- Email: [email protected]
Peculiarities of neural regulation of the thyroid, adrenocortical and testicular functions in old age
Mechanisms of Ageing and Development, 51 (I 990) 89--99
89
Elsevier Scientific Publishers Ireland Ltd.
PECULIARITIES OF NEURAL REGULATION OF THE THYROID, ADRENOCORTICAL AND TESTICULAR FUNCTIONS IN OLD AGE
VLADIMIR V. FROLKIS, EVGENI N. GORBAN and EVGENIJA V. MOROZ Expermental Department, Institute of Gerontology, VyshgorodskayaSt. 67, AMS USSR, 252655 Kiev-ll4 (U.S.S.R.) (Received April 21st, 1989)
SUMMARY
The age changes of neural control over the function of the thyroid, adrenal cortex and testicles were examined in adult (6 months) and old (28 months) male rats. In old age, there was a weakening of adrenergic control of thyroidogenesis, alphaadrenergic and M-cholinergic control of glucocorticoid function of the adrenal cortex and reduction of adrenergic and M-cholinergic influences in the regulation of steroidogenic function of the testicles.
Key words: Aging; Neural regulation; Hormones; Thyroid; Adrenal cortex; Testicles INTRODUCTION
Through their regulatory influences the nervous and endocrine systems provide for the organism's adaptation to extrinsic and intrinsic changes. The interrelationships between these two systems in the provision of adaptive-regulatory mechanisms are multifactoral. During aging these interrelationships alter significantly, decreasing the contribution of neural regulatory influences and increasing the role of humoral factors [ 1]. The neural control of the endocrine function is realized through two main channels, i.e., regulatory influences of the superior brain areas on the "hypothalamushypophysis-peripheral endocrine glands" system [2--6], and direct neurotrophic influences on the endocrine glands cell function [7--15]. At present there is extensive material on the significant role of changes in the hypothalamic-hypophyseal regulatory influences on endocrine glands regarding the mechanisms of aging of the whole organism [16--21]. However, the neural control of the endocrine function requires further study. The purpose of the present investigation was to study age changes in the neural control of the thyroid, adrenal cortex and testicles under conditions of selective blockage of its various links. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
90
MATERIALS AND METHODS
Materials Chemicals used in the experiments included thyrotropin (TSH), corticotropin (ACTH; Plant for Endocrine Preparations, Kaunas, U.S.S.R.), reserpin, atropin sulphate, anaprylin (propranolol; U.S.S.R.), dibenamin hydrochloride (N,N-dibenzyl-beta-chloroethyl-amine HCI; ICN Pharmaceutical Inc., U.S.A.). Medium 199 manufactured by the Institute of Polymyelitis and Viral Encephalitis (U.S.S.R.) was used for the incubation of isolated endocrine glands. Experiments were performed on adult (6 months) and old (28 months) male Wistar rats maintained on a standard diet and supplied by the Stolbovaja Laboratory for Experimental Animals of the U.S.S.R. Academy of Medical Sciences.
Administration of drugs The role of different links of adrenergic and cholinergic components of neural regulation was studied following administration (two times/day for 3 days) of reserpin (1.5 mg/kg) to empty the catecholamine depot in tissues; dibenamin (10 mg/kg) or anaprylin (10.0 mg/kg) to selectively switch off alpha- or beta-adrenergic components of neural regulation; atropin (10 mg/kg) to block M-cholinoreactive structures. The same doses of saline were administered to control animals. The next day following last administration the animals were killed by thoracotomy. Blood was collected to obtain serum. Blood serum samples were kept at - 30°C prior to estimation of hormone concentration.
Preparation of glands The thyroid and adrenals were removed and prepared. Each lobe of the thyroid was dried by filtering paper, weighed, dissected longitudinally along 3/4 of its length, fixed on a plastic bedding and placed in a plastic tube containing 1.0 ml of incubation medium. Each of the two adrenals were dissected in two parts, both parts of one adrenal were fixed on a plastic bedding and placed in plastic tube containing 1.0 ml of incubation medium.
Incubation of glands Isolated thyroid and adrenals were incubated at 37 °C for 4 h. The incubation medium was aerated by carbogen (5070 CO 2 and 95070 02). One lobe of each thyroid was incubated in medium 199, while another one - - in medium 199, containing TSH (100 munits/ml). After incubation the medium was transferred to glass ampules, welded and kept at - 30°C prior to radioimmunological estimation of the thyroid hormones (TH). Each of the two adrenals of each animal were incubated separately. Incubation medium was changed each hour. A particular feature of incubation was that one of the adrenal pair was used as control and was incubated in medium 199 for 4 h, while
91 another one was incubated during the first hour in medium 199, during the second hour - - in medium 199, containing ACTH (10.0 munits/ml), and during the third and fourth hours - - in medium 199. Each portion of incubation medium was transferred to glass ampules, welded and kept at - 3 0 ° C prior to fluorimetric determination of 11-oxyketosteroids (11-OKS). Estimation o f hormones Blood ACTH, TSH, 11-OKS, thyroxine (T4), triiodothyronine (T3) and testosterone (T), as well as T 4, T 3 and 11-OKS in incubation medium were determined in isolated thyroid and adrenals. ACTH, TSH, T 4, T 3 and T were determined radioimmunologically. ACTH and T were determined using "ACTHK-PR" and "Testok" sets (Oris Industrie, France) and expressed in ng/l and nmol/l, respectively; TSH -- "RIA-mat TSH" (Mallinkrodt Diagnostica, F.R.G.) and expressed in mcI.U./l; T 4 and T 3 --"rio-T4-PG" and "rio-T3-PG" (Institute of Bioorganic Chemistry of Academy of Sciences of the Byelorussian SSR, U.S.S.R.) and expressed as nmol/1 (in the blood), and as pmol/ mg of thyroid tissue for a 4-h incubation (in the incubation medium). 11-OKS was determined fluorimetrically according to the corticosterone standard (Serva, U.S.A.). Blood 11-OKS was expressed as nmol/l. Hourly production of 11OKS by the isolated adrenals was expressed as pmol/mg adrenal tissue/h, while gross production of 11-OKS for a 3-h incubation (second, third and fourth hours) -as pmol/mg adrenal tissue. (Hormone production during the first hour was not taken into account in the estimation of gross 11-OKS production by the isolated adrenals). RESULTS Blood hormones and hormonogenesis of isolated thyroid and adrenals of intact animals The pattern of changes of blood hormones during ageing was different: ACTH increased, TSH and 11-OKS remained unchanged, TH and T decreased (Fig. 1). Basal (non-stimulated) TH production by the isolated thyroid expressed per unit of gland mass decreased significantly with age (Fig. 2). Age differences in TH production by the isolated thyroid retain against the background of thyroidogenesis stimulation by adding TSH to the incubation medium, while the increase of basal thyroidogenesis was statistically significant in the adult thyroid only (Fig. 2). Basal and ACTH-stimulated production of l l-OKS by the isolated adrenals remained unchanged with age (Fig. 3). Blood TSH, T4 and T3 and thyroidogenesis o f isolated thyroid at weakening of adrenergic influences Reserpin-induced emptying of the catecholamine depots in tissues produced an
92
~x~h
December
TSH
!, n:!," !, !, 0
R
100
A m
:] lBi i C
R
K
•
C
~"
[J
C
a-
/'3
A~'~H
11 .(IKS
:t 0
0
R
•
Fig. 1. Blood concentration of T S H ( m c l U / l ) , T 4 ( n m o l / l ) , T 3 ( n m o l / l ) , A C T H ( n g / l ) , 1 1 - O K S ( n m o l / I ) and testosterone (nmol/l) in adult and old male rats at weakening of adrenergic or cholinergic influences. White columns - - adult rats; black columns - - old rats; C - - c o n t r o l ; R - - r e s e r p i n ; A - - a t r o p i n ; a - alpha-adrenoblocker dibenamin; /? - - beta-adrenoblocker anaprylin; *Significant differences with control.
insignificant effect on blood TSH in all age groups (Fig. 1), significantly decreased blood T 4 in adult animals only, but had no effect o n T 3. Basal thyroidogenesis of isolated reserpin-treated thyroids and their response to TSH in vitro declined sharply in adult animals only compared to control (Fig. 2). Selective blockade of alpha- and beta-adrenergic structures produced no significant effect on blood TSH in both groups (Fig. 1), but similar to reserpinization, it sharply inhibited the thyroidogenesis in adult rats only. Blood T 4 concentration
93 December
gash aeropiu
T&
B
,i i,i
S
B
S
B
S
B
S
B
S
B
Contx'ol
S
B
S
AlphaB~aad_~-w~oblooke~ ad.~emoblookEe
,I i II ii ,I B S
B
S
B S
B
S
B
S
Croat:col T:
Control
Reserpi~
~ .50'
At ropi~
adrenoblocke~
':1II,I i,I ilii 11,01,0i B
S
B
S
B S
B
S
B$
B
B
S
S
B S
B
S
B S
a~l~enoblockez e
B S
II B
S
Fig. 2. T 4 and T 3 production by the isolated thyroid of adult and old male rats at weakening of adrenergic or cholinergic influences (pmol/mg thyroid tissue/4 h). White columns - - adult rats; black columns - old rats; B - - basal thyroidogenesis; S - - stimulated thyroidogenesis (100 munits TSH/ml); *Significant differences with control.
Rese..--,pJ.n
ga~.t:z,o l
.,7,00• 200" loo.
•
.ll
B
S
o B
S
B
S
B
S
Alpha-
~
DORa B B S
B S
B
S
B
S
B
S
I
S
Beta-
B
S
B
S
B
~-=
Fig. 3.1 I-OKS production by the isolated adrenals of adult and old male rats for a 3-h (2, 3 and 4 h) incubation at weakening of adrenergic or cholinergic influences (pmol/mg adrenal tissue). White columns - adult rats; black columns - - old rats; B - - basal steroidogenesis; S - - stimulated steroidogenesis (10 munits ACTH/ml); *Significant differences with control.
94 (Fig. 1) and basal and TSH-stimulated thyroidogenesis of isolated thyroids (Fig. 2) decreased significantly in adult rats only, as compared with controls.
Blood TSH, T~, T3 and thyroidogenesis of isolated thyroid at weakening of M-cholinergic influences Atropin had no effect on blood TSH and T H in any age group (Fig. 1). Thyroidogenesis of isolated atropin-treated thyroid increased sharply. There was a significant increase o f basal production o f T H by the isolated thyroids in both age groups, intensification o f TSH-stimulated production of T 4 by adult and old thyroids, of T 3 - - by the isolated thyroid of old animals, as well as marked trend towards rise of TSH-stimulated T 3 production by the isolated thyroids of adult vs. control animals (Fig. 2).
Blood ACTH, ll-OKS and steroidogenesis of isolated adrenals at weakening of adrenergic influences Blood A C T H and l l - O K S concentrations o f reserpin-treated animals of both ages were similar to those of controls (Fig. 1). The basal steroidogenesis of isolated adrenals of these animals and their response to A C T H were sharply inhibited in old rats only (Fig. 3). Blood A C T H concentration of adrenoblocker-treated animals of both ages differed insignificantly compared to control (Fig. 1). Blood l l-OKS (Fig. 1) and ACTH-stimulated steroidogenesis of isolated adrenals of adult rats treated with alpha-adrenoblocker dibenamin were significantly increased compared to control, while dibenamin-treated old rats showed a tendency towards the decrease of blood 11-OKS, as well as significant decrease of basal and ACTH-stimulated steroidogenesis of isolated adrenals compared to control (Fig. 3). Selective blockade of betaadrenoblockers had no effect on 1 I-OKS production in both age groups.
Blood A CTH, l l-OKS and steroidogenesis of isolated adrenals at weakening of M-cholinergic influences Chronic atropinization resulted in the increased blood A C T H and 1 I-OKS of adult rats, while in old rats these hormones remained unchanged (Fig. 1). Basal and ACTH-stimulated steroidogenesis o f isolated adrenals increased in adult atropin-treated animals, while in old rats it remained unchanged as compared with control (Fig. 3).
Blood Tat weakening of adrenergic of M-cholinergic influences Significant age differences were revealed in the role of tonic adrenergic impulsation in the provision of steroidogenic function of testicles. Emptying of tissue catecholamine depot by chronic reserpin administration resulted in marked decrease of blood T concentration in adult animals, while in old animals the T content remained unchanged (Fig. 1).
95 Essential age differences were registered in the contribution of M-cholinergic regulatory influences in the provision of steroidogenic function of testicles. Chronic administration of atropin significantly inhibited the T production in adult animals
only (Fig. I). DISCUSSION The data obtained showed the age-related weakening of different components of neurotrophic control of the thyroid, adrenocortical and testicular functions. The results of experiments with switching-off various links of adrenergic provision of thyroidogenesis (reserpin, alpha- and beta-blockers) enable to conclude that in adult rats both components of adrenergic neural influences contribute significantly to the maintenance of normal viability and to the modulation of thyroid reactivity, while during aging the adrenergic control of thyroid function weakens. Reserpin administration was shown to decrease T 4 in the peripheral blood, while T 3 remained unchanged compared to control (Fig. 1). These data closely correlate with unaltered T 3 and decreased T 4 secretion by the isolated thyroids of these animals (Fig. 2). Since the final stages of intrathyroid hormone production are represented by the oxidative condensation of iodotyrosines, the decrease of T 4 production by the isolated thyroid of reserpin-treated adult animals against the background of unchanged T 3 production may, obviously, indicate the disturbance of intrathyroid T 4 production at the stage of condensation of two molecules of diiodotyrosine. These results are in agreement with those described in the literature for the significant role of sympathetic neural impulses in the hormone-producing function of the thyroid, especially, at the final stage of intrathyroid hormonogenesis, thus signifying the weakening of conversion of iodotyrosines to iodotyronines following the thyroid sympatectomy [22]. The investigation of regulatory mechanisms of in vivo conversion of T 4 to T 3 in the canine thyroid showed the denervation-induced activation of intrathyroid T 4 deiodination [23]. This may indicate the potentiation of intrathyroid T 4 to T 3 conversion to be a cause for unaltered T 3 production by the isolated thyroid of adult animals against the background of decreased T 4 production, all these resulting from the reserpin-conditioned deficit of adrenergic influences. The blood T 3content is a value summing-up the two processes, i.e. intensity of T 3 secretion by the thyroid tissue and the level of T 4 monodeiodination in peripheral tissues. The contribution of mechanism of extrathyroid T 4 monodeiodination to the maintenance of blood T a level in different species varies significantly. Thus, in human being 12--20°70 of blood T 3 is secreted by the thyroid, while about 80~/0 is formed out of T 4 extrathyroidally. At the same time, in rats the contribution of this mechanism to the maintenance of blood T 3 level is sharply reduced (17~/0) [24]. It
96 may be assumed that the low specific weight of extrathyroid conversion of T 4 t o T3, peculiar for rat, in combination with stable processes of hormone production in the isolated thyroid prior t o T 3 synthesis and/or with the enhanced intrathyroid conversion of T 4 to T 3 are the factors which provide the stability of T 3 c o n t e n t in the peripheral blood of reserpin-treated adult rats vs. control. Sharp reduction of TSH-stimulated production of both studied TH by the isolated thyroids of adult reserpin-treated animals showed significant role of adrenergic neurotrophic influences in the maintenance of normal response of adult animal thyroid to tropic hormone. Atropin-induced blockade of M-cholinoreactive structures produced no significant effect on TSH and blood T 4 concentration in animals of both ages (Fig. 1), whereas both basal and TSH-stimulated secretion of T 4 by the isolated thyroid glands of these animals were sharply increased compared to control (Fig. 2). Lack of changes in the blood TH concentration of atropin-treated animals against the background of sharp activation of their secretion in vitro may be conditioned by the intensified extrathyroid metabolism and by their enhanced elimination from blood in the deficit of M-cholinergic influences. Opposite pattern of relationships between the level of hormone in the peripheral blood and the rate of its secretion by the gland was shown by the authors previously at the example of aldosterone: the peripheral blood aldosterone was found not to change with age in male rats, while the rate of its secretion by the gland was decreased significantly. Such discrepancy between hormone concentration and the rate of its secretion by the gland was linked with agerelated changes in metabolism and decelerated aldosterone elimination from the blood of old male rats [25]. The results obtained on isolated TH of atropin-treated animals suggest direct inhibiting effect of M-cholinergic tonic neural influences on hormone production in the thyroid tissue in animals of both ages. Significant age differences were found in the neurotrophic provision of steroidogenesis in the adrenal cortex. Obviously, the inhibition of steroidogenesis of isolated old rat adrenals, registered against the background of reserpin-induced emptying of tissue catecholamine depots resulted from the decreased range of adaptive capacities of old rat adrenals in the presence of deficit of adrenergic influences (Fig. 3). The results of experiments with switching-off separate links of adrenergic provision of steroidogenesis in the adrenal cortex suggest significant role played by the alpha-adrenergic mechanisms in the regulation of glucocorticoid production. Obviously, the alpha-adrenergic tonic influences on steroidogenesis in the adrenal cortex of intact adult rats are of inhibitory character; during aging they weaken or even take an opposite direction. The possibility that the increase of sensitivity of adrenal cortex to ACTH occurs in adult rats against the background of chronic administration of alpha-adrenoblockers should not be ruled out. This assumption can be supported by the fact that
97 the in vitro response of dibenamin-treated isolated adult adrenals to ACTH is sharply enhanced compared to control, while the basal 11-OKS production by these adrenals remains unchanged. Possible explanation may be that in vitro, when the adrenal misses the natural background of tropic hormone, there may be no differences in basal steroidogenesis of isolated adrenals of control and alpha-adrenoblocker-treated animals, even in case of increased sensitivity of the latter to ACTH. However, in vivo the high blood 11-OKS against the background of unaltered concentration of circulating ACTH may result from the increased sensitivity of the adrenal cortex to tropic hormone. Our data on substantial decrease of steroidogenesis of testicles by reserpininduced emptying of tissue catecholamine depots may testify, first, to significant activating role of tonic adrenergic impulsation in the maintenance of steroidogenesis in the testicles of adult rats, and, second, to weakening of the role of adrenergic influences in the regulation of steroidogenic function in old rat testicles (Fig. 1). Marked age-related differences were found in the contribution of M-cholinergic component of regulation of steroidogenesis in the adrenal cortex and testicles (Figs. 1, 3). Chronic atropinization resulted in the rise of blood ACTH and 11-OKS (Fig. 1) and activated basal and ACTH-stimulated steroidogenesis of isolated adrenals of adult rats only, having no effect on these indices in old animals (Fig. 3). The increased blood ACTH against the background of high 11-OKS suggests the possibility of a primary effect of the deficit of cholinergic impulsation on extra-adrenocortical mechanisms of regulation of glucocorticoid production, since if it is not the case, then the triggering of the negative feed-back control should inhibit the corticotropic function of the hypophysis against the background of high 11-OKS concentration in the blood. The fact that switching-off the M-cholinergic component of neural regulation significantly effects the function of the adrenal cortex and testicles in adult rats only supports the following two conclusions: (1) in intact adult rats the M-cholinergic component of steroidogenesis regulation plays significant role both in the adrenals and testicles, inhibiting the corticotropic function of adenohypophysis and steroidogenesis in the adrenal cortex, but stimulating the sex steroid production, and (2) lack of shift in the indices studied in old rats against the background of chronic atropin administration signifies essential age-related weakening of contribution of the Mcholinergic component of regulation of steroidogenic function of the adrenal cortex and testicles. The data obtained showed age-related weakening of different components of neural control of the thyroid, adrenocortical and testicular functions. Such weakening is assumed to cause age changes in hormonal secretion and response of peripheral target glands to tropic hormones. Interesting is the fact that the selective blockade of sympathetic and parasympathetic pathways revealed the differing pattern of changes in the thyroid, adrenocortical and testicular function. This suggests
98
that during aging the pattern of weakening of neural control over the function of different endocrine glands is different. There is reason to expect a certain relationship between the degree of autonomic innervation of the endocrine glands that has been formed in phylogenesis and the role of changes in neural control in age shifts of their function. ACKNOWLEDGEMENT
Gratitude is expressed to Alex Volkov who translated the original Russian text into English. REFERENCES V.V. Frolkis, Aging and life-prolongingprocesses, Springer-Verlag, Vienna-New York, 1982. K. Shizume and S. Okinaka, Control of thyroid function by the nervous system. In: Major problems in neuroendocrinology. Basel-N.Y., 1964, 286--306. 3 E.V. Naumenko, The central regulation o f the hypophyseal-adrenal complex. Nauka, Leningrad, 1971 (In Russian). 4 Ya.I. Azhipa, The nerves o f endocrine glands and mediators in regulation o f endocrine functions. A.D. Ado (ed.), Nauka, Moscow, 1981 (In Russian). 5 S.C. Woods, Interaction entre l'insulinosecretion et le systeme nerveux central. Journees annu. diabetoL HoteI-Dieu, 5--7 mai, 1983, Paris, 1983, pp. 27--33. 6 S. Feldman, Neural pathways mediating adrenocortical responses, Pt 2. Fed. Proc., 44 (1985) 169-175. 7 T. Aburaya and N. Hata, On the neural regulation of the adrenal cortex. Tohoku J. Exp. Med., 8 (1961) 142--148. 8 M.G. Amiragova, M.A. Berlina and I.N. Osipova, On the trophic function of the vegetative nerves of the thyroid gland. Bull. Exp. BioL, 64 (1967) NI0, 13--15 (In Russian). 9 D.S. Gann, Vagus mediated control of adrenal corticosteroid secretion in the dog. Am. J. Physiol., 221 (1971) 1004--1008. 10 R.N. Bergman and R.E. Miller, Direct enhancement of insulin secretion by vagal stimulation of the isolated pancreas. Am. J. PhysioL, 225 (1973) 481--486. 11 A. Melander, L.E. Ericson, F. Sundler and S.H. lngbar, Sympathetic innervation of the mouse thyroid and its significance in thyroid hormone secretion. Endocrinology, 94 (1974) 959--966. 12 A.V. Edvards, Adrenal medullary responses to splanchnic nerve stimulation in new-born calves. J. PhysioL (Gr. Brit.), 357 (1984)409--416. 13 M.W. Roy, K.C. Lee, M.S. Jones and R.E. Miller, Neural control of pancreatic insulin and somatostatin secretion. Endocrinology, 115 (1984) 770--775. 14 N. Kleitman and M.A. Holzwarth, Compensatory adrenal cortical growth is inhibited by sympathectomy. Am. J. Physiol., 248 (1985) E/261--E/263. 15 H.E. Romeo, R.J. Boado and D.P. Cardinali, Role of the sympathetic nervous system in the control of thyroid compensatory growth of normal and hypophysectomized rats. Neuroendocrinology, 40 (1985) 303--315. 16 V.V. Frolkis, N.V. Verzhikovskaya and G.V. Valueva, The thyroid and age. Exp. GerontoL, 8 (1973) 285--296. 17 R.C. Adelman, T.L. Klug, M.F. Obenrader and A. Kitahara, Regulation of hormone levels and activities during aging. Liver and aging. Amsterdam, 1978, 277--283. 18 T.L. Klug and R.C. Adelman, Altered hypothalamic-pituitary regulation of thyrotrophin in male rats during aging. Endocrinology, 104 (1979) 1136--1142. 19 A.V. Everitt, The neuroendocrine system and aging. Gerontology, 26 (1980) 108--119. I 2
99 20 21 22 23
24 25
J. Meites, Changes in neuroendocrine control of anterior pituitary function during aging. Neuroendocrinology, 34 (1982) 151-- 156. J. Meites, Neuroendocrinoiogy of aging. Plenum Press, New York, London, 1983. XVI. S.I. Chuprinova, Effect of the sympathetic nervous system on the hormonopoiesis in the thyroid. Bul. eksper. Biol. L Med., 64 (1967) N9, 21--24 (In Russian). Ya.Kh. Turakulov, T..P. Tashkhodzhaeva, R.B. Burikhanov, S.I. Ismailov and D.Sh. Shakhizirov, Intrathyroid thyroxin deiodination; the role of thyrotropic hormone and denervation in the process. Probl. Endokrinol., 32 (1986) NS, 72--76 (In Russian). C.S. Pittman, Hormone metabolism. In: L.J. De Groot (ed.), Endocrinology, VoL 1, Grune and Stratton, New York, San Francisco, London, 1979, pp. 365--672. V.V. Frolkis, N.S. Verkhratsky and L.V. Magdich, Regulation of aldosterone secretion in old rats. Gerontology, 31 (1985) N2, 84--94.
89
Elsevier Scientific Publishers Ireland Ltd.
PECULIARITIES OF NEURAL REGULATION OF THE THYROID, ADRENOCORTICAL AND TESTICULAR FUNCTIONS IN OLD AGE
VLADIMIR V. FROLKIS, EVGENI N. GORBAN and EVGENIJA V. MOROZ Expermental Department, Institute of Gerontology, VyshgorodskayaSt. 67, AMS USSR, 252655 Kiev-ll4 (U.S.S.R.) (Received April 21st, 1989)
SUMMARY
The age changes of neural control over the function of the thyroid, adrenal cortex and testicles were examined in adult (6 months) and old (28 months) male rats. In old age, there was a weakening of adrenergic control of thyroidogenesis, alphaadrenergic and M-cholinergic control of glucocorticoid function of the adrenal cortex and reduction of adrenergic and M-cholinergic influences in the regulation of steroidogenic function of the testicles.
Key words: Aging; Neural regulation; Hormones; Thyroid; Adrenal cortex; Testicles INTRODUCTION
Through their regulatory influences the nervous and endocrine systems provide for the organism's adaptation to extrinsic and intrinsic changes. The interrelationships between these two systems in the provision of adaptive-regulatory mechanisms are multifactoral. During aging these interrelationships alter significantly, decreasing the contribution of neural regulatory influences and increasing the role of humoral factors [ 1]. The neural control of the endocrine function is realized through two main channels, i.e., regulatory influences of the superior brain areas on the "hypothalamushypophysis-peripheral endocrine glands" system [2--6], and direct neurotrophic influences on the endocrine glands cell function [7--15]. At present there is extensive material on the significant role of changes in the hypothalamic-hypophyseal regulatory influences on endocrine glands regarding the mechanisms of aging of the whole organism [16--21]. However, the neural control of the endocrine function requires further study. The purpose of the present investigation was to study age changes in the neural control of the thyroid, adrenal cortex and testicles under conditions of selective blockage of its various links. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
90
MATERIALS AND METHODS
Materials Chemicals used in the experiments included thyrotropin (TSH), corticotropin (ACTH; Plant for Endocrine Preparations, Kaunas, U.S.S.R.), reserpin, atropin sulphate, anaprylin (propranolol; U.S.S.R.), dibenamin hydrochloride (N,N-dibenzyl-beta-chloroethyl-amine HCI; ICN Pharmaceutical Inc., U.S.A.). Medium 199 manufactured by the Institute of Polymyelitis and Viral Encephalitis (U.S.S.R.) was used for the incubation of isolated endocrine glands. Experiments were performed on adult (6 months) and old (28 months) male Wistar rats maintained on a standard diet and supplied by the Stolbovaja Laboratory for Experimental Animals of the U.S.S.R. Academy of Medical Sciences.
Administration of drugs The role of different links of adrenergic and cholinergic components of neural regulation was studied following administration (two times/day for 3 days) of reserpin (1.5 mg/kg) to empty the catecholamine depot in tissues; dibenamin (10 mg/kg) or anaprylin (10.0 mg/kg) to selectively switch off alpha- or beta-adrenergic components of neural regulation; atropin (10 mg/kg) to block M-cholinoreactive structures. The same doses of saline were administered to control animals. The next day following last administration the animals were killed by thoracotomy. Blood was collected to obtain serum. Blood serum samples were kept at - 30°C prior to estimation of hormone concentration.
Preparation of glands The thyroid and adrenals were removed and prepared. Each lobe of the thyroid was dried by filtering paper, weighed, dissected longitudinally along 3/4 of its length, fixed on a plastic bedding and placed in a plastic tube containing 1.0 ml of incubation medium. Each of the two adrenals were dissected in two parts, both parts of one adrenal were fixed on a plastic bedding and placed in plastic tube containing 1.0 ml of incubation medium.
Incubation of glands Isolated thyroid and adrenals were incubated at 37 °C for 4 h. The incubation medium was aerated by carbogen (5070 CO 2 and 95070 02). One lobe of each thyroid was incubated in medium 199, while another one - - in medium 199, containing TSH (100 munits/ml). After incubation the medium was transferred to glass ampules, welded and kept at - 30°C prior to radioimmunological estimation of the thyroid hormones (TH). Each of the two adrenals of each animal were incubated separately. Incubation medium was changed each hour. A particular feature of incubation was that one of the adrenal pair was used as control and was incubated in medium 199 for 4 h, while
91 another one was incubated during the first hour in medium 199, during the second hour - - in medium 199, containing ACTH (10.0 munits/ml), and during the third and fourth hours - - in medium 199. Each portion of incubation medium was transferred to glass ampules, welded and kept at - 3 0 ° C prior to fluorimetric determination of 11-oxyketosteroids (11-OKS). Estimation o f hormones Blood ACTH, TSH, 11-OKS, thyroxine (T4), triiodothyronine (T3) and testosterone (T), as well as T 4, T 3 and 11-OKS in incubation medium were determined in isolated thyroid and adrenals. ACTH, TSH, T 4, T 3 and T were determined radioimmunologically. ACTH and T were determined using "ACTHK-PR" and "Testok" sets (Oris Industrie, France) and expressed in ng/l and nmol/l, respectively; TSH -- "RIA-mat TSH" (Mallinkrodt Diagnostica, F.R.G.) and expressed in mcI.U./l; T 4 and T 3 --"rio-T4-PG" and "rio-T3-PG" (Institute of Bioorganic Chemistry of Academy of Sciences of the Byelorussian SSR, U.S.S.R.) and expressed as nmol/1 (in the blood), and as pmol/ mg of thyroid tissue for a 4-h incubation (in the incubation medium). 11-OKS was determined fluorimetrically according to the corticosterone standard (Serva, U.S.A.). Blood 11-OKS was expressed as nmol/l. Hourly production of 11OKS by the isolated adrenals was expressed as pmol/mg adrenal tissue/h, while gross production of 11-OKS for a 3-h incubation (second, third and fourth hours) -as pmol/mg adrenal tissue. (Hormone production during the first hour was not taken into account in the estimation of gross 11-OKS production by the isolated adrenals). RESULTS Blood hormones and hormonogenesis of isolated thyroid and adrenals of intact animals The pattern of changes of blood hormones during ageing was different: ACTH increased, TSH and 11-OKS remained unchanged, TH and T decreased (Fig. 1). Basal (non-stimulated) TH production by the isolated thyroid expressed per unit of gland mass decreased significantly with age (Fig. 2). Age differences in TH production by the isolated thyroid retain against the background of thyroidogenesis stimulation by adding TSH to the incubation medium, while the increase of basal thyroidogenesis was statistically significant in the adult thyroid only (Fig. 2). Basal and ACTH-stimulated production of l l-OKS by the isolated adrenals remained unchanged with age (Fig. 3). Blood TSH, T4 and T3 and thyroidogenesis o f isolated thyroid at weakening of adrenergic influences Reserpin-induced emptying of the catecholamine depots in tissues produced an
92
~x~h
December
TSH
!, n:!," !, !, 0
R
100
A m
:] lBi i C
R
K
•
C
~"
[J
C
a-
/'3
A~'~H
11 .(IKS
:t 0
0
R
•
Fig. 1. Blood concentration of T S H ( m c l U / l ) , T 4 ( n m o l / l ) , T 3 ( n m o l / l ) , A C T H ( n g / l ) , 1 1 - O K S ( n m o l / I ) and testosterone (nmol/l) in adult and old male rats at weakening of adrenergic or cholinergic influences. White columns - - adult rats; black columns - - old rats; C - - c o n t r o l ; R - - r e s e r p i n ; A - - a t r o p i n ; a - alpha-adrenoblocker dibenamin; /? - - beta-adrenoblocker anaprylin; *Significant differences with control.
insignificant effect on blood TSH in all age groups (Fig. 1), significantly decreased blood T 4 in adult animals only, but had no effect o n T 3. Basal thyroidogenesis of isolated reserpin-treated thyroids and their response to TSH in vitro declined sharply in adult animals only compared to control (Fig. 2). Selective blockade of alpha- and beta-adrenergic structures produced no significant effect on blood TSH in both groups (Fig. 1), but similar to reserpinization, it sharply inhibited the thyroidogenesis in adult rats only. Blood T 4 concentration
93 December
gash aeropiu
T&
B
,i i,i
S
B
S
B
S
B
S
B
S
B
Contx'ol
S
B
S
AlphaB~aad_~-w~oblooke~ ad.~emoblookEe
,I i II ii ,I B S
B
S
B S
B
S
B
S
Croat:col T:
Control
Reserpi~
~ .50'
At ropi~
adrenoblocke~
':1II,I i,I ilii 11,01,0i B
S
B
S
B S
B
S
B$
B
B
S
S
B S
B
S
B S
a~l~enoblockez e
B S
II B
S
Fig. 2. T 4 and T 3 production by the isolated thyroid of adult and old male rats at weakening of adrenergic or cholinergic influences (pmol/mg thyroid tissue/4 h). White columns - - adult rats; black columns - old rats; B - - basal thyroidogenesis; S - - stimulated thyroidogenesis (100 munits TSH/ml); *Significant differences with control.
Rese..--,pJ.n
ga~.t:z,o l
.,7,00• 200" loo.
•
.ll
B
S
o B
S
B
S
B
S
Alpha-
~
DORa B B S
B S
B
S
B
S
B
S
I
S
Beta-
B
S
B
S
B
~-=
Fig. 3.1 I-OKS production by the isolated adrenals of adult and old male rats for a 3-h (2, 3 and 4 h) incubation at weakening of adrenergic or cholinergic influences (pmol/mg adrenal tissue). White columns - adult rats; black columns - - old rats; B - - basal steroidogenesis; S - - stimulated steroidogenesis (10 munits ACTH/ml); *Significant differences with control.
94 (Fig. 1) and basal and TSH-stimulated thyroidogenesis of isolated thyroids (Fig. 2) decreased significantly in adult rats only, as compared with controls.
Blood TSH, T~, T3 and thyroidogenesis of isolated thyroid at weakening of M-cholinergic influences Atropin had no effect on blood TSH and T H in any age group (Fig. 1). Thyroidogenesis of isolated atropin-treated thyroid increased sharply. There was a significant increase o f basal production o f T H by the isolated thyroids in both age groups, intensification o f TSH-stimulated production of T 4 by adult and old thyroids, of T 3 - - by the isolated thyroid of old animals, as well as marked trend towards rise of TSH-stimulated T 3 production by the isolated thyroids of adult vs. control animals (Fig. 2).
Blood ACTH, ll-OKS and steroidogenesis of isolated adrenals at weakening of adrenergic influences Blood A C T H and l l - O K S concentrations o f reserpin-treated animals of both ages were similar to those of controls (Fig. 1). The basal steroidogenesis of isolated adrenals of these animals and their response to A C T H were sharply inhibited in old rats only (Fig. 3). Blood A C T H concentration of adrenoblocker-treated animals of both ages differed insignificantly compared to control (Fig. 1). Blood l l-OKS (Fig. 1) and ACTH-stimulated steroidogenesis of isolated adrenals of adult rats treated with alpha-adrenoblocker dibenamin were significantly increased compared to control, while dibenamin-treated old rats showed a tendency towards the decrease of blood 11-OKS, as well as significant decrease of basal and ACTH-stimulated steroidogenesis of isolated adrenals compared to control (Fig. 3). Selective blockade of betaadrenoblockers had no effect on 1 I-OKS production in both age groups.
Blood A CTH, l l-OKS and steroidogenesis of isolated adrenals at weakening of M-cholinergic influences Chronic atropinization resulted in the increased blood A C T H and 1 I-OKS of adult rats, while in old rats these hormones remained unchanged (Fig. 1). Basal and ACTH-stimulated steroidogenesis o f isolated adrenals increased in adult atropin-treated animals, while in old rats it remained unchanged as compared with control (Fig. 3).
Blood Tat weakening of adrenergic of M-cholinergic influences Significant age differences were revealed in the role of tonic adrenergic impulsation in the provision of steroidogenic function of testicles. Emptying of tissue catecholamine depot by chronic reserpin administration resulted in marked decrease of blood T concentration in adult animals, while in old animals the T content remained unchanged (Fig. 1).
95 Essential age differences were registered in the contribution of M-cholinergic regulatory influences in the provision of steroidogenic function of testicles. Chronic administration of atropin significantly inhibited the T production in adult animals
only (Fig. I). DISCUSSION The data obtained showed the age-related weakening of different components of neurotrophic control of the thyroid, adrenocortical and testicular functions. The results of experiments with switching-off various links of adrenergic provision of thyroidogenesis (reserpin, alpha- and beta-blockers) enable to conclude that in adult rats both components of adrenergic neural influences contribute significantly to the maintenance of normal viability and to the modulation of thyroid reactivity, while during aging the adrenergic control of thyroid function weakens. Reserpin administration was shown to decrease T 4 in the peripheral blood, while T 3 remained unchanged compared to control (Fig. 1). These data closely correlate with unaltered T 3 and decreased T 4 secretion by the isolated thyroids of these animals (Fig. 2). Since the final stages of intrathyroid hormone production are represented by the oxidative condensation of iodotyrosines, the decrease of T 4 production by the isolated thyroid of reserpin-treated adult animals against the background of unchanged T 3 production may, obviously, indicate the disturbance of intrathyroid T 4 production at the stage of condensation of two molecules of diiodotyrosine. These results are in agreement with those described in the literature for the significant role of sympathetic neural impulses in the hormone-producing function of the thyroid, especially, at the final stage of intrathyroid hormonogenesis, thus signifying the weakening of conversion of iodotyrosines to iodotyronines following the thyroid sympatectomy [22]. The investigation of regulatory mechanisms of in vivo conversion of T 4 to T 3 in the canine thyroid showed the denervation-induced activation of intrathyroid T 4 deiodination [23]. This may indicate the potentiation of intrathyroid T 4 to T 3 conversion to be a cause for unaltered T 3 production by the isolated thyroid of adult animals against the background of decreased T 4 production, all these resulting from the reserpin-conditioned deficit of adrenergic influences. The blood T 3content is a value summing-up the two processes, i.e. intensity of T 3 secretion by the thyroid tissue and the level of T 4 monodeiodination in peripheral tissues. The contribution of mechanism of extrathyroid T 4 monodeiodination to the maintenance of blood T a level in different species varies significantly. Thus, in human being 12--20°70 of blood T 3 is secreted by the thyroid, while about 80~/0 is formed out of T 4 extrathyroidally. At the same time, in rats the contribution of this mechanism to the maintenance of blood T 3 level is sharply reduced (17~/0) [24]. It
96 may be assumed that the low specific weight of extrathyroid conversion of T 4 t o T3, peculiar for rat, in combination with stable processes of hormone production in the isolated thyroid prior t o T 3 synthesis and/or with the enhanced intrathyroid conversion of T 4 to T 3 are the factors which provide the stability of T 3 c o n t e n t in the peripheral blood of reserpin-treated adult rats vs. control. Sharp reduction of TSH-stimulated production of both studied TH by the isolated thyroids of adult reserpin-treated animals showed significant role of adrenergic neurotrophic influences in the maintenance of normal response of adult animal thyroid to tropic hormone. Atropin-induced blockade of M-cholinoreactive structures produced no significant effect on TSH and blood T 4 concentration in animals of both ages (Fig. 1), whereas both basal and TSH-stimulated secretion of T 4 by the isolated thyroid glands of these animals were sharply increased compared to control (Fig. 2). Lack of changes in the blood TH concentration of atropin-treated animals against the background of sharp activation of their secretion in vitro may be conditioned by the intensified extrathyroid metabolism and by their enhanced elimination from blood in the deficit of M-cholinergic influences. Opposite pattern of relationships between the level of hormone in the peripheral blood and the rate of its secretion by the gland was shown by the authors previously at the example of aldosterone: the peripheral blood aldosterone was found not to change with age in male rats, while the rate of its secretion by the gland was decreased significantly. Such discrepancy between hormone concentration and the rate of its secretion by the gland was linked with agerelated changes in metabolism and decelerated aldosterone elimination from the blood of old male rats [25]. The results obtained on isolated TH of atropin-treated animals suggest direct inhibiting effect of M-cholinergic tonic neural influences on hormone production in the thyroid tissue in animals of both ages. Significant age differences were found in the neurotrophic provision of steroidogenesis in the adrenal cortex. Obviously, the inhibition of steroidogenesis of isolated old rat adrenals, registered against the background of reserpin-induced emptying of tissue catecholamine depots resulted from the decreased range of adaptive capacities of old rat adrenals in the presence of deficit of adrenergic influences (Fig. 3). The results of experiments with switching-off separate links of adrenergic provision of steroidogenesis in the adrenal cortex suggest significant role played by the alpha-adrenergic mechanisms in the regulation of glucocorticoid production. Obviously, the alpha-adrenergic tonic influences on steroidogenesis in the adrenal cortex of intact adult rats are of inhibitory character; during aging they weaken or even take an opposite direction. The possibility that the increase of sensitivity of adrenal cortex to ACTH occurs in adult rats against the background of chronic administration of alpha-adrenoblockers should not be ruled out. This assumption can be supported by the fact that
97 the in vitro response of dibenamin-treated isolated adult adrenals to ACTH is sharply enhanced compared to control, while the basal 11-OKS production by these adrenals remains unchanged. Possible explanation may be that in vitro, when the adrenal misses the natural background of tropic hormone, there may be no differences in basal steroidogenesis of isolated adrenals of control and alpha-adrenoblocker-treated animals, even in case of increased sensitivity of the latter to ACTH. However, in vivo the high blood 11-OKS against the background of unaltered concentration of circulating ACTH may result from the increased sensitivity of the adrenal cortex to tropic hormone. Our data on substantial decrease of steroidogenesis of testicles by reserpininduced emptying of tissue catecholamine depots may testify, first, to significant activating role of tonic adrenergic impulsation in the maintenance of steroidogenesis in the testicles of adult rats, and, second, to weakening of the role of adrenergic influences in the regulation of steroidogenic function in old rat testicles (Fig. 1). Marked age-related differences were found in the contribution of M-cholinergic component of regulation of steroidogenesis in the adrenal cortex and testicles (Figs. 1, 3). Chronic atropinization resulted in the rise of blood ACTH and 11-OKS (Fig. 1) and activated basal and ACTH-stimulated steroidogenesis of isolated adrenals of adult rats only, having no effect on these indices in old animals (Fig. 3). The increased blood ACTH against the background of high 11-OKS suggests the possibility of a primary effect of the deficit of cholinergic impulsation on extra-adrenocortical mechanisms of regulation of glucocorticoid production, since if it is not the case, then the triggering of the negative feed-back control should inhibit the corticotropic function of the hypophysis against the background of high 11-OKS concentration in the blood. The fact that switching-off the M-cholinergic component of neural regulation significantly effects the function of the adrenal cortex and testicles in adult rats only supports the following two conclusions: (1) in intact adult rats the M-cholinergic component of steroidogenesis regulation plays significant role both in the adrenals and testicles, inhibiting the corticotropic function of adenohypophysis and steroidogenesis in the adrenal cortex, but stimulating the sex steroid production, and (2) lack of shift in the indices studied in old rats against the background of chronic atropin administration signifies essential age-related weakening of contribution of the Mcholinergic component of regulation of steroidogenic function of the adrenal cortex and testicles. The data obtained showed age-related weakening of different components of neural control of the thyroid, adrenocortical and testicular functions. Such weakening is assumed to cause age changes in hormonal secretion and response of peripheral target glands to tropic hormones. Interesting is the fact that the selective blockade of sympathetic and parasympathetic pathways revealed the differing pattern of changes in the thyroid, adrenocortical and testicular function. This suggests
98
that during aging the pattern of weakening of neural control over the function of different endocrine glands is different. There is reason to expect a certain relationship between the degree of autonomic innervation of the endocrine glands that has been formed in phylogenesis and the role of changes in neural control in age shifts of their function. ACKNOWLEDGEMENT
Gratitude is expressed to Alex Volkov who translated the original Russian text into English. REFERENCES V.V. Frolkis, Aging and life-prolongingprocesses, Springer-Verlag, Vienna-New York, 1982. K. Shizume and S. Okinaka, Control of thyroid function by the nervous system. In: Major problems in neuroendocrinology. Basel-N.Y., 1964, 286--306. 3 E.V. Naumenko, The central regulation o f the hypophyseal-adrenal complex. Nauka, Leningrad, 1971 (In Russian). 4 Ya.I. Azhipa, The nerves o f endocrine glands and mediators in regulation o f endocrine functions. A.D. Ado (ed.), Nauka, Moscow, 1981 (In Russian). 5 S.C. Woods, Interaction entre l'insulinosecretion et le systeme nerveux central. Journees annu. diabetoL HoteI-Dieu, 5--7 mai, 1983, Paris, 1983, pp. 27--33. 6 S. Feldman, Neural pathways mediating adrenocortical responses, Pt 2. Fed. Proc., 44 (1985) 169-175. 7 T. Aburaya and N. Hata, On the neural regulation of the adrenal cortex. Tohoku J. Exp. Med., 8 (1961) 142--148. 8 M.G. Amiragova, M.A. Berlina and I.N. Osipova, On the trophic function of the vegetative nerves of the thyroid gland. Bull. Exp. BioL, 64 (1967) NI0, 13--15 (In Russian). 9 D.S. Gann, Vagus mediated control of adrenal corticosteroid secretion in the dog. Am. J. Physiol., 221 (1971) 1004--1008. 10 R.N. Bergman and R.E. Miller, Direct enhancement of insulin secretion by vagal stimulation of the isolated pancreas. Am. J. PhysioL, 225 (1973) 481--486. 11 A. Melander, L.E. Ericson, F. Sundler and S.H. lngbar, Sympathetic innervation of the mouse thyroid and its significance in thyroid hormone secretion. Endocrinology, 94 (1974) 959--966. 12 A.V. Edvards, Adrenal medullary responses to splanchnic nerve stimulation in new-born calves. J. PhysioL (Gr. Brit.), 357 (1984)409--416. 13 M.W. Roy, K.C. Lee, M.S. Jones and R.E. Miller, Neural control of pancreatic insulin and somatostatin secretion. Endocrinology, 115 (1984) 770--775. 14 N. Kleitman and M.A. Holzwarth, Compensatory adrenal cortical growth is inhibited by sympathectomy. Am. J. Physiol., 248 (1985) E/261--E/263. 15 H.E. Romeo, R.J. Boado and D.P. Cardinali, Role of the sympathetic nervous system in the control of thyroid compensatory growth of normal and hypophysectomized rats. Neuroendocrinology, 40 (1985) 303--315. 16 V.V. Frolkis, N.V. Verzhikovskaya and G.V. Valueva, The thyroid and age. Exp. GerontoL, 8 (1973) 285--296. 17 R.C. Adelman, T.L. Klug, M.F. Obenrader and A. Kitahara, Regulation of hormone levels and activities during aging. Liver and aging. Amsterdam, 1978, 277--283. 18 T.L. Klug and R.C. Adelman, Altered hypothalamic-pituitary regulation of thyrotrophin in male rats during aging. Endocrinology, 104 (1979) 1136--1142. 19 A.V. Everitt, The neuroendocrine system and aging. Gerontology, 26 (1980) 108--119. I 2
99 20 21 22 23
24 25
J. Meites, Changes in neuroendocrine control of anterior pituitary function during aging. Neuroendocrinology, 34 (1982) 151-- 156. J. Meites, Neuroendocrinoiogy of aging. Plenum Press, New York, London, 1983. XVI. S.I. Chuprinova, Effect of the sympathetic nervous system on the hormonopoiesis in the thyroid. Bul. eksper. Biol. L Med., 64 (1967) N9, 21--24 (In Russian). Ya.Kh. Turakulov, T..P. Tashkhodzhaeva, R.B. Burikhanov, S.I. Ismailov and D.Sh. Shakhizirov, Intrathyroid thyroxin deiodination; the role of thyrotropic hormone and denervation in the process. Probl. Endokrinol., 32 (1986) NS, 72--76 (In Russian). C.S. Pittman, Hormone metabolism. In: L.J. De Groot (ed.), Endocrinology, VoL 1, Grune and Stratton, New York, San Francisco, London, 1979, pp. 365--672. V.V. Frolkis, N.S. Verkhratsky and L.V. Magdich, Regulation of aldosterone secretion in old rats. Gerontology, 31 (1985) N2, 84--94.