Effects of Adrenergic Blocking Agents on Basal

and Stimulated Thyroid Function

By GERALDBURKE

Thyrotropin (TSH) effects on thyroid adenyl cyclase inactivation for the in- are believed mediated via activation of hibitory effect of these drugs on stim- adenyl cyclase. Since adrenergic block- ulated thyroid glucose oxidation. PPL ing agents inhibit thyroid adenyl cyclase, and PH, 10-4 M, stimulated basal phos- we studied their effects on glucose oxida- pholipogenesis. Submaximal concentra- tion and phospholipogenesis in sheep tions of these drugs augmented maximal thyroid slices. Propranolol (PPL) and effective concentrations of TSH or di- phentolamine (PH), 10‘4 M, abolished butyryl cyclic-AMP in s3P incorporation TSH- and dibutyryl cyclic-AMP-induced studies indicating different mechanisms of increases in glucose oxidation but lesser thyroid stimulation. These data also indi- [physiologicI concentrations of PPL and cate that phospholipogenesis in thyroid PH, i.e., 10.” M, were without effect. is unrelated to adenyl cyclase activation

Basal glucose oxidation was not appre- or endocytosis. This study suggests due

ciably altered by these drugs. Both PPL caution in the use of adrenergic block-

and PH (5 X 10-J M) abolished TSH- ing agents to study the role of adenyl

and dibutyryl cyclic-AMP-induced endo- cyclase in thyroid stimulation. (Me-

cytosis within the thyroid cell. These tabolism 18: No. 11, November, 96 l-967,

data suggest a mechanism other than 1969)

R ECENT STUDIEF suggest that hormonal stimulation of the thyroid is mediated via activation of adenyl cyclase with resultant production of

cyclic-AMP [C-AMP]. Levey et al.” recently reported that pharmacologic doses of adrenergic blocking drugs inhibit thyroid adenyl cyclase and suggested that USC of such drugs would facilitate investigation of the role of cyclic nucleotide in thyroid function. We wish to report the effects of alpha and beta adrenergic blocking drugs on basal and stimulated thyroid function in order to point out the non-specificity and ambiguity associated with their use in the study of the adenyl cyclase-C-AMP system in thyroid.

MATERIALS AND METHODS

Bovine TSH (2 USP U/mg.) was a gift from the Endocrinology Study Section, NIH. N,-2’-0-dibutyryl C-AMP (DBC) was purchased from Schwarz BioResearch Inc.. Orangeburg, N.Y. DL-propranolol (DL-PPL) and D-propranolol (D-PPL) were gifts from Ayerst Laboratories Inc., New York, and phentolamine (PH) was donated by Winthrop Laboratories, New York.

Sheep thyroid slices were prepared and incubated as previously described.i3s A 2-hr.

From rhe Radioisotope Laboratory, Departmerlt of Medicine, Michael Reese Hospital trnd Medicul Center. Chicago, Ill.

Supported by USPHS Grant AM 11136 from the National Institute of Arthritis rlrrd Metrrholic Diseases.

Received for publication April 30, 1969. GERALD BURKE, M.D., CM.: Director, Radioisotope Zxboratory. Michael Reese Ho.ypital

and Medic.al Center, Chicago, 111.

METABOLISM, VOL. 18, No. 11 (NOVEMBER), 1969 961

962 GERALD BURKE

Table 1.Effects of Adrenergic Blocking Agents on Basal Glucose Oxidation and Phospholipogenesis in Sheep Thyroid Slices

Substance

Metabolic Pathway - Glucose-1-T oxidation s2P incorporation

(cpm/mg./2 hr.*) (cpm/mg./2 hr.‘)

KRB 223 f 28 PPL, 10-s M 201 -c 17 PPL, lo-5M 198 f 21 PPL, lo-4M 223 & 16 PPL, 5 X IO-4 M 234 I!I 19 PPL, 7.5 X 10-4 M 260 -+ 41

PH, 10-s M 203 2 19 PH, lo-5M 220 + 17 PH, 10-4 M 238 2 21 PH, 5 X 10-4 M 215 -r- 18 PH, 7.5 X 10-4 M 215 f 23

* Results are mean + SEM of triplicate determinations. t Significantly greater than KRB control (p < 0.01).

606 2 26 581 -c- 22 617 L 28 831 % 25 t

1350+41 t 1312 + 40 i

641 2 23 623 f 26 869 * 29 t

1200 zb 41 i 1306242:

_

incubation period was used in both glucose-l-rQZ oxidation and ssP incorporation into phospholipid studies with TSH and DBC. Phospholipids were isolated according to the procedure described by Kogl and Van Deenen.9

Substances to be tested were dissolved in Krebs Ringer bicarbonate (KRB) buffer, pH 7.4. TSH was used in a concentration of 500 mu/ml. in glucose oxidation studies and at 50-100 mu/ml. in ssP incorporation experiments. DBC was used in concentrations of 100-500 pg/ml. Adrenergic blocking agents were tested at concentrations ranging from 1 x 10-a M to 7.5 x lo-4 M.

Results were expressed as counts of 14COs or ssP/min./mg. of tissue (wet weight)/120 min. of incubation.

For statistical analysis of in vitro studies, paired t-tests were performed on the dif- ferences in the logarithms of the absolute values obtained in the absence and in the presence of thyroid stimulator, cyclic nucleotide or blocking agent. This is equivalent to testing whether the average ratio of values [e.g., +TSH/-TSH] is greater than unity.le The data (absolute values) obtained in these experiments were also evaluated by a l-factorial, l-nested analysis of variance.11

Slices, 0.5 mm. thick, were used in metabolic studies as previously detailed.i.8 TO study effects of TSH or DBC on endocytosis in the same experiment, slices 1.0 to 2.0 mm. thick were used, since thin slices showed numerous disrupted follicles. Tissue was fixed for 18 hours at 4” C in Bouin’s solution, embedded in paratlin and stained by the periodic acid-Schiff procedure with hematoxylin counterstain.

RESULTS

Eflects of PPL* and PH on Basal Glucose Oxidation and Phospholipogenesis in Sheep Thyroid Slices

Adrenergic blocking agents at 10.” to 7.5 X 10e4M had a variable and sta- tistically insignificant effect, overall, on basal glucose oxidation. In a total of 18 experiments, glucose oxidation was unaffected in 9, reduced in 6 and augmented in 3. There was no difference in the respective effects of PPL or PH in these experiments. Responsivity of tissue was validated in each experiment by con- sistent TSH stimulation of glucose oxidation.

_ .~__~_ *Unless otherwise specified, PPL will refer to the racemic mixture of this drug.

ADRENERGIC BLOCKING AGENTS 963

Table 2.-Time Course of Phospholipogenesis Induced by Adrenergic Blocking Agents in Sheep Thyroid Slices

Incubation Period SP Incorporation (cpm/mg./2 hr.*) ___--.

KRB PPL. 5 x 10.‘M PH. 5 x lo-’ ti ._~. _._ I5 min. 38k 4 88? 5t 79 t 5 .:

30 min. 88& 6 212 t 17 i 194? 16 f

60 min. 226 * 15 533 f 29 i 490 ?I 27 :

120 min. 588 rt 31 1358 c 52 : 1329 z 44 i-

+ Results are mean k SEM of triplicate determinations. -! Significantly greater than control (p <: 0.01).

Table 3.-Effects of Adrenergic Blocking Agents on Stimulated Glucose Oxidation in Sheep Thyroid Slices

Thyroid Stimulator

Adrenergic Blocking

Agent ~~ ~~~~__

Glucose-l-W Oxidation

(cpm/mg./l, hr.*) p vs. Corresponding Control

-- - 359 k 29 -

-- PPL, 10-4 M 371 k 23 -

-- PH, 10-J M 317226 -

TSH, 500 rnU/ml - 562 -r- 34 .:- -

TSH, 500 mlJ/ml PPL, 10-4 M 403 k 28 < 0.01

TSH. 500 mU/mZ PH, lo-” M 3X2 2 29 < 0.01

DBC. 500 /~g/ml - 486 -+ 25 + -

DBC. 500 pg/ml PPL, 10-4 M 365 k 33 < 0.02 DBC, 500 /~g/rnI PH, 10-4 M 384 -r- 27 < 0.02

__~ * Results are mean -c- SEM of triplicate determinations. i Significantly greater than KRB control (p < 0.01 ).

When slices from the same gland as used in a glucose-l-*%! experiment were incubated with adrenergic blockers and “2P-orthophosphate, consistently signif- icant stimulation of phospholipogenesis was observed. Alpha and beta blockers were of comparable potency in this regard. The results of representative ex- periments with glucose+“C and ““P-orthophosphate are detailed in Table I. Thyroid slices incubated continously at 37” C with 3zP and PPL or PH at 10.*M for periods ranging from 1.5 minutes to 2 hours consistently showed augmented phospholipogenesis at every time period under study. A representa- tive experiment (of a total of 4) is detailed in Table 2. Although basal phos- pholipogenesis increased over time? the per cent increment seen with PPL or PH remained fairly constant.

Eflects of PPL and PH on Stimulated Glucose Oxidation

In a series of 9 experiments, PPL and PH at 1 to 5 YI lo4 M consistently abolished the increase in glucose oxidation induced by TSH and DBC whether or not basal glucose oxidation was reduced. A representative study is detailed in Table 3. A similar effect was seen when the slices were preincubated for 30 minutes with adrenergic blocking agent at 37” C, washed and then incubated with glucose-l-14C and TSH or DBC in the absence of a blocking drug. PPL and PH at lo-” M and 10m5 M were without effect on stimulated glucose oxidation.

964 GERALD BURKE

Table 4.-Combined Effects of PPL and TSH on Pbospholipogenesis in Sheep Thyroid Slices

Per cent of Control (KRB; Substance

“P Incorporation cpm/mg./Z hr.*)

KRB 732 k 34 - PPL, 5 X 10-4 M 2143 +- 86 293 PPL, 1 X 10-a M 1180&45 161 TSH, 50 mu/ml. 1317 * 51 180 TSH, 250 mu/ml. 1296 k 42 177 PPL, 5 X 10-A M

+ 2143 rt 94 293 TSH, 50 mu/ml. PPL, 1 X 10-4 M

+ 1726 I!I 63 236 TSH, 50 mu/ml.

* Results are mean 2 SEM of triplicate determinations.

Fig. 1 .-Prevention of TSH-induced colloid droplet formation by PPL. Thyroid slices were preincubated with Krebs buffer (left) or PPL, 5 X 1O-1 M (right) for 30 minutes as 37” C, washed and then incubated with TSH, 500 mU./ml., and buffer for 2 hours at 37” C. Tissue was fixed for 18 hours at 4” C in Bouin’s solution, embedded in paraffin and stained by the periodic acid-Schiff procedure with hema- toxylin counterstain (magnification X 375).

Combined Eflects of Adrenergic Blocking Agents and Thyroid Stimulators on Phospholipogenesis

In a series of 8 experiments, thyroid slices were incubated for 30 minutes at 37’ C with 32P-orthophosphate and then incubated for an additional 2 hours with adrenergic blocking agent and maximal doses of thyroid stimulator or cyclic

ADRENERGIC BLOCKING AGENTS 065

Table K-Effects of Adrenergic Blocking Agents on Glucose Oxidation and Endocytosis in Sheep Thyroid Slices”

Substance

KRB TSH, 500 mu/ml. DBC, 500 ag./ml. PPL. 5 X lo-1 M PH, 5 X IO--i M TSH. 500 mU/ml.

in PPL, 5 X 10-a M TSH. 500 mu/ml.

1~ PH, 5 X IO-4 M DBC. 500 Gg./ml.

-1 PPL, 5 X lo--+ M DBC, 500 Pg./ml.

-!

PH. 5 X IO--1 M

Glucose-12°C Oxidation Number of Intracellular (cpm/w./2 hr.t) Colloid Droplets/25 follicles t

316t21 <5 547 -t 26 0 742 429 -+ 23 § 659 322 ZII 17 <F 310 -+- 16 <s

337 -r- 22 0

309 ?I 14 <5

299 k 22 <5

307 k 16 <-’

* Effecls of TSH, DBC, etc., on endocytosis were studied concomitantly with studies of their effects on glucose oxidation in slices from the same gland as described in text.

i- Mean k SEM of triplicate determinations. 1: Mean of duplicate determinations. I Significantly greater than control (p <: 0.02 to p < 0.01 ).

nucleotide. When maximal stimulatory concentrations of adrenergic blocking drugs and of TSH or DBC were combined, no additive effect was seen. When maximal doses of TSH or DBC were added to submaximal concentrations of adrenergic blockers, consistent and statistically significant additive effects were observed. A representative experiment with TSH and PPL is detailed in Table 4.

Eflects of Adrenergic Blocking Agents on Endocytosis

Onaya et al.]? recently reported that PPL prevents colloid droplet formation induced by TSH and DBC in dog thyroid and suggested that PPL may be a lysosomc stabilizer in addition to any effect on adenyl cyclase. We have con- firmed these findings with both PPL and PH (5 >I 10~” M) in sheep thyroid slices. The inhibitory effects of PPL and PH on TSH- and DBC-induced endo- cytosis are detailed in Table 5 and illustrated in Fig. 1. Both these drugs abolished TSH- and DBC-stimulated glucose-I-‘“C oxidation in slices from the same gland in the same 2-hour incubation.

Dichlorisoproterenol (purchased from Aldrich Chemical Co., Milwaukee, Wise. ), another beta adrenergic blocking drug and D-propranolol, which has I / 100th the beta blocking activity of DL-propranololl:! had the same effects on glucose oxidation and phospholipogenesis as the other drugs tested.

DISCUSSION

The concentrations of adrenergic blocking agents required for inhibition of stimulated glucose oxidation or stimulation of basal phospholipogenesis in

966 GERALD BURKE

this study are approximately 100 times greater than those which produce alpha

or beta adrenergic blockade,l”,‘” indicating that these substances are not acting

as alpha or beta blocking agents but by another mechanism. That D-propranolol

had the same effects as the racemic mixture despite marked differences in their

beta blocking activity reinforces this premise.

The finding that adrenergic blocking agents abolished stimulation of glucose

oxidation induced by TSH and DBC (in contradistinction to the findings of

Levey et al.” wherein DBC stimulation was unaffected) and the demonstrated

inhibition of TSH- and DBC-induced colloid droplet formation by PPL and PH,

suggest that a mechanism other than inactivation of adenyl cyclase” may under-

lie the inhibitory effects of these drugs on stimulated glucose oxidation. Given

the pharmacologic doses of adrenergic blockers necessary to obtain these in-

hibitory effects, one must also consider the possibility that the doses used arc

blocking C-AMP effects as well as adenyl cyclase as has been suggested by

studies with ACTH and norepinephrine on lipolysis”’ and with epinephrinc-in-

duced hyperglycemia.”

The consistent stimulation of thyroidal phospholipid synthesis by adrenergic

blocking agents during continuous incubations is in direct contrast to the mar-

ginal effect of these drugs on glucose oxidation. Altman et al.lh previously pre-

sented data indicating that these two effects are not interrelated, and recent

findings in our laboratory* confirm these conclusions. Although the present studies (Table 4) indicate differing mechanisms for stimulation of thyroidal

phospholipogenesis by TSH and DBC on the one hand, and adrenergic block-

ing agents on the other, the precis e mechanism underlying the stimulatory ef-

fects of these drugs on phospholipid synthesis must remain speculative. Levey

et al.” also noted in their study that PPL and PH stimulated phospholipid

synthesis. These data and the demonstrated additive effects of TSH or DBC

and adrenergic blocking agents on :IL’P incorporation into phospholipids in the

present study indicate that inhibition of adenyl cyclase or of colloid droplet

formation does not prevent hormonal stimulation of phospholipid synthesis in

thyroid. These studies suggest that phospholipogenesis in thyroid is quite un-

related to adenyl cyclase activation or to endocytosis.

The effects of adrenergic blocking drugs on thyroid are clearly complex.

The data reported herein suggest that use of these drugs to study the role of

the adenyl cyclase-C-AMP system in the mediation of thyroid stimulation must

be viewed more critically than has been the case heretofore.

ACKNOWLEDGMENTS

The author gratefully acknowledges the technical assistance of Mrs. Vera Kertes

and Mrs. Adrienne Remer. Miss Irene Goldberger. Department of Pathology. prepared

the histologic sections.

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*We have recently demonstrated that PPL and PH, 10-4 M. inhibit TSH activation of

adenyl cyclase in sheep thyroid homogenates thus confirming the findings of Levey et al. in canine and bovine thyroid.

ADRENERGIC BLOCKING AGENTS 967

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