Inhibition of Thyroid Adenylate Cyclase by Thyroid Hormone: A Possible Locus for the "Short-Loop" Negative Feedback Phenomenon1 YOCHANAN FRIEDMAN, MICHAEL LANG, AND GERALD BURKE
ECENT findings by us (1) and others (2, 3) suggest the existence of a thyroid hormone-mediated "short-loop" negative feedback mechanism directly regulating thyroid function. There has, however, been disagreement as to whether such negative feedback is exerted prior to or following TSH-induced generation of thyroid cyclic 3',5'-adenosine monophosphate [cAMP] (3,4). We now present evidence that thyroid
Received September 2, 1976. Supported by a Grant (AM 17561) from the U.S.P.H.S. 1 Presented in part to the 58th Annual Meeting of the Endocrine Society, San Francisco, California, June 23-25, 1976. Abbreviations used in this paper: cAMP, cyclic adenosine 3'-5'monophosphate; (Bu)2cAMP, N6-2'O-dibutyryl cyclic adenosine 3',5'-monophosphate; PGE2, prostaglandin E2; PGF^, prostaglandin F ^ ; T3, 3,5,3'-triiodothyronine; T4, L-thyroxine; rT3, 3,3',5'triiodothyronine.
Hospital,
35%; F~-induced activity was unaffected. Although
ABSTRACT. The effect of thyroid hormone on bovine thyroid plasma membrane adenylate cyclase activity was studied in order to determine if the locus of the previously observed "short-loop" negative feedback in the thyroid (J Clin Invest 57: 745, 1976) is the adenylate cyclase enzyme system. Adenylate cyclase activity was measured in bovine thyroid plasma membrane. This preparation was responsive to TSH (100 mU/ml), PGE2 (5 x 10"»M) and F" (10~ 2 M) with increases in activity of 70%, 50%, and 1,000% over basal (1.3 ± 0.5 pmol cAMP formed/10 min/mg protein), respectively. L-Thyroxine (T4), 3,5,3'-triiodothyronine (T3), D-thyroxine, 3,5,3'-triiodothyroacetic acid, and 3,3',5'-triiodothyronine inhibited basal adenylate cyclase activity by 25-35%; in contrast, only T4, T3, and D-T4 inhibited TSH- and PGE 2 -stimulated adenylate cyclase activity. F~ stimulation was unaffected. Lubrol-PX, a non-ionic detergent, abolished TSH-induced adenylate cyclase activity, but plasma membrane so treated was still responsive to PGE2 and F~. Thyroid hormone inhibited both basal and PGE2induced activity in Lubrol-treated membrane by
R
of Medicine, Cook County
both
GTP
(3 x 10~ 4 M)
and
ITP
(1 x 10" 4 M)
enhanced basal adenylate cyclase activity in intact plasma membrane, the inhibitory effect of thyroid hormone was still seen. Thyroid hormone did not alter plasma membrane ATP'ase activity. T3 (5 fig) was shown to inhibit (by 78-97%) both TSH and dibutyryl cAMP [(Bu)2cAMP] stimulation of rat thyroid ornithine decarboxylase activity (ODC). Additionally, T 3 pretreatment of the McKenzie bioassay mouse led to a dose-related reduction in both TSH- and (Bu)2cAMP-stimulated thyroid hormone release. Conclusions: 1) Thyroid hormone directly inhibits thyroid adenylate cyclase activity; this effect is not exerted at the TSH receptor or the catalytic site of the enzyme, but may involve the "coupler" subunit of the enzyme. 2) Thyroid hormone directly inhibits (Bu)2cAMP stimulation of thyroid function. 3) Thus, circulating thyroid hormones may regulate thyroid function by a "shortloop" feedback mechanism(s) effected prior to as well as following generation of thyroidal cAMP. (Endocrinology 101: 858, 1977).
hormone directly inhibits thyroid gland adenylate cyclase activity, but also blocks stimulatory effects on exogenous dibutyrl cAMP [(Bu)2cAMP] as well. Materials and Methods Tissue preparation A "crude" plasma membrane fraction was prepared by a modification of the method of Wolff and Jones (5). Bovine thyroid glands were obtained fresh from the slaughterhouse and kept on ice from the time of removal up to the time of tissue preparation (always less than 2 h postsacrifice). The thyroids were trimmed of excess fat and fascia and were finely minced. The minced thyroid (—12 g) was homogenized in 4 vol of cold 0.25M sucrose + 0 . 0 0 1 M EDTA, pH 7.3, in a "polytron" homogenizer (Brinkman) and then centrifuged at 2500 x g for 10 min in an IEC B-20 refrigerated centrifuge. The result-
858
Downloaded from https://academic.oup.com/endo/article-abstract/101/3/858/2618062 by Nagoya University user on 13 January 2019
Division of Endocrinology and Metabolism, Department Chicago, Illinois 60612
INHIBITION OF THYROID ADENYLATE CYCLASE
Preparation of "Solubilized" adenylate cyclase Two methods were used to prepare solubilized adenylate cyclase. The first is based on Levey's procedure (6) wherein the crude plasma membrane fraction was isolated in the usual fashion, and then "solubilized" by the addition of Lubrol-PX to a final concentration of 0.05%. The enzyme solution was kept at 4 C for 30 min with intermittent vortexing, after which the mixture was centrifuged at 35,000 rpm (100,000 x g) in the ultracentrifuge (Beckman-Model L-65B). The resulting supernatant was designated as "solubilized" adenylate cyclase. (Note: adenylate cyclase activity was assayed immediately after the centrifugation since it was observed that waiting until the next day resulted in a loss of activity when Lubrol-PX was present.) The second method involves the preparation of plasma membrane directly in Lubrol-PX. The finely minced thyroid tissue was homogenized in 0.25M sucrose + 0 . 0 0 1 M EDTA, pH 7.3, containing 0.1% Lubrol-PX. The mixture was kept at 4 C for 30 min with intermittent vortexing, after which the regular procedure for isolation of the plasma membrane fraction was carried out (see above). This procedure is similar to that employed by Yamashita and Field (7) for treatment of thyroid plasma membrane with phospholipases A and C. The plasma membrane obtained by this procedure was no longer responsive to TSH (see Results), and yielded a preparation with more stable adenylate cyclase activity, since the Lubrol-PX is removed by washing during the procedure. Adenylate cyclase assay Adenylate cyclase activity was assayed at 37 C for 10 min according to a modification of the method by Wolff and Jones (5). The incubation mixture (final vol 110 jxl) contained: 40 mM
Tris-HCl, pH 7.5; 5 mM MgCl2; 10 mM theophylline; 4 mM cAMP; 0.1% bovine serum albumin (BSA); 10 mM creatine phosphate; 35 \xg creatine phosphokinase (160 U/mg); 2 fiM [a-32P]ATP (unless stated otherwise). The reaction was initiated by the addition of plasma membrane (~250 fMg) and the reaction was terminated by boiling the incubation mixture for 4 min. The assay for [32P]cAMP thus formed was performed by the combined methods of Ramachandran (8) and Krishna and Birnbaumer (9) as described by Sato et al. (10). The thyroid hormones and their analogs were dissolved in a minimal amount of NaOH before addition to the assay mixture. Control flasks contained an equivalent concentration of NaOH and the results are corrected for any solvent effect (ca., 5% reduction in basal enzyme activity). Results are expressed as pmol of cAMP formed/10 min/mg protein. Other assays Ornithine decarboxylase (ODC) was assayed according to the method of Yu et al. (1), using an incubation temperature of 44 C. It was found (data not shown) that this temperature is optimal for ODC activity under our assay conditions. Five rats were used for each determination. 3,5,3 '-triiodo-L-thyronine (T3; 5 fig) was injected sc 16 h before TSH (2U) or (Bu)2cAMP (40 mg) + aminophylline (10 mg) was given ip. Four hours later the animals were sacrificed, the thyroids removed, and ODC activity was assayed. The results are expressed as pmol 14CO2 formed/ g tissue/30 min incubation. 131 I release in the mouse was measured by the method of McKenzie (11). The mice had been kept on a low-iodine diet for two weeks prior to use and were pretreated with varying amounts of T3 (0.25 fig to 20 fig) given sc 24 h and again 4 h (ip) before TSH or (Bu)2cAMP administration. The animals were bled 4 h after the second T3 treatment (0 h), injected with 2.5 mg (Bu)2cAMP + 0.15 mg aminophylline or 0.5 mU TSH, and bled again 3 h later. The results are expressed as (3 h cpm/0 h cpm) x 100. Protein was measured by the method of Lowry et al. (12). Total and free T3 and T4 concentrations in the incubation medium were measured by methods previously detailed elsewhere (13). All data were analyzed for statistical significance by Student's t test unless otherwise stated.
Downloaded from https://academic.oup.com/endo/article-abstract/101/3/858/2618062 by Nagoya University user on 13 January 2019
ing pellet was then resuspended in 4 vol of 0.25M sucrose + 0.001M EDTA, pH 7.3, with a loosefitting Dounce glass homogenizer (Kontes) and centrifuged at 150 x g for 10 min to remove the cellular debris. The supernatant was then removed and centrifuged at 2500 x g for 10 min. The resulting pellet (designated as "crude plasma membrane") was resuspended in 0.04M Tris-HCl, pH 7.8 (usually 1.2 ml), and used for the adenylate cyclase assay. The protein concentration in this suspension was between 10 and 15 mg/ml.
859
FRIEDMAN, LANG AND BURKE
860
Endo • 1977 Vol 101 • No 3
E O
Effect of prostaglandins E2 and F2a on plasma membrane adenylate cyclase activity PGE
i67
io6
id5
to"3
[ P G ] Molar FIG. 1. Effects of prostaglandins (PGE2 and PGF2a) on adenylate cyclase activity in bovine thyroid plasma membrane. The vertical bars indicate 1 SD.
The following materials were used: TSH in the form of Thytropar® was purchased from Armour Pharmaceutical; [a-32P]ATP (tetraethylammonium salt, 10 Ci/mmol), from ICN Pharmaceuticals, Inc., and phosphocreatine, Lubrol-PX, ITP, GTP, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, L-thyronine, L-thyroxine (T4), D-thyroxine (D-T 4 ), 3,5,3'-triiodo-Lthyronine (T3), 3,5,3'-triiodothyroacetic acid (TRIAC), and creatine phosphokinase (160 U/mg), from Sigma Chemical Co. Prostaglandin E 2 (PGE2) and Prostaglandin F ^ (PGFa,) were kindly supplied by Dr. John Pike (Upjohn Co.). Reverse T3 (rT3; 3,3',5'-triiodo-L-thyronine) was kindly supplied by Warner-Lambert Co.
Results Effect of TSH on adenylate cyclase activity Various sub-cellular "marker" enzymes (5) were assayed in the crude plasma membrane fraction; it was found that this fraction
PGE 2 and PGF 2a effectively increased adenylate cyclase activity (Fig. 1). PGE 2 was the more potent stimulator and an increase in activity was observed at 1 x 10~5M concentration, whereas PGF 2a was ineffective at concentrations less than 1 x 10~4M. At 5 x 10~4M (the highest concentration tested), PGE 2 increased enzyme activity by 60% and PGF 2a by 30%. Effect of T3 on basal, TSH-, and prostaglandin E2-stimulated adenylate cyclase activity Figure 2 shows the effect of various doses of T3, added to the incubation medium, on basal as well as TSH- and PGE2-stimulated adenylate cyclase activity (similar results were obtained with T4 (data not shown)). T3 inhibited basal enzyme activity in a doserelated manner, reducing [32P]cAMP formaation by 15% (P < .025) at 4 fig (5 x 10" 5 M), and by 50% at 20 fig (2 x 10" 4 M). Iodide at 10 fig (5 x 10" 4 M) had no effect on adenylate cyclase activity. (In other experiments, I~ was tested at 20 /xg/ml and no effect was observed.) In addition, T3 (20 fig) had no significant effect on adenylate cyclase activity in plasma membrane from other tissues. Plasma membrane ATPase was also unaffected by T3 and T4 (data not shown). As can be seen from Fig. 2, TSH-stimulated adenylate cyclase activity was also
Downloaded from https://academic.oup.com/endo/article-abstract/101/3/858/2618062 by Nagoya University user on 13 January 2019
gave an enrichment by a factor of 4 over the whole homogenate when plasma membrane marker enzymes (5'-nucleotidase, ATPase, and alkaline phosphatase) were assayed (data not shown). TSH increases adenylate cyclase activity in a dose-related manner from 10 to 100 mU/ ml (50% increase over control), after which no further increase is seen. Similar membrane preparations from liver, kidney, lung, and spleen failed to give a TSH response (data not shown).
INHIBITION OF THYROID ADENYLATE CYCLASE
861
pmoles cAMP formed / 1 0 min/mg protein I
2
|
Basal
| | T S H (lOOmU/ml)
§|PGE2(5xl0*4M)
TSH(IOOmU/ml) • T3 lug • T 3 4ug • TjlOug +T320/ig • I" 10/xg
^S^^^
ECENT findings by us (1) and others (2, 3) suggest the existence of a thyroid hormone-mediated "short-loop" negative feedback mechanism directly regulating thyroid function. There has, however, been disagreement as to whether such negative feedback is exerted prior to or following TSH-induced generation of thyroid cyclic 3',5'-adenosine monophosphate [cAMP] (3,4). We now present evidence that thyroid
Received September 2, 1976. Supported by a Grant (AM 17561) from the U.S.P.H.S. 1 Presented in part to the 58th Annual Meeting of the Endocrine Society, San Francisco, California, June 23-25, 1976. Abbreviations used in this paper: cAMP, cyclic adenosine 3'-5'monophosphate; (Bu)2cAMP, N6-2'O-dibutyryl cyclic adenosine 3',5'-monophosphate; PGE2, prostaglandin E2; PGF^, prostaglandin F ^ ; T3, 3,5,3'-triiodothyronine; T4, L-thyroxine; rT3, 3,3',5'triiodothyronine.
Hospital,
35%; F~-induced activity was unaffected. Although
ABSTRACT. The effect of thyroid hormone on bovine thyroid plasma membrane adenylate cyclase activity was studied in order to determine if the locus of the previously observed "short-loop" negative feedback in the thyroid (J Clin Invest 57: 745, 1976) is the adenylate cyclase enzyme system. Adenylate cyclase activity was measured in bovine thyroid plasma membrane. This preparation was responsive to TSH (100 mU/ml), PGE2 (5 x 10"»M) and F" (10~ 2 M) with increases in activity of 70%, 50%, and 1,000% over basal (1.3 ± 0.5 pmol cAMP formed/10 min/mg protein), respectively. L-Thyroxine (T4), 3,5,3'-triiodothyronine (T3), D-thyroxine, 3,5,3'-triiodothyroacetic acid, and 3,3',5'-triiodothyronine inhibited basal adenylate cyclase activity by 25-35%; in contrast, only T4, T3, and D-T4 inhibited TSH- and PGE 2 -stimulated adenylate cyclase activity. F~ stimulation was unaffected. Lubrol-PX, a non-ionic detergent, abolished TSH-induced adenylate cyclase activity, but plasma membrane so treated was still responsive to PGE2 and F~. Thyroid hormone inhibited both basal and PGE2induced activity in Lubrol-treated membrane by
R
of Medicine, Cook County
both
GTP
(3 x 10~ 4 M)
and
ITP
(1 x 10" 4 M)
enhanced basal adenylate cyclase activity in intact plasma membrane, the inhibitory effect of thyroid hormone was still seen. Thyroid hormone did not alter plasma membrane ATP'ase activity. T3 (5 fig) was shown to inhibit (by 78-97%) both TSH and dibutyryl cAMP [(Bu)2cAMP] stimulation of rat thyroid ornithine decarboxylase activity (ODC). Additionally, T 3 pretreatment of the McKenzie bioassay mouse led to a dose-related reduction in both TSH- and (Bu)2cAMP-stimulated thyroid hormone release. Conclusions: 1) Thyroid hormone directly inhibits thyroid adenylate cyclase activity; this effect is not exerted at the TSH receptor or the catalytic site of the enzyme, but may involve the "coupler" subunit of the enzyme. 2) Thyroid hormone directly inhibits (Bu)2cAMP stimulation of thyroid function. 3) Thus, circulating thyroid hormones may regulate thyroid function by a "shortloop" feedback mechanism(s) effected prior to as well as following generation of thyroidal cAMP. (Endocrinology 101: 858, 1977).
hormone directly inhibits thyroid gland adenylate cyclase activity, but also blocks stimulatory effects on exogenous dibutyrl cAMP [(Bu)2cAMP] as well. Materials and Methods Tissue preparation A "crude" plasma membrane fraction was prepared by a modification of the method of Wolff and Jones (5). Bovine thyroid glands were obtained fresh from the slaughterhouse and kept on ice from the time of removal up to the time of tissue preparation (always less than 2 h postsacrifice). The thyroids were trimmed of excess fat and fascia and were finely minced. The minced thyroid (—12 g) was homogenized in 4 vol of cold 0.25M sucrose + 0 . 0 0 1 M EDTA, pH 7.3, in a "polytron" homogenizer (Brinkman) and then centrifuged at 2500 x g for 10 min in an IEC B-20 refrigerated centrifuge. The result-
858
Downloaded from https://academic.oup.com/endo/article-abstract/101/3/858/2618062 by Nagoya University user on 13 January 2019
Division of Endocrinology and Metabolism, Department Chicago, Illinois 60612
INHIBITION OF THYROID ADENYLATE CYCLASE
Preparation of "Solubilized" adenylate cyclase Two methods were used to prepare solubilized adenylate cyclase. The first is based on Levey's procedure (6) wherein the crude plasma membrane fraction was isolated in the usual fashion, and then "solubilized" by the addition of Lubrol-PX to a final concentration of 0.05%. The enzyme solution was kept at 4 C for 30 min with intermittent vortexing, after which the mixture was centrifuged at 35,000 rpm (100,000 x g) in the ultracentrifuge (Beckman-Model L-65B). The resulting supernatant was designated as "solubilized" adenylate cyclase. (Note: adenylate cyclase activity was assayed immediately after the centrifugation since it was observed that waiting until the next day resulted in a loss of activity when Lubrol-PX was present.) The second method involves the preparation of plasma membrane directly in Lubrol-PX. The finely minced thyroid tissue was homogenized in 0.25M sucrose + 0 . 0 0 1 M EDTA, pH 7.3, containing 0.1% Lubrol-PX. The mixture was kept at 4 C for 30 min with intermittent vortexing, after which the regular procedure for isolation of the plasma membrane fraction was carried out (see above). This procedure is similar to that employed by Yamashita and Field (7) for treatment of thyroid plasma membrane with phospholipases A and C. The plasma membrane obtained by this procedure was no longer responsive to TSH (see Results), and yielded a preparation with more stable adenylate cyclase activity, since the Lubrol-PX is removed by washing during the procedure. Adenylate cyclase assay Adenylate cyclase activity was assayed at 37 C for 10 min according to a modification of the method by Wolff and Jones (5). The incubation mixture (final vol 110 jxl) contained: 40 mM
Tris-HCl, pH 7.5; 5 mM MgCl2; 10 mM theophylline; 4 mM cAMP; 0.1% bovine serum albumin (BSA); 10 mM creatine phosphate; 35 \xg creatine phosphokinase (160 U/mg); 2 fiM [a-32P]ATP (unless stated otherwise). The reaction was initiated by the addition of plasma membrane (~250 fMg) and the reaction was terminated by boiling the incubation mixture for 4 min. The assay for [32P]cAMP thus formed was performed by the combined methods of Ramachandran (8) and Krishna and Birnbaumer (9) as described by Sato et al. (10). The thyroid hormones and their analogs were dissolved in a minimal amount of NaOH before addition to the assay mixture. Control flasks contained an equivalent concentration of NaOH and the results are corrected for any solvent effect (ca., 5% reduction in basal enzyme activity). Results are expressed as pmol of cAMP formed/10 min/mg protein. Other assays Ornithine decarboxylase (ODC) was assayed according to the method of Yu et al. (1), using an incubation temperature of 44 C. It was found (data not shown) that this temperature is optimal for ODC activity under our assay conditions. Five rats were used for each determination. 3,5,3 '-triiodo-L-thyronine (T3; 5 fig) was injected sc 16 h before TSH (2U) or (Bu)2cAMP (40 mg) + aminophylline (10 mg) was given ip. Four hours later the animals were sacrificed, the thyroids removed, and ODC activity was assayed. The results are expressed as pmol 14CO2 formed/ g tissue/30 min incubation. 131 I release in the mouse was measured by the method of McKenzie (11). The mice had been kept on a low-iodine diet for two weeks prior to use and were pretreated with varying amounts of T3 (0.25 fig to 20 fig) given sc 24 h and again 4 h (ip) before TSH or (Bu)2cAMP administration. The animals were bled 4 h after the second T3 treatment (0 h), injected with 2.5 mg (Bu)2cAMP + 0.15 mg aminophylline or 0.5 mU TSH, and bled again 3 h later. The results are expressed as (3 h cpm/0 h cpm) x 100. Protein was measured by the method of Lowry et al. (12). Total and free T3 and T4 concentrations in the incubation medium were measured by methods previously detailed elsewhere (13). All data were analyzed for statistical significance by Student's t test unless otherwise stated.
Downloaded from https://academic.oup.com/endo/article-abstract/101/3/858/2618062 by Nagoya University user on 13 January 2019
ing pellet was then resuspended in 4 vol of 0.25M sucrose + 0.001M EDTA, pH 7.3, with a loosefitting Dounce glass homogenizer (Kontes) and centrifuged at 150 x g for 10 min to remove the cellular debris. The supernatant was then removed and centrifuged at 2500 x g for 10 min. The resulting pellet (designated as "crude plasma membrane") was resuspended in 0.04M Tris-HCl, pH 7.8 (usually 1.2 ml), and used for the adenylate cyclase assay. The protein concentration in this suspension was between 10 and 15 mg/ml.
859
FRIEDMAN, LANG AND BURKE
860
Endo • 1977 Vol 101 • No 3
E O
Effect of prostaglandins E2 and F2a on plasma membrane adenylate cyclase activity PGE
i67
io6
id5
to"3
[ P G ] Molar FIG. 1. Effects of prostaglandins (PGE2 and PGF2a) on adenylate cyclase activity in bovine thyroid plasma membrane. The vertical bars indicate 1 SD.
The following materials were used: TSH in the form of Thytropar® was purchased from Armour Pharmaceutical; [a-32P]ATP (tetraethylammonium salt, 10 Ci/mmol), from ICN Pharmaceuticals, Inc., and phosphocreatine, Lubrol-PX, ITP, GTP, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine, L-thyronine, L-thyroxine (T4), D-thyroxine (D-T 4 ), 3,5,3'-triiodo-Lthyronine (T3), 3,5,3'-triiodothyroacetic acid (TRIAC), and creatine phosphokinase (160 U/mg), from Sigma Chemical Co. Prostaglandin E 2 (PGE2) and Prostaglandin F ^ (PGFa,) were kindly supplied by Dr. John Pike (Upjohn Co.). Reverse T3 (rT3; 3,3',5'-triiodo-L-thyronine) was kindly supplied by Warner-Lambert Co.
Results Effect of TSH on adenylate cyclase activity Various sub-cellular "marker" enzymes (5) were assayed in the crude plasma membrane fraction; it was found that this fraction
PGE 2 and PGF 2a effectively increased adenylate cyclase activity (Fig. 1). PGE 2 was the more potent stimulator and an increase in activity was observed at 1 x 10~5M concentration, whereas PGF 2a was ineffective at concentrations less than 1 x 10~4M. At 5 x 10~4M (the highest concentration tested), PGE 2 increased enzyme activity by 60% and PGF 2a by 30%. Effect of T3 on basal, TSH-, and prostaglandin E2-stimulated adenylate cyclase activity Figure 2 shows the effect of various doses of T3, added to the incubation medium, on basal as well as TSH- and PGE2-stimulated adenylate cyclase activity (similar results were obtained with T4 (data not shown)). T3 inhibited basal enzyme activity in a doserelated manner, reducing [32P]cAMP formaation by 15% (P < .025) at 4 fig (5 x 10" 5 M), and by 50% at 20 fig (2 x 10" 4 M). Iodide at 10 fig (5 x 10" 4 M) had no effect on adenylate cyclase activity. (In other experiments, I~ was tested at 20 /xg/ml and no effect was observed.) In addition, T3 (20 fig) had no significant effect on adenylate cyclase activity in plasma membrane from other tissues. Plasma membrane ATPase was also unaffected by T3 and T4 (data not shown). As can be seen from Fig. 2, TSH-stimulated adenylate cyclase activity was also
Downloaded from https://academic.oup.com/endo/article-abstract/101/3/858/2618062 by Nagoya University user on 13 January 2019
gave an enrichment by a factor of 4 over the whole homogenate when plasma membrane marker enzymes (5'-nucleotidase, ATPase, and alkaline phosphatase) were assayed (data not shown). TSH increases adenylate cyclase activity in a dose-related manner from 10 to 100 mU/ ml (50% increase over control), after which no further increase is seen. Similar membrane preparations from liver, kidney, lung, and spleen failed to give a TSH response (data not shown).
INHIBITION OF THYROID ADENYLATE CYCLASE
861
pmoles cAMP formed / 1 0 min/mg protein I
2
|
Basal
| | T S H (lOOmU/ml)
§|PGE2(5xl0*4M)
TSH(IOOmU/ml) • T3 lug • T 3 4ug • TjlOug +T320/ig • I" 10/xg
^S^^^
