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Effects of long-acting thyroid stimulator and prostaglandin antagonists on adenyl cyclase activity in isolated bovine thyroid cells
Life Sciences Vol. 10, Part II, pp . 969-981, 1971 . Printed in Great Britain
Pergamon Press
EFFECTS OF LANG-ACTING THYROID STIMULATOR AND PROSTAGLANDIN ANTAGONISTS ON ADENYL CYCIASE ACTIVITY IN ISOLATED BOVINE THYROID CELLS
Gerald Burke and Seiya Sato Division of Endocrinology and Nuclear Medicine, Department of Medicine, Michael Reese Hospital and Medical Center, and the Department of Medicine, University of Chicago Pritzker School of Medicine, Chicago, Illinois 60616, (Received 24 Ma.y 1971 ; in final form 26 July 1971)
Summary We studied the effects of long-acting thyroid stimulator [LATS] and two unrelated compounds reported to be prostaglandin antagonists, 7-oxa-l3-proatynoic acid [PY1] end polyphloretin phosphate [PPP] on adenyl cyçlase activity in isolated bovine thyroid cells [ITC], LATS-IgG stimulated ITC adenyl cyclase in a dose-related manner? the timecourse of IATS-induced cyclase activation was comparable to that observed with thyrotropin [TSH] [onset at 5 min, maximal at 7,5 to 10 min] PY1, 5 pg/ml [= 1,4 x 10 -5M] and PPP, 5 pg/ml [= 3,3 x 10 - gM], significantly inhibited the stimulatory effects of prostaglandin E2, TSH and IATS on ITC adenyl cyclase but did not modify basal cyclase activity, PPP, 0,5 mq, inhibited the effects of both TSH and IATS on mouse thyroid radioiodine release in vivo in an apparently competitive manner but did not modify dibutyryl cyclic AMP effects thereon, These studies suggest that activation of a prostaglandin receptor site may be an early step in the action of both TATS and TSH on thyroid, wE have recently reported (1,2) that activation of a thyroidal prostaglandin receptor site may be an essential requirement in the action of TSH to stimulate cyclic AMP formation and hormonogenesi4
The studies described herein suggest that a similar mechanism may obtain for the long-acting thyroid stimulator [LATS] of Graves' disease, Materials and Methods Bovine thyroid cells were isolated for culture by an intermittent trypsinization technique (3,4), The cell yield from BO to 100 gm of thyroid tissue ranged from 1,0 to 1,8 ml [12-20 x 106 cells/ml],
TSH [in the form of Thytropa~] was purchased from Armour Pharmaceutical Co, PGE2 was generously supplied by Dr . John Pike, The Upjohn Co, The prostaglandin "antagonists" (5,6j, 7-oxa-13 prostynoic acid [PY1] and polyphloretin phosphate [PPP] were gifts from Dr, Josef Fried of the University of Chicago and Dr, B, Ffögberg, 969
97 0
Aaenyl Cyclase of Bovine Thyroid Celle
Vol. 10, No . 17
Aktiebolaget Leo, Halsingborg, Sweden, respectively,
TATS-positive sera were donated by two patients with untreated hyperthyroidism of Graves' disease, [0,5 ml of unfractionated serum fzom each patient gave an 18-hr response index of 3989 ± 352 and 2264 ± 242, respectively, in the mouse bioassay
.] . The method of Haumstark et al (8) was used for isolation of IgG from plasma using DEAE~ephadex A-50, Five mg of the IgG
fractions so obtained yielded an 18-hr bioassay response index of 3920 ± 381 and 1883 ± 159, respectively, [Since the effects of these IgG fractions on adenyl cyclase activation did not differ significantly *, no attempt has been made to distinguish between the data obtained with one or another fraction in "Results",], Normal IgG [prepared by the method of Baumstark et al (8)
from pooled normal human serum was used as a control for LATSIgG in each experiment, Measurement of adenyl cyclaae - [B- 14 C] Adenine,
50 to 55 mCi/mmole, was purchased from Schwarz-Mann BioReaearch, Inc, One ml of an aqueous solution [100 ~Ci/ml] of the nucleotide was
passed through an AG SOW-x2 [H +], 100-200 mesh [0,7 x 4 cm] column ; the resin wes washed with 15 ml water followed by 80 ml O,1M aqueous ammonia, The eluted product [comprising a sharply delineated radioactive peak between 2 and 8 ml of elutes] was concentrated to dryness in vacuo and then dissolved in meter to
give a concentration of 100 pCi/ml, The procedure of Humes et al (9) for measuring adenyl cyclase in intact cells was used with minor modifications, Fifty Nl isolated thyroid cells were placed in 1 ml of Krebs Ringer bicarbonate 14 buffer [KRH], pH 7,4, containing 1,0 pCi purified [8- C] adenine [2 x 10 -5 M adenine], 0,19 bovine serum albumin and 0,006M glucose, This preparation was "preincubated" under 95% 02 - 5% Cot in 80 x 16 mm covered sterile plastic tubes for 2 hr at 37 C in a Dubnoff metabolic shaker, The tubes were then centrifuged at 500 Xg for
5 min and the supernate discarded, The tubes containing the sedimented cell suspension were then placed in an ice-bath, 0,5 ml ice-cold KRB containing theophylline, 10 -2 M, and hormone and/or t We have previously commented ( Endocrinology 84 : 1063,1969) on the limited correlation between the in vivo ~d in vitro thyroidstimulating properties of LATS,
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agent as 1ndi~ted in "Results" wns added' and the tubes gassed and resealed, The cells were reavepended by Vortex prior to a l0-
min incubation [see "Results"] at 37 C, The enzymic reactions were terninated by the addition of 0,2 ml of 0,6M trichloracetic acid [TCA] and 0,05 ml of an aqueous solution ["nucleotide solution"] containing O,O1M each of ATP, ADP, 5'AMP, cyclic AMP, adenosine. and adenine to facilitate recovery of the labeled nucleotides, This treatment resulted in cell ~,yais ; insoluble material was removed by centrifuging for 10 min at 500 Xg, The resulting pellet was washed with 0,2 ml 2~6 TCA, recentrifuged for 20 min
and the supernate added to the initial TCA extract, The combined aupernates were neutralized with 0,1 ml 2M tris-HC1 buffer, pH 8,0 [resultant pH 7,4 to 7,6] and passed through an AG 509-x2 [H +], 100-200 mesh [0,7 x 3,5 cm] column, which was then eluted with water, The initial 2 ml effluent was discarded and the
cyclic AMP was collected in the subsequent 5 ml fraction, After treatment with 0,2 ml each of the aqueous 5% zinc sulfate and 0,3 N Ha(OH) 2 , the slurry was centrifuged for 5 min at 500 Xg,
The resulting aupernate was again subjected to this coprecipita-
tion procedure and then lyophilized to dryness, The residue, consisting primarily of 14 C-cyclic AMP, was 8lssolved in 1,0 ml water and the radioactivity of an 0,5 ml aliquot measured in Bray's solution (10), Another 0,5 ml aliquot was relyophilized, dissolved in 0,05 ml nucleotide solution [0,005M] and applied to precoated polyethylene-imine [PEI]-impregnated cellulose thin layer sheets [cellulose PEI-F, J.T, Haker Chemical Co,J and developed for 40 min in 0,3M LiCl (11), The cyclic AMP spot was visualised by
ultraviolet light, cut out and counted in 10 ml toluene scintillation fluid, Formation of 14 C-cyclic AMP was calculated as follows t TSH and LATS were dissolved in the requisite medium or buffer prior to addition to the appropriate tube or flask, One m4 of PGE2 or PY was dissolved in 0,1 ml of absolute ethanol, To this was added ~,9 ml Na2C03 [0,2 mg/ml, pH 7], This stock solution was diluted with buffer or medium to give the desired concentration of prostaglandin and/or antagonist in the tube, Each control Vessel contained a quantity of ethanol-carbonate diluent equivalent to that placed in the test vessel containing the maximum concentration of PGE2 and/or PYl in any given experiment, Stock PPP solution was prepared by dissolving 1 gm PPP in 44 ml 0,1 N NsOH which yielded a clear solution of pH _- 7,
97 2
Adenyl Cyclase of Bovine Thyroid Cells 14 C~yclic
AMP [cpm] = cpm HaSO4 supernate x
Vol . 10, No . 17
cyclic AMP spot [cpm]
total chromatogram radioactivity [cpm] These values were converted to picomoles 14 C-cyclic AMP formed/106 cells using the specific activity of the [8- 14 C] adenine [20 picomolea (8- 14 C) adenine -_ 1000 cpm] and correcting for the actual number of cells used [12-20 x 106 cells/ml] in
any given experiment, All studies were done in triplicate or duplicate, TATS bioassay was performed using a minor modification of the McKenzie method (7)r all test materials were administered
intraperitoneally, Statistical analysis of data was performed by paired t-test (12) or, where appropriate, by Dunnet's test which permits comparison of several experimental groups with a single control (13 ),
*= cpm in 0,5 ml aliquot x 2, Results
Time-course of TATS-induced adenyl Cyclase activation As esn be seen from Fig, 1, both TSH [100 mU/ml] and IATS[0,5 mg/ml] effected significant activation of adenyl Cyclase IgG in isolated bovine thyroid cells during incubation periods
ranging from 5 to 15 min, [Although shorter incubation periods (1 and 2,5 min) were attempted, these proved technically difficult and no consistent TSH or IATS effect was observed,], In each instance maximum stimulation was obtained between 7,5 and 10 mint in all experiments to be detailed hereafter, a 10-min incubation period was employed, Aa can be seen from Fig, l, 14 Ccyclic AMP levels in untreated cells or in cells incubated with
normal IgG did not change significantly over time indicating that a "steady state" had been achieved and that the specific activity of the cyclic AMP yielding ATP pool was probably constant,
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8 Q w 0
0 a a
U U Û â O U â
TIME (min) FIG, 1 Time-course of TSH [100 mU/ml] and IATS-IgG [0,5 mq/ml] activation of adenyl cyclase in isolated bovine thyroid cells, 8ach point represents the moan ± STja of 4 experiments, each experimental determination performed in dupli~te, control ~ untreated cells, Normal IqG = cells incubated with normal IgG, 0,5 m9/ml,
TATS-IgG dose response curve The response of bovine isolated thyroid cells to TATS [and TSH] varied somewhat from experiment to experiment, Nonetheless, a dose-related IATS-IqG effect was consistently observed [FIG, 2],
974
Ade~l-Cyclase od Bovine Thyroid Cells
0 O w O
120
lL
80
U
60
U 'Û
40
a
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NORMAL IqG
O g Ôr 0.05
0.13
ß2S
IgG (mg/m l)
0~5
1 .0
FIG, 2 Dose-response curve, IATS-IgG activation of adenyl cyclaae in isolated bovine thyroid cells, Incubation time = 10 min, Each point represents the mean + 1 S~, of 4 experiments, each experimental determination performed i-n duplicate,
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Adenyl Cyclase of Bovine Thyroid Cells
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PICOMOLES ~4C-CYCLIC AMP FORMED/10` cells o
xo
4o
so
eo
loo
X20
CONTROL Normal IqG, QSmghni PGE=, 2.5 x 10'~M TSH, 10pmU/ml LATS IqG, 0.5 mq/ml PY 5p.q/ml PGE =, 2.5 x 10'' M+ PY 5 Y~q/ml TSH, 100mU/ml+ PY 5pq/ml LATS IqG, 0.5mghnl+ PY 5pq/ml FIG . 3 Effects of PYl on basal and stimulated adenyl cyclase activity in isolated bovine thyroid cells, Incubation time = 10 min Each value represents the mean ± 1 S~of 6 experiments, each experimental determination performed in duplicate . * denotes values significantly [p< 0,01] greater than controls ** denotes values signifi~ntly [p < 0,01] less than corresponding stimulator alone,
978
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Adenyl Cyclase od Bovine Thyroid Cells
PICOMOLES "C-CYCLIC AMP FORMED/108 cells 0
20
40
60
80
100
CONTROL Normal IqG 0.5 mq/ml PGE =, 2 .5 x 10'~M TSH, 50mU/ml LATS IqG, 0.5mq/ml PPP, 5~aq/ml PGE=, 2 .5 x 10-~M + PPP, 5Y.q/ml TSH, 50mU/ml + PPP, 5 fcq/ml LATS IpG,0.5mq/ml+ PPP,5~q/ml
FIG, 4 Effects of PPP on basal and stimulated adenyl cyclase activity in isolated bovine thyroid cells, Incubation time _- 10 min, Each value represents the mean ± 1 SF~ of 6 experiments, each experimental determination performed in duplicate, * denotes values significantly [p < 0,01] greater than controls ** denotes values significantly [p < 0,02 to p < 0,01] less than corresponding stimulator alone, As can be seen from FIGs, 3 and 4, PY 1 , 5 +ag/ml, [= 1,4 x 1~M]
and PPP , 5 leg/ml [= 3,3 x 10 -8M] significantly inhibited PGE2 , TSH and IATS effects on adenyl cyclase activity in isolated bovine thyroid cells, PY 1, 5-20 lug/ml, had weak non-dose related agonist properties when tested alone [maximum 15-20% increase over control]s PPP, 1-25 pg/ml, exhibited variable and statistically nonsignificant inhibitory effects on basal cyclase activity, In order to confirm that these f indinga were not peculiar to
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Adenyl Cyclase of Bovine Thyroid Cells
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the isolated bovine thyroid cell adenyl cyclase system, a series
of 3 mouse bioassay experiments [measuring thyroidal radioiodine release (7 )] were performed with TSH, IATS [whole plasma] and PPP*, PPP, 0,1 to 1,0 mg/mouse alone was without effer_t on blood radioactivity, As can be seen from FIGs, 5 and 6, PF~P, 0, 5 !~T/ mouse, significantly inhibited the effects of both TSH and LATST in each instance, the slope of the intermediate dose portion of the log-concentration effect curve was not changed in the presence; of the antagonist, while antagonism was ultimately blunted ôr surmounted by increasing the concentration of the
agonist [0,75 mU TSH, 1 :2 dilution of IATS plasma], In contrast, the response to dibutyryl cyclic AMP [DECAMP], 2,5 mq/mouse, was unaffected by PPP, 0,5 mg/mouse [6 hr zesoonse index, DBc.AMP alone, 465 ± 83 vs, DECAMP + PPP,
535 ± 92,],
TSH, TATS and PPP were administered intraoeritoneally, total volume -- 0,5 ml/substance(s) administered,
17ßO 7500
~Ô E
X Z
1250 1000 750
W
tn
W
500 2ßO O~i. 0
0.10
02ß
TSH (mU)
Q50
0 .7ß
FIG, 5 Effects of PPP, 0,5 mg/mouse, on TSH-induced mouse thyroid radioiodine release, Each point represents the mean ± 1 SF~,of 3 experiments, 6 mice/material(s) assayed in each experiment, Oomparison between TSH [e-i] and TSH + PPP [~- - -e] were, in each instance, made in the same experiment,
978
Adenyl Cyclase of Bovine Thyroid Cells
Vol . 10, No . 17
zaoo z
i +z
7~fi
~ 12.5
zs
LATS (Serum Dilution x 100)
F IG,, 6 Effect of PPP, (0,5 mg/mouse], on LATS-induced mouse thyroid radioiodine release, Each point represents the mean ± 1 SFm of 3 experiments, 6 mice/material(s) assayed in each experiment, Comparisons between TATS [~ ~] and TATS + PPP [~ - - ~] were in each instance, made in the same experiment, Dü cuss ion
We have recently reported (2) that PYl , a compound structurally related to the proataglandins (5 ), inhibits both PGE and TSH effects on adenyl cyclase activation and cyclic AMP formation in thyroid, These findings raised the possibility that there might be a single TSH-related prostaglandin receptor in thyroid, activation of which is requisite to TSH stimulation of
hormonogeneais in the gland (2), [Kuehl et al (14) had earlier advanced a similar concept for the action of luteinizing hormone
on the ovary .], Since the results of many previous studies (15-22) support the postulate that the sites of action of TSH and LATS on thyroid are similar or identical, we now examined the effects of PY 1 and PPP on TATS activation of thyroid adenyl cyclase, PPP, a phosphorylated polyanionic derivative of phloridzin, has been shown (6, 23-26) to antagonize the effects of prostaglandins E and F on a variety of physiologic systems, Stimulatory effects of IATS on thyroid adenyl cyclase have now
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Adegyl Cyclase of Bovine Thyroid Cells
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been demonstrated in three species - ovine, bovine and canine thyroid (27-29 ), Although there is some disagreement within these
studies ae to the latency of IATS action relative to that of TSB, it is apparent that the increase in labeled cyclic AMP formation represents adenyl cyclase activation rather than phoaphodieaterase inhibition (28,29), Similar evidence has been reported (30) in
thyroid adenyl cyclase studies with proataglandins E and F, Given these Eindinge and the addition of the potent phoaphodieaterase inhibitor, theophylline, to each incubation mixture, it would seem reasonable to assume that any changes in 14 C-cyclic AMP levels in the present study reflect changes in adenyl cyclaae activity only, Thus, it may be concluded Erom the studies reported herein that two unrelated prostaglandin "antagonists" , PY l and PPP, inhibit the effects of IATS [as well as those of PGE2 and TSB] on thyroid adenyl cyclase, Although attempts to characterize the nature of this antagonism in in vitro cell studies have, to date, been unsuccessful, the mouse bioassay studies do shed some additional light on the problem. Therein, the parallel shift in thyroid stimulator-induced dose-response curves with PPP and the demonstration that higher stimulator concentrations overcome PPP antagonism [see FI3a, 5 and 6], fulfill the criteria of
Stephenaon (31) for competitive antagoniam, Clearly however, additional in vitro studies with both PY 1 and PPP will be required ** , In any event, the to support these preliminary conclusions, mouse bioassay studies indicate that the antagonism exhibited bY PY 1 and PPP to TSH and TATS is not peculiar to the in vitro
isolated bovine thyroid cell system, The results of these in vivo studies are consistent with earlier findings (30) wherein we
reported that proataglandina E and F augmented mouse thyroid radioiodine release in vivo but when combined with the more potent TSH
or LATS, reduced the stimulatory effect significantly below that observed with either TSH or IATS alone, The lack of PPP antagonism to DHcAMP in the mouse bioassay is important in several ways, First,
it excludes a non-specific
"toxic" effect as the basis for PPP antagonism to TSH and IATS, Second, [along with the lack of "PPP only" effect on blood radioactivity],
it suggests that the PPP-induced changes in blood
*ie, prostaglandin antagonists in some systems, *The limited availability of PY1 precludes in vivo [is, mouse bioassay] studies with this compound,
980
Adenyl Cyclase of Bovine Thyroid Cells
Vol . 10, No . 17
radioactivity truly reflect changes in thyroidal radioiodine
release rather than PPP-induced changes in clearance of labeled thyroid hormone from the blood (32 ), Finally, it suggests, as have our previous in vitro studies with PY l (1,2) that PPP antagonistea stimulated thyroidal hormonogenesis as a direct
consequence of inhibition of adenyl Cyclase activation without involving steps subsequent thereto,
The studies reported herein support the concept that activation of a prostaglandin adenyl Cyclase receptor Bite may be an early step in the action of both LATS and TSH on thyroid, Açknowledgments This study was supported by a grant [AM 11136] from the National Institute of Arthritis and Metabolic Diseases, USPHS, The authors gratefully acknowledge the technical assistance of L, Wang and C, Lonergan, References 1, G, BURKE, K, KOWAISKI and D, BABIARZ, Life Sci , 10 ; 513 (1971), 2, G, BURKE, S, SATO, K, K04~LSKI, D. HABZARZ and M, SZABO, Program 53rd Meeting Endocr, Soc p A-140, ,Tune 24-26 (1971), 3, M,M, EDMUNDS, V,V, ROw and R, VOLPE, 480 (1970),
J_ Clin, Endocr ,
4, G, BURKE and K. KOWALSKI, Life Sci , 10 : 361 (1971) . 5, J, FRIED, T.S, SANTHANAKRISHNAN, J. HIMIZU, C,H, LIN, S,H, FORD, B, RUBIN and E,O, GRIGAS, Nature 233 : 208 (1969), 6, K,E, EAKINS, S,M,M, KARIM and J,D, MILLER, Brit . J. Pharmacol , 39s 556 (1970), 7, G, BURi~, J. Lab, Clin Med , 69 : 713 (1967), 8, J,S, BAUMSTARK, R,J, IAFFIN and W,A,BARDAWIL, Arch . Biochem , 108" 514 (1964), 9, J,L, HUMES, M, ROUNBEHLER and F,A, KUEHL, Jr Anal, Biochem, 32 : 210 (1969), 10, G,A, BRAY, Anal . Biochem, 1s 279 (1960), 11, H,-F, BÂR and O . HECHTER, Anal . Biochem, 29s 476 (1969), 12, W,J, DIXON and F,J, MASSEY, Jr Introduction to Statistical Analysis, McGraw Hill, New York, 124-128 (1957), 13, C, W, DÜNNET, J. Amer . Statist . Aa90C . 50 : 1096 (1955),
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14,
F .A . KUEHL, Jr J,L, HUMES, J. TARNOFF, V.J, CIRILLO and E,A, ffAM ., Science 169s 883 (1970),
15,
T,W, SCOTT, B.F, GOOD and K,A, FERGUSON, Endocrinology 79 : 949 (1966),
16,
Y, SHISHIBA, D,H. SOLOMON and G,N, BEALL, Endocrinology 80s 957 (1967),
17,
G, BURKE, J. Clin . Endocr , 28s 286 (1968),
18,
Y, OCHI and L, J. DeGROOT, Biochim. Biophys, Acta 170s 198 (1968),
19,
G, BURKE, Endocrinology 83s 1210 (1968),
20,
G, BURKE, Metabolism 18s 720 (1969),
21,
T, ONAYA and D,H . SOIAMON, Endocrinology 85 : 1010 (1969),
22,
Y, SHISHIBA, D.H, SOIAMON and W,D, UArVIDSON, Endocrinology 86s 183 (1970),
23,
K.E, EAKINS and S,M .M, KARIM,
24,
K,E, EAKINS, J,D . MILLER and S,M,M, KARIM, J_ Pharmacol, Exp, Therap , 176s 441 (1971),
25,
R, VILLANUEVA, L, HINDS, R,L . KATZ and K,E, EAKINS, Fed . Proc , 30, 626abs (1971),
26,
R,A . BETHEL and K,E, EAKINS, Fed . Proc , 30s 626abs (1971),
27,
G, BURKE, Clin . Res , 17s 521 (1969),
28,
G,S, LEVEY and I, PASTAN, Life Sci, 9s 67 (1970),
29,
T, KANEKO, U . ZOR and J,B. FIELD, Metabolism 19s 430 (1970),
30,
G . BURKE, Am . J. Physiol, 218 : 1445 (1970),
31,
R,P, STEPHENSON, Brit, J, Pharmacol , 11s 379 (1965),
32,
D .H, SOLOMON, G,N, BEALL and I,J . CHOPRA, J. Clin, Endocr , 31s 603 (1970),
Life Sci , 9 : 1 (1970),
Pergamon Press
EFFECTS OF LANG-ACTING THYROID STIMULATOR AND PROSTAGLANDIN ANTAGONISTS ON ADENYL CYCIASE ACTIVITY IN ISOLATED BOVINE THYROID CELLS
Gerald Burke and Seiya Sato Division of Endocrinology and Nuclear Medicine, Department of Medicine, Michael Reese Hospital and Medical Center, and the Department of Medicine, University of Chicago Pritzker School of Medicine, Chicago, Illinois 60616, (Received 24 Ma.y 1971 ; in final form 26 July 1971)
Summary We studied the effects of long-acting thyroid stimulator [LATS] and two unrelated compounds reported to be prostaglandin antagonists, 7-oxa-l3-proatynoic acid [PY1] end polyphloretin phosphate [PPP] on adenyl cyçlase activity in isolated bovine thyroid cells [ITC], LATS-IgG stimulated ITC adenyl cyclase in a dose-related manner? the timecourse of IATS-induced cyclase activation was comparable to that observed with thyrotropin [TSH] [onset at 5 min, maximal at 7,5 to 10 min] PY1, 5 pg/ml [= 1,4 x 10 -5M] and PPP, 5 pg/ml [= 3,3 x 10 - gM], significantly inhibited the stimulatory effects of prostaglandin E2, TSH and IATS on ITC adenyl cyclase but did not modify basal cyclase activity, PPP, 0,5 mq, inhibited the effects of both TSH and IATS on mouse thyroid radioiodine release in vivo in an apparently competitive manner but did not modify dibutyryl cyclic AMP effects thereon, These studies suggest that activation of a prostaglandin receptor site may be an early step in the action of both TATS and TSH on thyroid, wE have recently reported (1,2) that activation of a thyroidal prostaglandin receptor site may be an essential requirement in the action of TSH to stimulate cyclic AMP formation and hormonogenesi4
The studies described herein suggest that a similar mechanism may obtain for the long-acting thyroid stimulator [LATS] of Graves' disease, Materials and Methods Bovine thyroid cells were isolated for culture by an intermittent trypsinization technique (3,4), The cell yield from BO to 100 gm of thyroid tissue ranged from 1,0 to 1,8 ml [12-20 x 106 cells/ml],
TSH [in the form of Thytropa~] was purchased from Armour Pharmaceutical Co, PGE2 was generously supplied by Dr . John Pike, The Upjohn Co, The prostaglandin "antagonists" (5,6j, 7-oxa-13 prostynoic acid [PY1] and polyphloretin phosphate [PPP] were gifts from Dr, Josef Fried of the University of Chicago and Dr, B, Ffögberg, 969
97 0
Aaenyl Cyclase of Bovine Thyroid Celle
Vol. 10, No . 17
Aktiebolaget Leo, Halsingborg, Sweden, respectively,
TATS-positive sera were donated by two patients with untreated hyperthyroidism of Graves' disease, [0,5 ml of unfractionated serum fzom each patient gave an 18-hr response index of 3989 ± 352 and 2264 ± 242, respectively, in the mouse bioassay
.] . The method of Haumstark et al (8) was used for isolation of IgG from plasma using DEAE~ephadex A-50, Five mg of the IgG
fractions so obtained yielded an 18-hr bioassay response index of 3920 ± 381 and 1883 ± 159, respectively, [Since the effects of these IgG fractions on adenyl cyclase activation did not differ significantly *, no attempt has been made to distinguish between the data obtained with one or another fraction in "Results",], Normal IgG [prepared by the method of Baumstark et al (8)
from pooled normal human serum was used as a control for LATSIgG in each experiment, Measurement of adenyl cyclaae - [B- 14 C] Adenine,
50 to 55 mCi/mmole, was purchased from Schwarz-Mann BioReaearch, Inc, One ml of an aqueous solution [100 ~Ci/ml] of the nucleotide was
passed through an AG SOW-x2 [H +], 100-200 mesh [0,7 x 4 cm] column ; the resin wes washed with 15 ml water followed by 80 ml O,1M aqueous ammonia, The eluted product [comprising a sharply delineated radioactive peak between 2 and 8 ml of elutes] was concentrated to dryness in vacuo and then dissolved in meter to
give a concentration of 100 pCi/ml, The procedure of Humes et al (9) for measuring adenyl cyclase in intact cells was used with minor modifications, Fifty Nl isolated thyroid cells were placed in 1 ml of Krebs Ringer bicarbonate 14 buffer [KRH], pH 7,4, containing 1,0 pCi purified [8- C] adenine [2 x 10 -5 M adenine], 0,19 bovine serum albumin and 0,006M glucose, This preparation was "preincubated" under 95% 02 - 5% Cot in 80 x 16 mm covered sterile plastic tubes for 2 hr at 37 C in a Dubnoff metabolic shaker, The tubes were then centrifuged at 500 Xg for
5 min and the supernate discarded, The tubes containing the sedimented cell suspension were then placed in an ice-bath, 0,5 ml ice-cold KRB containing theophylline, 10 -2 M, and hormone and/or t We have previously commented ( Endocrinology 84 : 1063,1969) on the limited correlation between the in vivo ~d in vitro thyroidstimulating properties of LATS,
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agent as 1ndi~ted in "Results" wns added' and the tubes gassed and resealed, The cells were reavepended by Vortex prior to a l0-
min incubation [see "Results"] at 37 C, The enzymic reactions were terninated by the addition of 0,2 ml of 0,6M trichloracetic acid [TCA] and 0,05 ml of an aqueous solution ["nucleotide solution"] containing O,O1M each of ATP, ADP, 5'AMP, cyclic AMP, adenosine. and adenine to facilitate recovery of the labeled nucleotides, This treatment resulted in cell ~,yais ; insoluble material was removed by centrifuging for 10 min at 500 Xg, The resulting pellet was washed with 0,2 ml 2~6 TCA, recentrifuged for 20 min
and the supernate added to the initial TCA extract, The combined aupernates were neutralized with 0,1 ml 2M tris-HC1 buffer, pH 8,0 [resultant pH 7,4 to 7,6] and passed through an AG 509-x2 [H +], 100-200 mesh [0,7 x 3,5 cm] column, which was then eluted with water, The initial 2 ml effluent was discarded and the
cyclic AMP was collected in the subsequent 5 ml fraction, After treatment with 0,2 ml each of the aqueous 5% zinc sulfate and 0,3 N Ha(OH) 2 , the slurry was centrifuged for 5 min at 500 Xg,
The resulting aupernate was again subjected to this coprecipita-
tion procedure and then lyophilized to dryness, The residue, consisting primarily of 14 C-cyclic AMP, was 8lssolved in 1,0 ml water and the radioactivity of an 0,5 ml aliquot measured in Bray's solution (10), Another 0,5 ml aliquot was relyophilized, dissolved in 0,05 ml nucleotide solution [0,005M] and applied to precoated polyethylene-imine [PEI]-impregnated cellulose thin layer sheets [cellulose PEI-F, J.T, Haker Chemical Co,J and developed for 40 min in 0,3M LiCl (11), The cyclic AMP spot was visualised by
ultraviolet light, cut out and counted in 10 ml toluene scintillation fluid, Formation of 14 C-cyclic AMP was calculated as follows t TSH and LATS were dissolved in the requisite medium or buffer prior to addition to the appropriate tube or flask, One m4 of PGE2 or PY was dissolved in 0,1 ml of absolute ethanol, To this was added ~,9 ml Na2C03 [0,2 mg/ml, pH 7], This stock solution was diluted with buffer or medium to give the desired concentration of prostaglandin and/or antagonist in the tube, Each control Vessel contained a quantity of ethanol-carbonate diluent equivalent to that placed in the test vessel containing the maximum concentration of PGE2 and/or PYl in any given experiment, Stock PPP solution was prepared by dissolving 1 gm PPP in 44 ml 0,1 N NsOH which yielded a clear solution of pH _- 7,
97 2
Adenyl Cyclase of Bovine Thyroid Cells 14 C~yclic
AMP [cpm] = cpm HaSO4 supernate x
Vol . 10, No . 17
cyclic AMP spot [cpm]
total chromatogram radioactivity [cpm] These values were converted to picomoles 14 C-cyclic AMP formed/106 cells using the specific activity of the [8- 14 C] adenine [20 picomolea (8- 14 C) adenine -_ 1000 cpm] and correcting for the actual number of cells used [12-20 x 106 cells/ml] in
any given experiment, All studies were done in triplicate or duplicate, TATS bioassay was performed using a minor modification of the McKenzie method (7)r all test materials were administered
intraperitoneally, Statistical analysis of data was performed by paired t-test (12) or, where appropriate, by Dunnet's test which permits comparison of several experimental groups with a single control (13 ),
*= cpm in 0,5 ml aliquot x 2, Results
Time-course of TATS-induced adenyl Cyclase activation As esn be seen from Fig, 1, both TSH [100 mU/ml] and IATS[0,5 mg/ml] effected significant activation of adenyl Cyclase IgG in isolated bovine thyroid cells during incubation periods
ranging from 5 to 15 min, [Although shorter incubation periods (1 and 2,5 min) were attempted, these proved technically difficult and no consistent TSH or IATS effect was observed,], In each instance maximum stimulation was obtained between 7,5 and 10 mint in all experiments to be detailed hereafter, a 10-min incubation period was employed, Aa can be seen from Fig, l, 14 Ccyclic AMP levels in untreated cells or in cells incubated with
normal IgG did not change significantly over time indicating that a "steady state" had been achieved and that the specific activity of the cyclic AMP yielding ATP pool was probably constant,
Vol. 10, No . 17
Ade~rl Cyc~ase of Bovine Thyroid Celle
973
8 Q w 0
0 a a
U U Û â O U â
TIME (min) FIG, 1 Time-course of TSH [100 mU/ml] and IATS-IgG [0,5 mq/ml] activation of adenyl cyclase in isolated bovine thyroid cells, 8ach point represents the moan ± STja of 4 experiments, each experimental determination performed in dupli~te, control ~ untreated cells, Normal IqG = cells incubated with normal IgG, 0,5 m9/ml,
TATS-IgG dose response curve The response of bovine isolated thyroid cells to TATS [and TSH] varied somewhat from experiment to experiment, Nonetheless, a dose-related IATS-IqG effect was consistently observed [FIG, 2],
974
Ade~l-Cyclase od Bovine Thyroid Cells
0 O w O
120
lL
80
U
60
U 'Û
40
a
Vol . 10, No . 17
NORMAL IqG
O g Ôr 0.05
0.13
ß2S
IgG (mg/m l)
0~5
1 .0
FIG, 2 Dose-response curve, IATS-IgG activation of adenyl cyclaae in isolated bovine thyroid cells, Incubation time = 10 min, Each point represents the mean + 1 S~, of 4 experiments, each experimental determination performed i-n duplicate,
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Adenyl Cyclase of Bovine Thyroid Cells
975
PICOMOLES ~4C-CYCLIC AMP FORMED/10` cells o
xo
4o
so
eo
loo
X20
CONTROL Normal IqG, QSmghni PGE=, 2.5 x 10'~M TSH, 10pmU/ml LATS IqG, 0.5 mq/ml PY 5p.q/ml PGE =, 2.5 x 10'' M+ PY 5 Y~q/ml TSH, 100mU/ml+ PY 5pq/ml LATS IqG, 0.5mghnl+ PY 5pq/ml FIG . 3 Effects of PYl on basal and stimulated adenyl cyclase activity in isolated bovine thyroid cells, Incubation time = 10 min Each value represents the mean ± 1 S~of 6 experiments, each experimental determination performed in duplicate . * denotes values significantly [p< 0,01] greater than controls ** denotes values signifi~ntly [p < 0,01] less than corresponding stimulator alone,
978
Vol. 10, No . 17
Adenyl Cyclase od Bovine Thyroid Cells
PICOMOLES "C-CYCLIC AMP FORMED/108 cells 0
20
40
60
80
100
CONTROL Normal IqG 0.5 mq/ml PGE =, 2 .5 x 10'~M TSH, 50mU/ml LATS IqG, 0.5mq/ml PPP, 5~aq/ml PGE=, 2 .5 x 10-~M + PPP, 5Y.q/ml TSH, 50mU/ml + PPP, 5 fcq/ml LATS IpG,0.5mq/ml+ PPP,5~q/ml
FIG, 4 Effects of PPP on basal and stimulated adenyl cyclase activity in isolated bovine thyroid cells, Incubation time _- 10 min, Each value represents the mean ± 1 SF~ of 6 experiments, each experimental determination performed in duplicate, * denotes values significantly [p < 0,01] greater than controls ** denotes values significantly [p < 0,02 to p < 0,01] less than corresponding stimulator alone, As can be seen from FIGs, 3 and 4, PY 1 , 5 +ag/ml, [= 1,4 x 1~M]
and PPP , 5 leg/ml [= 3,3 x 10 -8M] significantly inhibited PGE2 , TSH and IATS effects on adenyl cyclase activity in isolated bovine thyroid cells, PY 1, 5-20 lug/ml, had weak non-dose related agonist properties when tested alone [maximum 15-20% increase over control]s PPP, 1-25 pg/ml, exhibited variable and statistically nonsignificant inhibitory effects on basal cyclase activity, In order to confirm that these f indinga were not peculiar to
Vol. 10, No . 17
Adenyl Cyclase of Bovine Thyroid Cells
977
the isolated bovine thyroid cell adenyl cyclase system, a series
of 3 mouse bioassay experiments [measuring thyroidal radioiodine release (7 )] were performed with TSH, IATS [whole plasma] and PPP*, PPP, 0,1 to 1,0 mg/mouse alone was without effer_t on blood radioactivity, As can be seen from FIGs, 5 and 6, PF~P, 0, 5 !~T/ mouse, significantly inhibited the effects of both TSH and LATST in each instance, the slope of the intermediate dose portion of the log-concentration effect curve was not changed in the presence; of the antagonist, while antagonism was ultimately blunted ôr surmounted by increasing the concentration of the
agonist [0,75 mU TSH, 1 :2 dilution of IATS plasma], In contrast, the response to dibutyryl cyclic AMP [DECAMP], 2,5 mq/mouse, was unaffected by PPP, 0,5 mg/mouse [6 hr zesoonse index, DBc.AMP alone, 465 ± 83 vs, DECAMP + PPP,
535 ± 92,],
TSH, TATS and PPP were administered intraoeritoneally, total volume -- 0,5 ml/substance(s) administered,
17ßO 7500
~Ô E
X Z
1250 1000 750
W
tn
W
500 2ßO O~i. 0
0.10
02ß
TSH (mU)
Q50
0 .7ß
FIG, 5 Effects of PPP, 0,5 mg/mouse, on TSH-induced mouse thyroid radioiodine release, Each point represents the mean ± 1 SF~,of 3 experiments, 6 mice/material(s) assayed in each experiment, Oomparison between TSH [e-i] and TSH + PPP [~- - -e] were, in each instance, made in the same experiment,
978
Adenyl Cyclase of Bovine Thyroid Cells
Vol . 10, No . 17
zaoo z
i +z
7~fi
~ 12.5
zs
LATS (Serum Dilution x 100)
F IG,, 6 Effect of PPP, (0,5 mg/mouse], on LATS-induced mouse thyroid radioiodine release, Each point represents the mean ± 1 SFm of 3 experiments, 6 mice/material(s) assayed in each experiment, Comparisons between TATS [~ ~] and TATS + PPP [~ - - ~] were in each instance, made in the same experiment, Dü cuss ion
We have recently reported (2) that PYl , a compound structurally related to the proataglandins (5 ), inhibits both PGE and TSH effects on adenyl cyclase activation and cyclic AMP formation in thyroid, These findings raised the possibility that there might be a single TSH-related prostaglandin receptor in thyroid, activation of which is requisite to TSH stimulation of
hormonogeneais in the gland (2), [Kuehl et al (14) had earlier advanced a similar concept for the action of luteinizing hormone
on the ovary .], Since the results of many previous studies (15-22) support the postulate that the sites of action of TSH and LATS on thyroid are similar or identical, we now examined the effects of PY 1 and PPP on TATS activation of thyroid adenyl cyclase, PPP, a phosphorylated polyanionic derivative of phloridzin, has been shown (6, 23-26) to antagonize the effects of prostaglandins E and F on a variety of physiologic systems, Stimulatory effects of IATS on thyroid adenyl cyclase have now
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Adegyl Cyclase of Bovine Thyroid Cells
979
been demonstrated in three species - ovine, bovine and canine thyroid (27-29 ), Although there is some disagreement within these
studies ae to the latency of IATS action relative to that of TSB, it is apparent that the increase in labeled cyclic AMP formation represents adenyl cyclase activation rather than phoaphodieaterase inhibition (28,29), Similar evidence has been reported (30) in
thyroid adenyl cyclase studies with proataglandins E and F, Given these Eindinge and the addition of the potent phoaphodieaterase inhibitor, theophylline, to each incubation mixture, it would seem reasonable to assume that any changes in 14 C-cyclic AMP levels in the present study reflect changes in adenyl cyclaae activity only, Thus, it may be concluded Erom the studies reported herein that two unrelated prostaglandin "antagonists" , PY l and PPP, inhibit the effects of IATS [as well as those of PGE2 and TSB] on thyroid adenyl cyclase, Although attempts to characterize the nature of this antagonism in in vitro cell studies have, to date, been unsuccessful, the mouse bioassay studies do shed some additional light on the problem. Therein, the parallel shift in thyroid stimulator-induced dose-response curves with PPP and the demonstration that higher stimulator concentrations overcome PPP antagonism [see FI3a, 5 and 6], fulfill the criteria of
Stephenaon (31) for competitive antagoniam, Clearly however, additional in vitro studies with both PY 1 and PPP will be required ** , In any event, the to support these preliminary conclusions, mouse bioassay studies indicate that the antagonism exhibited bY PY 1 and PPP to TSH and TATS is not peculiar to the in vitro
isolated bovine thyroid cell system, The results of these in vivo studies are consistent with earlier findings (30) wherein we
reported that proataglandina E and F augmented mouse thyroid radioiodine release in vivo but when combined with the more potent TSH
or LATS, reduced the stimulatory effect significantly below that observed with either TSH or IATS alone, The lack of PPP antagonism to DHcAMP in the mouse bioassay is important in several ways, First,
it excludes a non-specific
"toxic" effect as the basis for PPP antagonism to TSH and IATS, Second, [along with the lack of "PPP only" effect on blood radioactivity],
it suggests that the PPP-induced changes in blood
*ie, prostaglandin antagonists in some systems, *The limited availability of PY1 precludes in vivo [is, mouse bioassay] studies with this compound,
980
Adenyl Cyclase of Bovine Thyroid Cells
Vol . 10, No . 17
radioactivity truly reflect changes in thyroidal radioiodine
release rather than PPP-induced changes in clearance of labeled thyroid hormone from the blood (32 ), Finally, it suggests, as have our previous in vitro studies with PY l (1,2) that PPP antagonistea stimulated thyroidal hormonogenesis as a direct
consequence of inhibition of adenyl Cyclase activation without involving steps subsequent thereto,
The studies reported herein support the concept that activation of a prostaglandin adenyl Cyclase receptor Bite may be an early step in the action of both LATS and TSH on thyroid, Açknowledgments This study was supported by a grant [AM 11136] from the National Institute of Arthritis and Metabolic Diseases, USPHS, The authors gratefully acknowledge the technical assistance of L, Wang and C, Lonergan, References 1, G, BURKE, K, KOWAISKI and D, BABIARZ, Life Sci , 10 ; 513 (1971), 2, G, BURKE, S, SATO, K, K04~LSKI, D. HABZARZ and M, SZABO, Program 53rd Meeting Endocr, Soc p A-140, ,Tune 24-26 (1971), 3, M,M, EDMUNDS, V,V, ROw and R, VOLPE, 480 (1970),
J_ Clin, Endocr ,
4, G, BURKE and K. KOWALSKI, Life Sci , 10 : 361 (1971) . 5, J, FRIED, T.S, SANTHANAKRISHNAN, J. HIMIZU, C,H, LIN, S,H, FORD, B, RUBIN and E,O, GRIGAS, Nature 233 : 208 (1969), 6, K,E, EAKINS, S,M,M, KARIM and J,D, MILLER, Brit . J. Pharmacol , 39s 556 (1970), 7, G, BURi~, J. Lab, Clin Med , 69 : 713 (1967), 8, J,S, BAUMSTARK, R,J, IAFFIN and W,A,BARDAWIL, Arch . Biochem , 108" 514 (1964), 9, J,L, HUMES, M, ROUNBEHLER and F,A, KUEHL, Jr Anal, Biochem, 32 : 210 (1969), 10, G,A, BRAY, Anal . Biochem, 1s 279 (1960), 11, H,-F, BÂR and O . HECHTER, Anal . Biochem, 29s 476 (1969), 12, W,J, DIXON and F,J, MASSEY, Jr Introduction to Statistical Analysis, McGraw Hill, New York, 124-128 (1957), 13, C, W, DÜNNET, J. Amer . Statist . Aa90C . 50 : 1096 (1955),
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Adeiryl Cyclase of Bovine Thyroid Celle
981
14,
F .A . KUEHL, Jr J,L, HUMES, J. TARNOFF, V.J, CIRILLO and E,A, ffAM ., Science 169s 883 (1970),
15,
T,W, SCOTT, B.F, GOOD and K,A, FERGUSON, Endocrinology 79 : 949 (1966),
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Y, SHISHIBA, D,H. SOLOMON and G,N, BEALL, Endocrinology 80s 957 (1967),
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G, BURKE, J. Clin . Endocr , 28s 286 (1968),
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Y, OCHI and L, J. DeGROOT, Biochim. Biophys, Acta 170s 198 (1968),
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G, BURKE, Endocrinology 83s 1210 (1968),
20,
G, BURKE, Metabolism 18s 720 (1969),
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T, ONAYA and D,H . SOIAMON, Endocrinology 85 : 1010 (1969),
22,
Y, SHISHIBA, D.H, SOIAMON and W,D, UArVIDSON, Endocrinology 86s 183 (1970),
23,
K.E, EAKINS and S,M .M, KARIM,
24,
K,E, EAKINS, J,D . MILLER and S,M,M, KARIM, J_ Pharmacol, Exp, Therap , 176s 441 (1971),
25,
R, VILLANUEVA, L, HINDS, R,L . KATZ and K,E, EAKINS, Fed . Proc , 30, 626abs (1971),
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R,A . BETHEL and K,E, EAKINS, Fed . Proc , 30s 626abs (1971),
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G, BURKE, Clin . Res , 17s 521 (1969),
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G,S, LEVEY and I, PASTAN, Life Sci, 9s 67 (1970),
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T, KANEKO, U . ZOR and J,B. FIELD, Metabolism 19s 430 (1970),
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G . BURKE, Am . J. Physiol, 218 : 1445 (1970),
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R,P, STEPHENSON, Brit, J, Pharmacol , 11s 379 (1965),
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D .H, SOLOMON, G,N, BEALL and I,J . CHOPRA, J. Clin, Endocr , 31s 603 (1970),
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