

Assay substrate hydrolyzed

pmol/mg per min

Control 3.54±0.2 4.64±0.20.1 nmol cAMP 59.2 ±9.7 65.5±+2.410 nmol cAMP 3,1984492 2,426±328

Beef thyroid slices were incubated with or without TSH(50 mU/ml) for 120 min, washed, and incubated in buffer fora second incubation of 30 min. Slices were then homogenizedand assayed for phosphodiesterase activity. Each valuerepresents the mean4±SEMof triplicate determinations.

phodiesterase activity in thyroid slices during short in-cubations, the longer incubations utilized in the presentstudies might produce different results. The data in Ta-ble III indicate that prior exposure of thyroid slices toTSH and the attendant increase in cyclic AMP levelsdoes not increase phosphodiesterase activity when mea-sured just before the third incubation. This is true forboth the high and low Kmenzyme activities.

Refractoriness is associated with an inability of TSHto increase adenylate cyclase activity in homogenates oftissue previously exposed to the hormone (Table IV).TSH (1 and 10 mU) significantly increased adenylatecyclase activity in homogenates of thyroid slices whichhad not previously been exposed to the hormone. In con-trast, homogenates from slices incubated with TSHduring the first incubation did not respond with aug-mented adenylate cyclase activity when assayed with 1or 10 mUof TSH. Refractoriness was not related tochanges in the catalytic activity of the adenylate cyclase,since NaF (10 mM) stimulated enzyme activity equallyin homogenates prepared from thyroid slices incubatedwith or without TSH during the initial incubation.Furthermore, the increase in adenylate cyclase activityin homogenates induced by TSH was not impaired byprior exposure of thyroid slices to PGE1 (data notshown). Although guanosine triphosphate (GTP) (1mM) augmented basal adenylate cyclase activity in bothhomogenates, enzyme activtiy was significantly less inthe tissue that had previously been exposed to TSH.However, the percent stimulation was approximately thesame in both tissues. While TSH did not increaseadenylate cyclase activity in the TSH treated tissue, itdid augment enzyme activity when GTP was alsopresent. However, under these conditions enzyme ac-tivity was still not equivalent to that obtained in ho-mogenates that had not previously been exposed toTSH, but the percent stimulation was similar.


TABLE IVFailure of TSH to Stimulate Adenylate Cyclase Activity

during Refractoriness to TSH

First incubation

Control TSH

Adenylate cyclase activity[14C]ATP incorporated

Substance present in assay into ['4C]cAMP

cpm/mg per 10 minExperiment 1

Control 8.6 4±1.7 11.3 i±1.0TSH, 1 mUlO.)1 ml 13.8 40.5* 11.7 40.8TSH, 10 mU/0.11 ml 23.3 41.8t 12.6+1.4NaF, 10 mM 46.440.4t 47.2 ±0.8t

Experiment 2Control 30.8±+0.6 26.34±1.9TSH, 10 mU/O.11 ml 50.5+0.91 28.9±0.3GTP, 1 mM 52.5 41.2$ 43.91.5t§GTP, 1 mM+ TSH 10 mU/Oill ml 72.5 ±2.9t** 59.6 -2.9**1§NaF, 10 mM 145.8 L 1.5* 141.4 42.11

Beef thyroid slices were incubated with or without TSH (50 mU/ml) for120 min and washed. The second incubation was for 30 min. Slices werethen homogenized and assayed for adenylate cyclase activity. In someassays 1 mMGTP was also included. Each value represents the meanASEMof triplicate determinations.* P < 0.05 when compared to appropriate control.$ P < 0.01 when compared to appropriate control.I P < 0.01 when compared to 52.5 ±1.2.* P < 0.01 when compared to the same homogenate incubated only withGTP 10-3 M.5§ P < 0.05 when compared to 72.5 ±2.9.

DISCUSSIONInitial exposure of thyroid tissue from several speciesto TSH induces decreased responsiveness of the adenyl-ate cyclase-cyclic AMPsystem when the tissue is re-exposed to the hormone. Induction of refractoriness re-quires at least 30 min of incubation of the tissue withTSH. Although binding of TSH requires less time (12)and its peak effect on cyclic AMPis evident within 10min (1), the results suggest that refractoriness is notan immediate consequence of binding or elevated cyclicAMP levels but requires additional time for furthermetabolic changes to occur. The exact nature of suchmetabolic changes is not apparent, but they do not seemto be dependent upon new protein synthesis (Fig. 3).These results differ from those of DeVellis and Brookerwho reported that actinomycin D or acetoxycyclohexi-mide prevented refractoriness in rat glial tumor cellsinduced by norepinephrine (21). The refractorinesscould not be attributed to release of iodide, and its sub-sequent organification, or thyroid hormone, (Figs. 4and 5) although these substances can decrease the ac-tivation of adenylate cyclase by TSH (13-18). Preven-tion of organification of iodide abolishes such inhibition(13, 14) while in the present experiments, propylthio-uracil did not modify refractoriness induced by TSH.

These results do not exclude the possibility that in-organic iodide could cause refractoriness. In additionthe presence of T4 and T3 in the buffer during all threeincubations did not induce unresponsiveness of thyroidslices exposed to TSH only during the final incubation.

The refractoriness of thyroid, like several other tis-sues (2, 8, 9) differs from that reported for adipose tis-sue by Ho and Sutherland (4) since it was not reversedby repeated washing of the tissue. A second incubationwithout hormone for as long as 5 h did not abolish thedecreased responsiveness to TSH indicating that thephenomenon is not easily nor rapidly reversed. Al-though PGE1 produced diminished responsiveness tosubsequent stimulation by this hormone after a secondincubation of 2 h, it did not modify the response to TSHindicating some specificity for the induction of refrac-toriness. When the second incubation was prolonged to5 h in the absence of PGE,, the refractoriness of thyroidtissue to subsequent stimulation by this substance wasno longer apparent. This contrasts to the results withTSH since refractoriness persisted even after a 5-hsecond incubation. This difference could reflect the morepersistent binding of TSH compared to PGE1 which hasbeen previously demonstrated (12). The lack of hor-monal cross reactivity in producing refractoriness issimilar to that reported in guinea pig cerebral corticalslices (8), human astrocytoma cells (22), and humanlung fibroblasts (9) but different than that found inadipose tissue (2). Such discrepancies could reflectdifferences in the underlying mechanism responsible forrefractoriness.

The failure of TSH to stimulate adenylate cyclaseactivity in homogenates of thyroid slices previously in-cubated with TSH provides a basis for the refractori-ness. This unresponsiveness to TSH stimulation couldreflect several possibilities: (a) decreased binding ofTSH to its receptors because they are either alreadyoccupied or have been altered by TSH from the firstincubation, (b) the coupling mechanism between thehormone receptor and adenylate cyclase has been modi-fied, or (c) inhibition of the catalytic activity of adenyl-ate cyclase. The last possibility appears less likely sinceNaF and PGE1 stimulated adenylate cyclase activityequally in homogenates from slices previously incubatedwith or without TSH. Furthermore, GTP also aug-mented adenylate cyclase activity in homogenates fromtissue incubated initially with TSH although the abso-lute, but not the percentage, effect was somewhat lessthan that observed in control homogenates. The stimu-lation by GTP confirms the results previously reportedby Wolff and Cook (23). Although the effect of thisnucleotide has been attributed to an action at a nucleo-tide regulatory site, its exact relationship to the cata-lytic activity has not been elucidated (24). It has been


postulated that binding of GTP to hepatic adenylatecyclase results in a transition state of the enzyme whichcan then isomerize into a form with increased catalyticactivity. This latter process is accelerated by glucagon.Although the results obtained with both GTP and TSHsuggest that adenylate cyclase activity from tissue previ-ously incubated with TSH is no longer refractory, in-terpretation of such experiments is somewhat difficultbecause of the different base lines with GTP alone. Theamount of increase of activity induced by TSH andGTP compared to GTP alone in the two homogenateswas similar, but the total enzyme activities were differ-ent. If GTP does diminish the refractoriness of adenyl-ate cyclase to TSH, the mechanism is not apparent.Perhaps activation of the nucleotide regulatory siteovercomes the alteration of either the TSH binding orthe coupling step causing refractoriness. Wehave previ-ously reported that GTP potentiation of stimulation ofadenylate cyclase in thyroid plasma membranes by TSHwas associated with decreased binding of TSH (25)while Moore and Wolff found no change in hormonebinding (26). We have been unable to measure ade-quately binding of TSH to its receptors during refrac-toriness because the nonspecific binding using homoge-nates is too high to permit interpretation of the data.In adipose tissue an initial activation of adenylate cy-clase is essential for induction of refractoriness sincePGE1 and nicotinic acid, both inhibitors of adenylatecyclase activity in adipose tissue, abolished refractori-ness in adipose tissue induced by epinephrine (2).

The diminished cyclic AMP response to TSH wasnot due to increased phosphodiesterase activity. Assayswere done for both the high and low Km enzyme ac-tivities. Furthermore, refractoriness was demonstratedwith theophylline in the buffer. Although inhibition ofphosphodiesterase activity with isobutylmethylxanthinepartially reversed refractoriness of cerebral corticalslices to biogenic amines (8), this did not seem to con-tribute to this phenomenon in rat glial tumor cells oradipose tissue (3, 21).

The possibility exists that some other, as yet uniden-tified, substance accounts for the inhibition of stimula-tion of the adenylate cyclase-cyclic AMP system byTSH. The extensive washing of the slices and the useof fresh medium excludes total release of such an inhibi-tor into the buffer. Evidence for such an inhibitory sub-stance as an important component of the refractoryphenomenon has been presented in several other sys-tems. The feedback regulator from adipose tissue stud-ied by Ho et al. was released into the medium (3). Fain(27) and Schwabe et al. (28) suggested that refractori-ness to hormonal stimulation in adipose tissue might bemediated by release of adenosine into the medium. How-ever, in brain slices, adenosine prevents refractoriness

induced by biogenic amines (8). In the thyroid, effectsof adenosine are quite complex. Although it stimulatescyclic AMPaccumulation, it also partially inhibits thestimulation induced by TSH.2

The physiologic significance, if any, of such refrac-toriness to TSH stimulation remains unknown. It couldprovide a mechanism to modulate the action of TSH onthe thyroid. Such mechanisms would be important sincebiologically active TSH persists bound to thyroid tissue(12). Although the major feedback loop controlling thy-roid function is probably inhibition of TRH by thyroidhormones, other regulatory mechanisms may exist. Theobservation that chronic propylthiouracil treatment ofanimals with its attendant persistent elevation of TSHis still associated with goiter formation is not incom-patible with the present demonstration of refractorinessto TSH. Since the refractoriness is not complete, therecould still be expression of effects of TSH. However,this demonstration of refractoriness to TSH might be ofno physiologic significance and may only reflect the re-sults of in vitro incubations. This may well be the casein adipose tissue since Schimmel (2) reported that dur-ing the refractory period induced by epinephrine therewas no diminution of glycerol release or the lipolyticresponse to the hormone despite significant reduction ingeneration of cyclic AMP.

ACKNOWLEDGMENTSWe would like to thank Mary E. Kerins, Gail Bloom, andCheng-Ying Chou for their excellent technical assistance.We would also like to thank Constance Copetas for herinvaluable help in preparing the figures, and Barbara Shee-han and Susan Gehring for their outstanding help in pre-paring the manuscript.

This work was supported by U. S. Public Health Servicegrant AM06865.

2Kariya, T., and Field, B. J. Unpublished observations.

REFERENCES1. Zor, U., T. Kaneko, I. P. Lowe, G. Bloom, and J. B.

Field. 1969. Effect of thyroid-stimulating hormone andprostaglandins on thyroid adenyl cyclase activation andcyclic adenosine 3',5'-monophosphate. J. Biol. Chem.244: 5189-5195.

2. Schimmel, R. J. 1974. Responses of adipose tissue tosequential lipolytic stimuli. Endocrinology. 94: 1372-1380.

3. Ho, R. J., T. Russell, and A. Asakawa. 1975. The lastconversation with Dr. Earl W. Sutherland, Jr.: Thefeedback regulation of cyclic nucleotides. Metab. Clin.Exp. 24: 257-264.

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