J. Physiol. (1975), 250, pp. 261-273 With 9 text-figures Printed in Great Britain

261

EFFECT OF ADRENAL HORMONES ON THYROID SECRETION AND THYROID HORMONES ON ADRENAL SECRETION IN THE SHEEP

By IAN R. FALCONER AND F. JACKS* From the Department of Applied Biochemistry and Nutrition, University of Nottingham, School of Agriculture, Sutton Bonnington, Loughborough, Leics LE12 5RD, and the Department of Biochemistry and Nutrition, University of New England, Armidale, N.S.WK 2351, Australia

(Received 9 October 1974) SUMMARY

1. Previous work has shown that after stressful stimuli, sheep initially secrete increased amounts of thyroid hormone, at a time when adrenal secretion is also elevated. 2. This study was designed to evaluate (a) any short-term activation or inhibition of thyroid secretion by exogenous cortisol or ACTH administered in quantities comparable to those secreted after stress in sheep and (b) any short-term effect that exogenous thyroxine or triiodothyronine may have on the concentration of plasma cortisol in the sheep. 3. Thyroid activity was measured by determination of plasma protein bound 125I (PB1251) and total 125I in thyroid vein and mixed venous (jugular) blood. Plasma cortisol and thyroxine concentrations were measured by a competitive protein-binding assay at intervals for up to 5 hr after commencement of the experiment. 4. No evidence of an activation of thyroid secretion was found during cortisol or ACTH infusion, as monitored by thyroid vein PB125I. Similarly there was no evidence of any inhibition of thyroid function, as measured by continued secretion of thyroid hormones into thyroid vein blood. 5. No effect on plasma cortisol concentration due to thyroid hormone treatment was observed. 6. It was concluded that (a) elevated circulating corticosteroids in physiological concentrations have no short-term effects on thyroid activity in the sheep and (b) the short-term alterations in thyroid and adrenal cortical secretion observed during stress in the sheep could not be attributed to direct interaction of elevated thyroid hormone concentrations with adrenal cortical secretion. * Present address: A.R.C. Institute of Animal Physiology, Babraham, Cambridge.

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~I. R. FALCONER AND F. JACKS ThTTRODUCTI0OT

Investigations of the effects of pharmacological doses of ACTH and of adrenal corticosteroids in intact rats have provided evidence of suppression of 131I uptake into, and secretion from, the thyroid (Ingbar & Frienkel, 1956). Other studies have demonstrated changes in the concentration of thyroxine-binding proteins in the blood as a consequence of high dosages of prednisone (Oppenheimer & Werner, 1966), or abnormally high endogenous corticosteroid secretion (Burslem, Abad & Macleod, 1969). The normal physiological relationships between the adrenal and thyroid glands are, however, not clear, apart from recent evidence that there is no competition between the pituitary mechanisms involved in ACTH, growth hormone and TSH release in man (Besser, Ratcliffe, Kilborn, Ormston & Hall, 1971). During stress a reciprocal relationship has been suggested to operate between the thyroid and adrenal cortex, this concept being largely based on experiments with rodents and lagomorphs, in which stressful stimuli caused an inhibition of thyroid secretion (Brown-Grant, Harris & Reichlin, 1954). In sheep, however, exposure to a barking dog caused a stimulation of thyroid secretion (Falconer & Hetzel, 1964), implying that a marked difference in response may occur between species. To further evaluate the shortterm interaction between the secretion of adrenal steroids and short-term secretion of thyroid hormones the experiments described in this paper report (a) the results of infusions of ACTH and cortisol at physiological concentrations (Bassett & Hinks, 1969) on the secretion of thyroid hormones into the thyroid vein of sheep with permanently exteriorized thyroid glands (Falconer, 1963), and (b) the results of infusions of thyroxine and triiodothyronine on the concentration of plasma cortisol in the sheep. METHODS

Corti8ol and ACTH infusion experiments Ten adult ewes were prepared for study by surgical exteriorization of the thyroid gland, using the technique described by Falconer (1963). The result of this operation was that each animal possessed one lobe of the thyroid gland, connected to its original vascular supply, enclosed with the jugular vein and carotid artery in a permanent skin-covered tube projecting from the side of the neck. The opposing thyroid lobe was removed, and the carotid artery alone exteriorized into a second skin-covered tube. At least 1 year elapsed between surgery and experimental use of the animals, to allow full recovery and compensatory hypertrophy of the single remaining lobe of the thyroid to be completed before experimentation. The animals were permanently held indoors, and during the experimentation held in a controlled temperature room at 15-20o C. For the experiments on thyroid secretion described in this paper injections of 50 ,uc 1251 in 1-2 ml. Na~l solution (0-9 g/100 ml.) were given i.m. between 4 and 8

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days before blood sampling. Collection of thyroid vein blood, and hormone administration, were carried out as previously described (Falconer & Hetzel, 1964; Falconer, 1968). Thyroxine and triiodothyronine infusion experiments A group of six young adult ewes without surgical modification were used for this series of experiments. The animals were held permanently indoors, and were held in metabolism crates during experimentation in a controlled temperature room at 15-200 C. Cannulation of the jugular vein was carried out under local anaesthesia (0-5 ml. 2 % Xylocaine, Astra-Hewlett, Watford) and jugular blood samples collected and hormone injection given through this cannula as previously described (Falconer & Hetzel, 1964). Heart rate and arterial blood pressure measurement An electrocardiograph (Cardioline 3-channel, Associated Electrical Industries, London) was used to record heart rate, using two disk electrodes, one on the skin between the scapulae and the other over the lumbar vertebrae. These were connected to the arm and leg leads of the e.c.g. recorder. The recorder was operated at a speed of 2-5 cm/sec and mean heart rates calculated from the time for ten consecutive beats. Arterial blood pressure was measured using a sphygmomanometer cuff (infants size) wrapped round the carotid loop, and the systolic pressure observed from movement of the mercury meniscus of the manometer. A number of more sophisticated techniques were also tried, and discarded, in favour of the minimal disturbance of the animal and consistent results obtained by this simple method.

Analytical methods Total plasma 125I and protein-bound 125I were determined in an automatic welltype scintillation counter (Isotope Developments Ltd, Type 6052), using the precipitation techniques already described (Falconer, 1963). A total of 10,000 counts were recorded for each sample in order to reduce the S.D. of any single measurement to 1 % of the number of counts. Plasma cortisol was assayed by the technique of competitive protein binding, as described by Murphy (1967), using plasma from adult male dogs as a source of cortisol-binding protein. Recovery of cortisol added to plasma was 78-6 + 3-4 % (S.D.) on ten duplicate determinations. Plasma thyroxine was assayed by the similar method also described by Murphy (1965). Recovery of added thyroxine was 96-3+±5-2/ (S.D.) on ten duplicate determinations. The reproducibility within batches of a single cortisol assay was +10% (s.D.), and a single thyroxine assay + 9.6 % (S.D.). A complete series of assays from an experiment were assayed in one batch. Hormones and reagents. Cortisol was purchased from the Sigma Chemical Co. (St Louis, U.S.A.) and ACTH ('Acthar') from Armour Laboratories. Cortisol succinate was purchased from Glaxo (Greenford, Middx.). Reagents were Analytical Reagent grade, and obtained from B.D.H. (Poole). RESULTS

Effect of cortisol on thyroid secretion Six experiments were carried out in order to examine any direct effect of increased blood cortisol on thyroid secretion, or any indirect effect of elevated blood cortisol acting through the pituitary or hypothalamus on

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~I. R. FALCONER AND F. JACKS secretary activity of the thyroid. Elevation of plasma cortisol was done by the rapid i.v. injection of 10 mg cortisol succinate, followed by continuous i.v. infusion of 200 #tg cortisol succinate/min for 3 hr. Blood sampling was done at 20 mmn intervals, for 1 hr before, during and 30 min after cortisol infusion, collecting both thyroid vein and mixed venous blood samples. Anilytical procedure for these experiments was as previously described. Heart rate and blood pressure were also measured during the 5 hr period of the experiment at 20 min intervals. 10

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The results of the plasma cortisol measurements (including four experiments in which PB1251 was not measured) are shown in Fig. 1, demonstrating an approximately fourfold elevation of plasma cortisol concentration during the infusion. Plasma protein bound 125I (PB1251) determinations were carried out on the same thyroid vein blood samples, and are shown in Fig. 2. No increase in concentration of PB'125I was demonstrated during cortisol infusion. To evaluate the possibility of a cessation in thyroid output during cortisol infusion, comparison of mixed venous PB125JI and thyroid vein PB125I concentrations was made. In five of the six experiments PB125I concentrations were higher in the thyroid vein blood (average difference + 0.01 1 +0.0030/ dose/1OO ml. plasma, 0-01 > P > 0-001) than in mixed venous blood, and the remaining experiment showed an equal PB'125I concentration in blood from both sources. Thus in five of the six

THYROID-ADRENAL INTERACTIONS 265 experiments active secretion of 125I-labelled thyroid hormone was taking place during the cortisol infusion, and in one experiment no secretion was observed. Effect of ACTH on thyroid and adrenal secretion Thyroid vein blood samples were collected over a period of 4 hr at 20 min intervals, commencing about 30 min after cannulation of the vein. After collection of four samples, ACTH was infused i.v. at 66'7 m/z./min for 3 hr, and blood sampling continued until the end of infusion. Samples C

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266 I. R. FALCONER AND F. JACKS of mixed venous (jugular) blood were also taken. Heart rate and blood pressure were recorded at 20 min intervals throughout the 4 hr period. A total of eight experiments were carried out, and measurements of total and protein-bound plasma 125I, and plasma cortisol made. Fig. 3 shows the averaged results of the eight individual experiments in which plasma cortisol was measured during ACTH infusion. A uniformly increased concentration of plasma cortisol (7-8 ,ug/100 ml. plasma) was seen from 30 min after commencement of the infusion. The results of measurement of PB1251 in the same plasma samples are shown in Fig. 4, ._2 Cd

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Fig. 4. Effect of ACTH infusion (t-t) on the concentration of protein bound 125I in plasma from thyroid vein blood, calculated and plotted as for Fig. 2, eight expts.

and show that no activation of thyroid hormone release had occurred. Comparison of the concentration of PB125I in mixed venous and thyroid vein blood showed secretion of labelled hormone in seven out of eight experiments during ACTH infusion (average difference + 0018 + 0-004 % dose/100 ml. plasma, 0.01 > P > 0.001).

Effects of cortisol and ACTH infusion on heart rate and systolic blood pressure in sheep These measurements were carried out to provide basic data for sheep held under mildly stressful conditions in metabolism crates, in this case with cannulae inserted into their jugular veins and electrodes for e.c.g. measurements on their backs. Fig. 5 illustrates the mean heart rates and blood pressures of these sheep before and during cortisol and ACTH infusion.

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Effect of thyroxine on plasma cortisol concentration Jugular blood samples were collected through an indwelling cannula at 30 min intervals over a period of 4*5 hr, in a total of twelve experiments. In six of the experiments an i.v. injection of 500 ,tg sodium L-thyroxine was administered through the jugular cannula after collection of the fifth blood sample. Measurements of heart rate were made after each blood sample was collected.

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The results of thyroxine assay on these blood samples are shown in Fig. 6 for control sheep and for sheep given 500 ,tg L-thyroxine intravenously. The initial doubling of plasma thyroxine concentration immediately after injection decreased to approximately 1-6 times the original concentration at 1-5 hr after injection. Measurements of plasma cortisol on these blood samples are shown in Fig. 7. No evidence of an activation or inhibition of the secretion of cortisol was observed following thyroxine injection. In order to assess the possibility of an adrenocortical response to higher and more sustained concentrations of plasma thyroxine, a further six experiments were carried out in which 2-5 mg L-thyroxine were injected i.v. into the same adult ewes. Blood sampling was carried out at 30 min intervals for 5 hr, thyroxine being injected after the collection of four II-2

1. R. FALCONER AND F. JACKS

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269 THYROID-ADRENAL INTERACTIONS samples. Additionally, blood samples were taken at 28 and 53 hr after commencement of the experiment by jugular puncture. The results of these experiments are shown in Fig. 8. The initial elevation of plasma thyroxine concentration (upper graph) was approximately tenfold, and by 2 days after administration was still double the normal concentration of 6-8 ig/I100 ml. The lower graph on Fig. 8 shows the mean 80 E v

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plasma cortisol concentration up to 53 hr after thyroxine injection. It is apparent that the thyroxine treatment did not affect the concentration of circulating cortisol during the period of measurement. No effects on heart rate were detected during the 3 hr following the injection of thyroxine.

Effect of triiodothyronine on plasma cortisol concentration To assess the short-term effects of increased circulating triiodothyronine, intravenous administration of 250 gg sodium L-triiodothyronine was carried out in six sheep. The experimental design was the same as described above for thyroxine administration. The results of plasma cortisol measurement before and up to 53 hr after commencement of blood sampling

I. R. FALCONER AND F. JACKS 270 are shown in Fig. 9. No significant changes in plasma cortisol were demonstrated. No changes in heart rate were detected in the 3 hr after triiodothyronine injection. 4

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mean). DISCUSSION

To evaluate the relationship between the thyroid and adrenal glands in response to stress in the sheep it is necessary to isolate experimentally the effects that adrenal steroids and ACTH may have on the activity of the thyroid as well as the effects thyroid hormones have on adrenal secretion. Earlier investigations by Reid & Mills (1962) of the effect of various minor stresses to the sheep showed that cortisol secretion was rapidly stimulated. Initial mean concentrations of less than 1X2 zg/1100 ml. plasma in the undisturbed sheep were shown to rise to 2-7 4ug/100 ml. in sheep transported in a Land Rover for 2 hr. Similar changes were also reported by Bassett & Hinks (1969) following venepuncture in sheep. This rapid elevation of plasma cortisol as a result of stress in the sheep may influence the activity of the thyroid gland, either through a direct effect on the thyroid or through an influence exerted on the hypothalamus/ pituitary system, modulating the secretion of thyroid stimulating hormone

(TSH). In the experiments described in this paper, concentrations of cortisol were infused (Fig. 1) that simulated as closely as possible those observed experimentally during stress to the sheep (Reid, 1962; Reid & Mills, 1962; Bassett & Hinks, 1969). It was necessary to examine both the possibility of thyroid activation by cortisol, and of thyroid inhibition, since data

THYROID-ADRENAL INTERACTIONS 271 demonstrating both consequences of stress have been recorded (Falconer & Hetzel, 1964; Brow-n-Grant et al. 1954). The collection of thyroid vein blood from the undisturbed conscious sheep provides a rapid and sensitive method of assessing changes in thyroid activity. Sequential measurements of plasma PB1251 concentration demonstrate changes in rate of thyroid secretion with time, provided that blood flow through the gland and metabolic clearance rate of thyroxine do not vary. Comparison of mixed venous plasma PB125I with thyroid-vein plasma PB1251 supplies an additional indication of thyroid secretion. Figs. 2 and 4 show that a slight decrease in PB1251 concentration has occurred during the first 2 hr of the experiment in which cortisol or ACTH were infused. This has been described previously (Falconer, 1968), and is due to the return from a stress-induced increase in thyroid secretion following cannulation. No evidence of any activation of thyroid secretion has been observed during the cortisol infusion, and measurement of thyroid vein as compared with mixed venous plasma PB125I demonstrated a continued secretion of labelled thyroid hormone into thyroid vein blood, therefore providing no evidence for an inhibition of secretion by cortisol. Under normal conditions the elevation of blood cortisol concentration following stress in the sheep would be a result of endogenous ACTH secretion, which itself could exert an effect on thyroid function. To examine this possibility a series of ACTH infusions were carried out, which elevated blood cortisol to similar concentrations to those reported during stress (Fig. 3). As before, no evidence of secretary changes (Fig. 4) by the thyroid were observed during infusion. It can be concluded from these experiments that neither cortisol nor ACTH, administered in physiological quantities to sheep, modify the activity of the thyroid gland in a stimulatory or inhibitory manner. The observed activation of the thyroid by stressful stimuli in the sheep (Falconer & Hetzel, 1964; Falconer & Jacks, 1972) must therefore be independent of the simultaneous activation of the adrenal cortex. The experiments in which thyroxine or triiodothyronine were administered to sheep, and plasma cortisol measured, show no evidence of shortterm changes in adrenal cortical secretion. Even the unphysiologically high thyroxine concentrations resulting from i.v. injection of 2-5 mg sodium-L-thyroxine showed no response of plasma cortisol concentration. Because of the possibility that triiodothyronine might have a more rapid effect than thyroxine, due to its weaker binding to plasma proteins and its higher fractional disappearance rate (McConnon, Row & Volpe, 1971), triiodothyronine was also administered. The dose given i.v. (250 jig) was calculated on the basis of 5 times the daily production rate of a normal man of equivalent weight, since no data for sheep are available. The

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absence of any response in plasma cortisol concentration to triiodothyronine injection would indicate that neither the secretion of ACTH nor the activity of the adrenal cortex in response to the normal endogenous secretion of ACTH are affected (in the time interval observed) by elevated plasma triiodothyronine concentration. It can therefore be concluded that no short-term interaction between elevated concentrations of thyroid or adrenal cortical hormones occurs in the sheep. The short-term changes in plasma concentrations of thyroid and adrenal hormones, observed after stressful stimuli, may thus be attributed to higher levels of control than that of elevated circulating thyroid hormone affecting ACTH secretion, or elevated circulating cortisol affecting TSH secretion. The generous support of the Wellcome Trust is gratefully acknowledged.

REFERENCES

BASSETT, J. M. & Hnucs, H. T. (1969). Microdetermination of corticosteroids in ovine periferal plasma: effects of venipunctuire, corticotrophin, insulin and glucose. J. Endocr. 44, 387-403. BESSER, G. M., RATCLIFFE, J. G., KILBORN, J. R., ORMSTON, B. J. & HALL, R. (1971). Interaction between thyrotrophin corticotrophin and growth hormone secretion in man. J. Endoer. 51, 699-706. BRoww-GRANT, K., HARRis, G. W. & REICHLiN, S. (1954). The effect of emotional and physical stress on thyroid activity in the rabbit. J. Phy8iol. 126, 29-40. BURSLEM, W. A., ABAD, A. & MACLEOD, R. M. (1969). Influence of adrenal steroids on thyroid function and serum free thyroxine in tumour bearing rats. Proc. Soc. exp. Biol. Med. 130, 941-944. FALCONER, I. R. (1963). The exteriorization of the thyroid gland and measurement of its function. J. Endocr. 26, 241-247. FALCONER, I. R. (1968). The effect of vasopressin on hormone secretion and blood flow from the thyroid vein in sheep with exteriorized thyroids. J. Physriol. 199, 427-441. FALCONER, I. R. & HETZEL, B. S. (1964). Effect of emotional stress and TSH on thyroid vein hormone level in sheep with exteriorized thyroids. Endocrinology 75, 42-48. FALCONER, I. R. & JACKS, F. (1972). Response of the thyroid and adrenal glands of sheep to cold and fear. J. Physiol. 222, 86-87 P. INGBAR, S. H. & FRIENKEL, N. (1956). ACTH, cortisone and the metabolism of iodine. Metabolism 5, 652. McCCONON, J., Row, V. V. & VOLPA, R. (1971). Simultaneous comparative studies of thyroxine (T4) and triiodothyronine (T.) production rates in health and disease. In Further Advance8 in Thyroid Research, ed. FELLINGER, K. & HopERa, R. Vienna: Verlag der Wiener Medizinischen Akademie. MURPHY, B. E. P. (1967). Some studies of the protein binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein binding radioassay. J. dlin. Endocr. Metab. 27, 973-990.

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MURPHY, B. P. (1965). The determination of thyroxine by competitive proteinbinding analysis employing an anion-exchange resin and radiothyroxine. J. Lab. clin. Med. 66, 161-167. OPPENHEIMER, J. H. & WERNER, S. C. (1966). Effect of prednisone on thyroxinebinding proteins. J. clin. Endocr. 26, 715-721. REID, R. L. (1962). Studies on the carbohydrate metabolism of sheep. XV. The adrenal response to the climatic stresses of cold, wind and rain. Au8t. J. agric. Res. 13, 296-306. REID, R. L. & MILLS, S. C. (1962). Studies on the carbohydrate metabolism of sheep. XIV. The adrenal response to psychological stress. Aust. J. agric. Res. 13, 282-295.

Effect of adrenal hormones on thyroid secretion and thyroid hormones on adrenal secretion in the sheep.

J. Physiol. (1975), 250, pp. 261-273 With 9 text-figures Printed in Great Britain 261 EFFECT OF ADRENAL HORMONES ON THYROID SECRETION AND THYROID HO...
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