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Regulatory process in the mechanism of ageing
Exp. Geront. Vol. 3, pp. 113-123. Pergamon Press 1968. Printed in Great Britain
REGULATORY PROCESS IN T H E MECHANISM OF AGEING INTRODUCTORY LECTURE--VERBATIM REPORT V. V. FROLKIS Institute of Gerontology, Academy of Medical Sciences of USSR, Kiev
THE BASICconcept which I intend to present in this paper can be reduced to the statement that the process of ageing may be regarded as the sum of allometric changes in regulation at various levels of physiology. All the data collected in the course of our work seems to confirm the four premises of this concept. (1) Ageing is the sum of metabolic changes in the cells and shifts in the neurohormonal control mechanism. (2) The aUometric changes in the control mechanisms are responsible not only for the manifestations of falling metabolic rate and diminishing functional capacity associated with ageing, but also for the development of important adaptation mechanisms. Ageing cannot be regarded as a simple process of involution. (3) Age-linked changes in the regulatory and adaptive mechanism favour homeostasis but its maintenance becomes increasingly difficult in old age. (4) With ageing neural influences on tissues diminish while their sensitivity to humoral factors increases. The mechanism of regulation of vital processes is perfected in the course of phylogeny and ontogeny so that a very high degree of adaptation to environment is achieved. For this reason a study of age-linked changes of the regulatory mechanisms may help in our understanding of the intimate nature of the biological process of ageing. Regulation, adaptation and ageing represent three concepts and at the same time three most complex biological processes. The interaction of these, in our view, determines the direction and actual course of the age-linked evolution of the body. In previous communications we cited some evidence that age-linked changes take place in the regulatory mechanisms of effector systems in the heart, vessels, skeletal muscles, intestine, kidneys, in the regulatory mechanisms of the blood sugar level, intraocular pressure, intracellular energy exchanges and finally in the vegetative ganglia and receptor systems. In this paper I want to characterize some of the age-linked changes taking place (1) in the central control mechanisms of the central nervous and endocrine systems (2) at the molecular level of the metabolic mechanisms mediating the effector functions. We think that age-linked changes in the endocrine system can be summed up as follows: ageing is associated with a diminishing functional activity of endocrine glands but at the same time the sensitivity of the tissues, including the glands themselves, to hormones increases; in old age, the reactive capacity of the tissues to hormones diminishes. The tissue sensitivity to hormones was determined by finding the lowest 113
114
v.v.
FROLKIS
dose of the hormone capable of inducing metabolic and functional changes. The tissue reactive capacity on the other hand was inferred from the potential range of the metabolic and functional changes in response to stimulation of increasing intensity. So, for instance, the minimal dose of intravenous adrenaline capable of inducing a change of the arterial pressure in rabbits aged 1-1.5 yr and 4-4.5 yr is respectively 0.11 _+ 0.014 /zg/kg and 0.016 + 0.0015 /zg/kg. The fall of blood sugar after an intravenous injection of insulin 0.025 un./kg is 28.6 + 2.28 mg per cent and 14.0 ± 2.11 mg per cent in aged and mature rabbits respectively. An injection of adrenaline 5 /~g/kg in aged and in mature rabbits raises their respective blood sugar levels by 20.6 + 1.40 mg per cent and 6-5 + 1.87 mg per cent. The oestrus-inducing doses of oestradiol propionate for castrated rats aged 24-28 months and 8-10 months are, respectively, 1.60-1.7 and 2.0-2.2 i.u./kg. Cortisone 2 mg/kg produces a 1-5 to 2-fold increase of the phagocytic activity of leucocytes in aged rabbits, whereas in mature animals this function of white cells is practically unaffected by a dose of this order. With a dose of 5 mg/kg the reverse happens; aged animals fail to react and in mature ones oscillatory changes of phagocytic activity follow. The same pattern thus seems to emerge with different hormone used (adrenaline, insulin, cortisone, oestradiol propionate) and with different system followed (cardiovascular, reproductive, leucocytes, carbohydrate metabolism). With ageing the sensitivity of the tissues to hormones increases. In the presence of the declining functional activity of the endocrines this increase of tissue sensitivity to hormones represents an adaptive element in the age-linked changes affecting body control systems. It must be pointed out at that stage that the age-linked increase of tissue sensitivity to hormones includes also an increase of the sensitivity of endocrine glands to hormones produced by other endocrine glands. So thyrotropic hormone 0.5 un. (Fig. 1) produces only insignificant changes in the thyroid glands of mature rats, whereas in aged animals a similar dose induces a 27.3 per cent increase in gaseous exchanges, a 69 per cent increase in the weight of the gland and a 66 per cent increase in the height of the thyroid epithelium. As the tissue sensitivity to hormonal action increases with age, the reactivity of the tissues, i.e. the potential range of metabolic and functional changes taking place in (o)
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I15
REGULATORY PROCESS IN THE M E C H A N I S M OF AGEING
response to large doses of hormones, falls. The increase of tissue respiration rate in the thyroid and of the weight of the gland following an injection of 1.0 unit of thyrotropic hormone (Fig. 1) are bigger in mature than in aged animals. Blood sugar levels recorded after large doses of adrenaline or insulin reveal similar patterns. After an intravenous injection of insulin 1.5 un./kg blood sugar fell by 45.5 +__2.96 mg per cent and 57.5 + 3.56 mg per cent in aged and mature rabbits respectively, and after large doses of adrenaline (50 Fg/kg) by 33.2 _ 2.21 mg per cent and 84.2 + 9.4 mg per cent respectively. A highly significant element in the development of adaptation mechanisms within the endocrine system is represented by changes in the feed-back connections. Changes in the levels of hormones in blood and tissues influence all the subsequent links in the endocrine control system. The important link in this mechanism of age-induced changes of regulation is shift in sensitivity of the nerve centres. In aged animals smaller amounts of hormones are required to induce changes in the electrical activity of the brain. Insulin 0.0005 un./100 g produces more conspicuous changes of cerebral electrical activity in an aged compared with a mature adult animal (Fig. 2). In the case of the aged rat the hippocampus and amygdaloid area show synchronization of the theta-rhythm and isolated spikes within 3 min after the injection. The periodicity of rhythm-synchronization in the limbic system is associated with a slowing of (a) '
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116
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activity in the mesencephalic reticular system and posterior hypothalamus. In the 8-10th min the tracings show slow-amplitude rhythms in the anterior part of the hypothalamus, desynchronization in the reticular formation and in the cortex, evidence of inhibition of hippocampal activity. In the mature rat a similar dose of insulin produces in the 3rd min only the first phase of change in electrical activity of the brain (the theta-rhythm of the limbic system and a minor slowing down of the activity of the hippocampus and of the mesencephalie reticular formation.) The complex interconnections of the central nervous and endocrine systems are largely determined at the hypothalamo-pituitary level. Ageing is accompanied by changes in various aspects of the production and release of neurosecretory material. All phases of the neurosecretory cycle can be demonstrated in the supraoptic and paraventricular nuclei of 8-10-months-old rats, with evidence of formation and release of neurosecretory material. In rats aged 26-28 months all segments of the neurosecretory tract show predominance of the processes of accumulation of neurosecretions while their release is delayed. 45F
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R E G U L A T O R Y PROCESS I N T H E M E C H A N I S M O F A G E I N G
117
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metabolism and various other functions. Homeostasis is maintained in spite of the fact that the functional reliability of the system declines. In the complex process of ageing of the body as a whole an important part is played by changes taking place in nervous centres. By securing adaptation to the environment the nervous centres are an important factor in predetermining longevity. However, when age-linked changes take place in the C.N.S. itself, they produce serious disruption in other organs and systems. According to the data collected in our laboratory the C.N.S. changes include weakening of the subordinating influence of the higher centres of the C.N.S. on the lower ones, an increase of the sensitivity of C.N.S. structures to humoral factors and regulatory alterations in the paths of various centro-peripheral influences. In aged rats the thresholds of inhibitory and facilitatory corticospinal and reticulospinal influences are raised. To demonstrate these influences against the background of spinal reflex activity, the higher centres must be stimulated with relatively stronger currents. For instance, in order to demonstrate inhibition of spino-cerebral reflexes in aged animals the motor cortex (areas 4 and 6) requires a stimulus of 1.2 + 0.12 V whereas in mature animals a current of 0.53 + 0.05 V is adequate. The thresholds of inhibitory reticulo-spinal impulses (produced by stimulation of the reticular formation of the mid-brain) in aged and mature animals are respectively 0.53 + 0.036 V and 0.3 + 0.014V, and those of facilitatory reticulo-spinal impulses 0.55 +_ 0-059V and 0.3 + 0.03V. The fact that ageing is associated with a weakening of the subordinating influence of higher centres is further confirmed by our data on the course of spinal shock after chordotomy. We have shown in 1959 that the first phase in the development of spinal shock is associated with over-strong stimulation and the second one, much longer, with
118
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FIG. 5. Sensitivity of central structures to the humoral influences increases in old age. In old animals intravenous (a) and intraperitoneal (b) injections of the lesser dose of various substances cause the changes in the brain electrical activity. a-rabbits, b-rats. elimination of subordinating impulses. Chordotomy at the level of 6th dorsal vertebra was performed in rabbits and rats of various ages. A more profound depression of spinal functions was recorded in aged animals who also showed an earlier recovery of the initial level of excitability of the cord in the 2nd phase. In mature animals the decrement of spinal excitability was less steep in the initial few hours after chordotomy but was followed at a later stage by inhibition of the reflex activity of the brain. We attribute the rapid recovery of spinal function after chordotomy as observed in aged animals to a weakening of the subordinating influence of the higher centres of the brain on the lower centres. Further confirmation of this view is provided by the results of the experiments in which functional block of the spine was induced by cold. The resulting damage to the spine was much less severe than on section and the resulting inhibition of spinal function was much less pronounced in aged animals. These findings are not applicable to all central connections. We were unable to demonstrate any changes in the threshold of excitability on stimulation of amygdala when recording the activity of the hippocampus. Changes of intracentral connections in old age are accompanied by an increased sensitivity of C.N.S. structures to humoral factors. In aged rabbits smaller doses of active compounds are required to induce alterations in the electrical activity of various cerebral structures. That this is not due to increased permeability of blood-brain barrier was clearly demonstrated in a special series of experiments where the barrier was bypassed by introducing the compounds in question into the cerebral ventricles and directly into the individual structures. Under those conditions in aged animals smaller amounts of active substances were required to induce changes of bioelectrical activity. The threshold doses on intraventricular administration to old and mature rabbits were respectively: for adrenaline 0-22 + 0.03 and 1-70 + 0.61 mmkg/kg, for noradrenaline 0.22 + 0.33 and 2.36 + 0.88 mmkg/kg, for acetylcholine 0.09 _ 0.02 and 0.34 +__0.05
REGULATORY
PROCESS IN T H E M E C H A N I S M
OF AGEING
119
mmkg/kg. The changes of sensitivity of individual C.N.S. structures are not uniform. Consequently administration of these substances to the aged produce variable changes in neurodynamics and in the interrelationships of central structures. The increased sensitivityof neural structures to humoral stimuli may to some extent compensate for the weakening of tonic impulses transmitted along specific and nonspecific ascending paths. It must be pointed out, however, that in very old or senile animals this increase of sensitivityto humoral factors is much less evident. Alterations of the functional state of the C.N.S. arc reflectedin the functions of other organs and systems. Estimation of the threshold current required to induce changes in cardiovascular and respiratory functions when applied to hypothalamic nuclei and the limbic system has shown a weakening of the C.N.S. control in old age. However, no uniformity could be demonstrated in the pattern of changes occurring in various centroperipheral connections. In aged rats changes in arterialpressure can be induced by bipolar stimulation of the anterior part of the hypothalamus with currents of 3-0-3.5 V. In mature rats a current of 0.35-0.8 V is sufficient. The difference could not be demonstrated on bipolar stimulation of the posterior part of the hypothalamus. Bipolar stimulation, however, involves a great number of structures. In subsequent experiments we used local unipolar electrical stimulation and were able to demonstrate that the excitability of individual nuclei varies with age, but that the variation is not uniform; the excitability of some nuclei increases and that of others diminishes. For instance, in order to induce a liminal pressor reaction to stimulation of medial and lateral mammillary nuclei of the posterior part of hypothalamus currents of 0.054 + 0.0089 mA and 0"024 + 0.0002 mA were required in aged and mature rats respectively. The liminal vasopressor reaction to stimulation of the posterior hypothalamic nucleus requires currents of 0.046 + 0.053 mA in old and of 0.081 + 0-0055 mA in mature rats. When the stimulus is applied to the paraventricular nucleus of the anterior part of the hypothalamus the liminal pressor reaction is obtained when the current reaches 0.148 + 0.0442 mA and 0-080 + 0.0198 mA in the aged and in the mature animals respectively. Stimulation of the lateral pre-optic zone with currents of 0.030 + 0.006 mA in aged and 0.025 + 0.0031 mA in mature animals produce liminal pressor reaction. All these allometric changes in the sensitivities of various C.N.S. structures lead to qualitative as well as quantitative changes in the central regulatory mechanisms of various physiological systems. The weakening of centroperipheral connections in old age produces a situation where C.N.S. shifts may fail to be reflected at the periphery. It is well known, for instance, that the excitatory wave associated with a convulsion involves the vagal centre and produces bradycardia. In mature rabbits bradycardia failed to occur in 3 of 14 fits. On injection of subliminal doses of convulsants bradycardia failed to develop in 12 of 23 experiments on mature rabbits. In aged rabbits 9 of 14 experimentaUy induced fits were not accompanied by bradycardia. The changes in the C.N.S. functions described above, whether they affect intercentral or centro-peripheral connections, are an important factor in the alterations of the metabolic and overall functional control of the ageing animal and in the reduction of its adaptive powers. At the same time the intimate mechanism of age-linked changes in the regulatory processes depends on alterations in metabolism at the molecular level. Let us examine next therefore the metabolic basis of the regulatory processes. Cholinergic effects on tissue are connected with the synthesis and hydrolysis of
120
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acetylcholine and the reactivity of choline-sensitive receptors. We investigated agelinked changes in the metabolism of acetylcholine in the heart, skeletal muscles, vegetative ganglia, intestine and endocrine glands. In old age the concentration of acetylcholine in the heart falls (Fig. 6) because of the increased rate of hydrolysis and diminished rate of synthesis. The latter is largely due to the reduced activity of the choline acetylase which activates the transfer of the acetyl group from coenzyme A to choline. But Cholinesterase activity falls simultaneously, and the overall rate of acetylcholine hydrolysis is reduced. The reduction in the rate of hydrolysis of the enzyme combined with diminished rate of its synthesis helps to maintain the cholinergic control of the cell. At an earlier stage of our investigation we demonstrated age-linked changes in the choline-sensitive receptors themselves. With advancing age the number of active groups in the receptor diminishes but their sensitivity to the mediator increases. A connection between these changes in the receptor and alterations in the reactive capacity of sulphydryl groups has been experimentally demonstrated.
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121
REGULATORY PROCESS IN THE MECHANISM OF AGEING
These data suggest the following mechanism of the age-linked changes of cholinergic impulses. Loss of nerve terminals and reduced rates of synthesis of acetylcholine weaken the nervous influences on tissues. The reduced intensity of hydrolysis of the mediator and changes in the choline-sensitive receptors enhance the sensitivity of the ceils to cholinergic humoral factors. Data on age-linked changes in the metabolism of catecholamines are presented in Fig. 7. The reduction in the amount of protein-bound noradrenaline and of the potential capacity of the tissues prolongs the action of exogenous adrenaline and thus enhances its effect in aged animals. This is associated with rearrangements in the various metabolic pathways of catecholamines. So the activity of monoaminooxidase increases with age in spite of the fact that activities of other metabolic pathways are reduced. Hormones play an important role in energy metabolism. Changes in the latter lead to functional alterations in the cell. Smaller amounts of mediators or hormones are required to induce changes of the cell energy metabolism in the aged animals compared with the mature ones. The increases in the rates of phosphorylase activity, glycolysis and glycogenolysis are more pronounced after injections of small doses of adrenaline in aged rats, and after large doses-in mature animals. How prominent the action of adrenaline is will depend to a large extent on the effect of the hormone on the mitochondrial processes of oxidative phosphorylation. A dissociation of oxidation from phosphorylation, with an increased rate of free oxidation, are responsible for the so-called "caloric" effect of adrenaline. Figure 9 shows that in aged rats an injection of adrenaline produces a considerable dissociation between oxidation and phosphorylation processes in the mitochondria of the cells of the heart. Thyroxine is an important factor in the regulation of cell energy metabolism. Our experiments have shown that small doses of thyroid hormone produce more prominent Laclate % 300 28O 26O 240 P in rn~/g Activity of tissue 220 5 of '~' phosphonilase 200
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glycogenolysis intensity is more pronounced in the hearts of old rats but the effect of high doses is more pronounced in adult rats.
122
v . v . FROL'KIS 8-12 months old rats
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FIG. 9. Uncoupling adrenaline effect at oxidative phosphorylation was more pronounced in the old rats heart. changes in the gaseous exchanges, in the intensity of tissue respiration of the liver and myocardium, more evident dissociation of oxidation from phosphorylation and more marked enhancement of glycolysis in aged rats. When large doses are given the effects are more prominent in mature animals. It appears therefore that in aged animals smaller amounts of hormones or mediators are required to produce metabolic changes. However, with higher doses the changes are more marked in mature animals. This explains the age-linked increase of tissue sensitivity to humoral factors and the depression of their reactivity. In an aged animal we are dealing therefore with a qualitatively different pattern of neurohormonal regulation. The enhanced tissue sensitivity to hormones along with depressed endocrine function, the increased tissue sensitivity to humoral factors along with depressed neural influences, the decrease of excitability in some of the neural structures and its increase in others, changes in the feed-back information from the receptors, etc. can be considered as specific instances of adaptation. Thanks to these allometric changes in the various control mechanisms, homeostasis is still maintained into old age. In the aged in various pathological situations the regulatory mechanisms are upset relatively easily by strong stimuli with consequent collapse of homeostasis. The results of our investigation show that in different periods of life different mechanisms are responsible for maintaining the indices of homeostasis at constant levels. For instance, different values for peripheral resistance and cardiac output concur in the maintenance of the blood pressure at the same level in the young and in the elderly. The maintenance of constant blood sugar level is achieved, as already indicated, by increasing the tissue sensitivity to insulin, adrenaline and cortisone,
REGULATORY PROCESS IN THE MECHANISM OF AGEING
123
by increasing the sensitivity of the receptors to variations in the level of blood sugar and by enhancing cellular glycolysis. In this way the outward immutability of a number of homeostatic indices in the aged masks quantitative and qualitative changes in the regulatory mechanisms involving complex and coordinated depression of some, and emergence of other adaptative reactions. Acknowledgements--Data presented in this paper were received due to research work of my collaborators : V. Bezrukov, L. Bogatskaya, T . Duplenko, E. Yenis, A. Murashina, N. Verkhratsky, N. Verzhykovskaya, V. Shevchuk, V. Sinitsky, S. T a n i n and J. Oschogoleva.
REGULATORY PROCESS IN T H E MECHANISM OF AGEING INTRODUCTORY LECTURE--VERBATIM REPORT V. V. FROLKIS Institute of Gerontology, Academy of Medical Sciences of USSR, Kiev
THE BASICconcept which I intend to present in this paper can be reduced to the statement that the process of ageing may be regarded as the sum of allometric changes in regulation at various levels of physiology. All the data collected in the course of our work seems to confirm the four premises of this concept. (1) Ageing is the sum of metabolic changes in the cells and shifts in the neurohormonal control mechanism. (2) The aUometric changes in the control mechanisms are responsible not only for the manifestations of falling metabolic rate and diminishing functional capacity associated with ageing, but also for the development of important adaptation mechanisms. Ageing cannot be regarded as a simple process of involution. (3) Age-linked changes in the regulatory and adaptive mechanism favour homeostasis but its maintenance becomes increasingly difficult in old age. (4) With ageing neural influences on tissues diminish while their sensitivity to humoral factors increases. The mechanism of regulation of vital processes is perfected in the course of phylogeny and ontogeny so that a very high degree of adaptation to environment is achieved. For this reason a study of age-linked changes of the regulatory mechanisms may help in our understanding of the intimate nature of the biological process of ageing. Regulation, adaptation and ageing represent three concepts and at the same time three most complex biological processes. The interaction of these, in our view, determines the direction and actual course of the age-linked evolution of the body. In previous communications we cited some evidence that age-linked changes take place in the regulatory mechanisms of effector systems in the heart, vessels, skeletal muscles, intestine, kidneys, in the regulatory mechanisms of the blood sugar level, intraocular pressure, intracellular energy exchanges and finally in the vegetative ganglia and receptor systems. In this paper I want to characterize some of the age-linked changes taking place (1) in the central control mechanisms of the central nervous and endocrine systems (2) at the molecular level of the metabolic mechanisms mediating the effector functions. We think that age-linked changes in the endocrine system can be summed up as follows: ageing is associated with a diminishing functional activity of endocrine glands but at the same time the sensitivity of the tissues, including the glands themselves, to hormones increases; in old age, the reactive capacity of the tissues to hormones diminishes. The tissue sensitivity to hormones was determined by finding the lowest 113
114
v.v.
FROLKIS
dose of the hormone capable of inducing metabolic and functional changes. The tissue reactive capacity on the other hand was inferred from the potential range of the metabolic and functional changes in response to stimulation of increasing intensity. So, for instance, the minimal dose of intravenous adrenaline capable of inducing a change of the arterial pressure in rabbits aged 1-1.5 yr and 4-4.5 yr is respectively 0.11 _+ 0.014 /zg/kg and 0.016 + 0.0015 /zg/kg. The fall of blood sugar after an intravenous injection of insulin 0.025 un./kg is 28.6 + 2.28 mg per cent and 14.0 ± 2.11 mg per cent in aged and mature rabbits respectively. An injection of adrenaline 5 /~g/kg in aged and in mature rabbits raises their respective blood sugar levels by 20.6 + 1.40 mg per cent and 6-5 + 1.87 mg per cent. The oestrus-inducing doses of oestradiol propionate for castrated rats aged 24-28 months and 8-10 months are, respectively, 1.60-1.7 and 2.0-2.2 i.u./kg. Cortisone 2 mg/kg produces a 1-5 to 2-fold increase of the phagocytic activity of leucocytes in aged rabbits, whereas in mature animals this function of white cells is practically unaffected by a dose of this order. With a dose of 5 mg/kg the reverse happens; aged animals fail to react and in mature ones oscillatory changes of phagocytic activity follow. The same pattern thus seems to emerge with different hormone used (adrenaline, insulin, cortisone, oestradiol propionate) and with different system followed (cardiovascular, reproductive, leucocytes, carbohydrate metabolism). With ageing the sensitivity of the tissues to hormones increases. In the presence of the declining functional activity of the endocrines this increase of tissue sensitivity to hormones represents an adaptive element in the age-linked changes affecting body control systems. It must be pointed out at that stage that the age-linked increase of tissue sensitivity to hormones includes also an increase of the sensitivity of endocrine glands to hormones produced by other endocrine glands. So thyrotropic hormone 0.5 un. (Fig. 1) produces only insignificant changes in the thyroid glands of mature rats, whereas in aged animals a similar dose induces a 27.3 per cent increase in gaseous exchanges, a 69 per cent increase in the weight of the gland and a 66 per cent increase in the height of the thyroid epithelium. As the tissue sensitivity to hormonal action increases with age, the reactivity of the tissues, i.e. the potential range of metabolic and functional changes taking place in (o)
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I15
REGULATORY PROCESS IN THE M E C H A N I S M OF AGEING
response to large doses of hormones, falls. The increase of tissue respiration rate in the thyroid and of the weight of the gland following an injection of 1.0 unit of thyrotropic hormone (Fig. 1) are bigger in mature than in aged animals. Blood sugar levels recorded after large doses of adrenaline or insulin reveal similar patterns. After an intravenous injection of insulin 1.5 un./kg blood sugar fell by 45.5 +__2.96 mg per cent and 57.5 + 3.56 mg per cent in aged and mature rabbits respectively, and after large doses of adrenaline (50 Fg/kg) by 33.2 _ 2.21 mg per cent and 84.2 + 9.4 mg per cent respectively. A highly significant element in the development of adaptation mechanisms within the endocrine system is represented by changes in the feed-back connections. Changes in the levels of hormones in blood and tissues influence all the subsequent links in the endocrine control system. The important link in this mechanism of age-induced changes of regulation is shift in sensitivity of the nerve centres. In aged animals smaller amounts of hormones are required to induce changes in the electrical activity of the brain. Insulin 0.0005 un./100 g produces more conspicuous changes of cerebral electrical activity in an aged compared with a mature adult animal (Fig. 2). In the case of the aged rat the hippocampus and amygdaloid area show synchronization of the theta-rhythm and isolated spikes within 3 min after the injection. The periodicity of rhythm-synchronization in the limbic system is associated with a slowing of (a) '
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116
v.v. FROLKIS
activity in the mesencephalic reticular system and posterior hypothalamus. In the 8-10th min the tracings show slow-amplitude rhythms in the anterior part of the hypothalamus, desynchronization in the reticular formation and in the cortex, evidence of inhibition of hippocampal activity. In the mature rat a similar dose of insulin produces in the 3rd min only the first phase of change in electrical activity of the brain (the theta-rhythm of the limbic system and a minor slowing down of the activity of the hippocampus and of the mesencephalie reticular formation.) The complex interconnections of the central nervous and endocrine systems are largely determined at the hypothalamo-pituitary level. Ageing is accompanied by changes in various aspects of the production and release of neurosecretory material. All phases of the neurosecretory cycle can be demonstrated in the supraoptic and paraventricular nuclei of 8-10-months-old rats, with evidence of formation and release of neurosecretory material. In rats aged 26-28 months all segments of the neurosecretory tract show predominance of the processes of accumulation of neurosecretions while their release is delayed. 45F
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R E G U L A T O R Y PROCESS I N T H E M E C H A N I S M O F A G E I N G
117
Nervous centers
-Weakening of introcentrol cOnnections - Increasing of the sensitivity to the humoral factors
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metabolism and various other functions. Homeostasis is maintained in spite of the fact that the functional reliability of the system declines. In the complex process of ageing of the body as a whole an important part is played by changes taking place in nervous centres. By securing adaptation to the environment the nervous centres are an important factor in predetermining longevity. However, when age-linked changes take place in the C.N.S. itself, they produce serious disruption in other organs and systems. According to the data collected in our laboratory the C.N.S. changes include weakening of the subordinating influence of the higher centres of the C.N.S. on the lower ones, an increase of the sensitivity of C.N.S. structures to humoral factors and regulatory alterations in the paths of various centro-peripheral influences. In aged rats the thresholds of inhibitory and facilitatory corticospinal and reticulospinal influences are raised. To demonstrate these influences against the background of spinal reflex activity, the higher centres must be stimulated with relatively stronger currents. For instance, in order to demonstrate inhibition of spino-cerebral reflexes in aged animals the motor cortex (areas 4 and 6) requires a stimulus of 1.2 + 0.12 V whereas in mature animals a current of 0.53 + 0.05 V is adequate. The thresholds of inhibitory reticulo-spinal impulses (produced by stimulation of the reticular formation of the mid-brain) in aged and mature animals are respectively 0.53 + 0.036 V and 0.3 + 0.014V, and those of facilitatory reticulo-spinal impulses 0.55 +_ 0-059V and 0.3 + 0.03V. The fact that ageing is associated with a weakening of the subordinating influence of higher centres is further confirmed by our data on the course of spinal shock after chordotomy. We have shown in 1959 that the first phase in the development of spinal shock is associated with over-strong stimulation and the second one, much longer, with
118
v . v . FROLKIS 125
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FIG. 5. Sensitivity of central structures to the humoral influences increases in old age. In old animals intravenous (a) and intraperitoneal (b) injections of the lesser dose of various substances cause the changes in the brain electrical activity. a-rabbits, b-rats. elimination of subordinating impulses. Chordotomy at the level of 6th dorsal vertebra was performed in rabbits and rats of various ages. A more profound depression of spinal functions was recorded in aged animals who also showed an earlier recovery of the initial level of excitability of the cord in the 2nd phase. In mature animals the decrement of spinal excitability was less steep in the initial few hours after chordotomy but was followed at a later stage by inhibition of the reflex activity of the brain. We attribute the rapid recovery of spinal function after chordotomy as observed in aged animals to a weakening of the subordinating influence of the higher centres of the brain on the lower centres. Further confirmation of this view is provided by the results of the experiments in which functional block of the spine was induced by cold. The resulting damage to the spine was much less severe than on section and the resulting inhibition of spinal function was much less pronounced in aged animals. These findings are not applicable to all central connections. We were unable to demonstrate any changes in the threshold of excitability on stimulation of amygdala when recording the activity of the hippocampus. Changes of intracentral connections in old age are accompanied by an increased sensitivity of C.N.S. structures to humoral factors. In aged rabbits smaller doses of active compounds are required to induce alterations in the electrical activity of various cerebral structures. That this is not due to increased permeability of blood-brain barrier was clearly demonstrated in a special series of experiments where the barrier was bypassed by introducing the compounds in question into the cerebral ventricles and directly into the individual structures. Under those conditions in aged animals smaller amounts of active substances were required to induce changes of bioelectrical activity. The threshold doses on intraventricular administration to old and mature rabbits were respectively: for adrenaline 0-22 + 0.03 and 1-70 + 0.61 mmkg/kg, for noradrenaline 0.22 + 0.33 and 2.36 + 0.88 mmkg/kg, for acetylcholine 0.09 _ 0.02 and 0.34 +__0.05
REGULATORY
PROCESS IN T H E M E C H A N I S M
OF AGEING
119
mmkg/kg. The changes of sensitivity of individual C.N.S. structures are not uniform. Consequently administration of these substances to the aged produce variable changes in neurodynamics and in the interrelationships of central structures. The increased sensitivityof neural structures to humoral stimuli may to some extent compensate for the weakening of tonic impulses transmitted along specific and nonspecific ascending paths. It must be pointed out, however, that in very old or senile animals this increase of sensitivityto humoral factors is much less evident. Alterations of the functional state of the C.N.S. arc reflectedin the functions of other organs and systems. Estimation of the threshold current required to induce changes in cardiovascular and respiratory functions when applied to hypothalamic nuclei and the limbic system has shown a weakening of the C.N.S. control in old age. However, no uniformity could be demonstrated in the pattern of changes occurring in various centroperipheral connections. In aged rats changes in arterialpressure can be induced by bipolar stimulation of the anterior part of the hypothalamus with currents of 3-0-3.5 V. In mature rats a current of 0.35-0.8 V is sufficient. The difference could not be demonstrated on bipolar stimulation of the posterior part of the hypothalamus. Bipolar stimulation, however, involves a great number of structures. In subsequent experiments we used local unipolar electrical stimulation and were able to demonstrate that the excitability of individual nuclei varies with age, but that the variation is not uniform; the excitability of some nuclei increases and that of others diminishes. For instance, in order to induce a liminal pressor reaction to stimulation of medial and lateral mammillary nuclei of the posterior part of hypothalamus currents of 0.054 + 0.0089 mA and 0"024 + 0.0002 mA were required in aged and mature rats respectively. The liminal vasopressor reaction to stimulation of the posterior hypothalamic nucleus requires currents of 0.046 + 0.053 mA in old and of 0.081 + 0-0055 mA in mature rats. When the stimulus is applied to the paraventricular nucleus of the anterior part of the hypothalamus the liminal pressor reaction is obtained when the current reaches 0.148 + 0.0442 mA and 0-080 + 0.0198 mA in the aged and in the mature animals respectively. Stimulation of the lateral pre-optic zone with currents of 0.030 + 0.006 mA in aged and 0.025 + 0.0031 mA in mature animals produce liminal pressor reaction. All these allometric changes in the sensitivities of various C.N.S. structures lead to qualitative as well as quantitative changes in the central regulatory mechanisms of various physiological systems. The weakening of centroperipheral connections in old age produces a situation where C.N.S. shifts may fail to be reflected at the periphery. It is well known, for instance, that the excitatory wave associated with a convulsion involves the vagal centre and produces bradycardia. In mature rabbits bradycardia failed to occur in 3 of 14 fits. On injection of subliminal doses of convulsants bradycardia failed to develop in 12 of 23 experiments on mature rabbits. In aged rabbits 9 of 14 experimentaUy induced fits were not accompanied by bradycardia. The changes in the C.N.S. functions described above, whether they affect intercentral or centro-peripheral connections, are an important factor in the alterations of the metabolic and overall functional control of the ageing animal and in the reduction of its adaptive powers. At the same time the intimate mechanism of age-linked changes in the regulatory processes depends on alterations in metabolism at the molecular level. Let us examine next therefore the metabolic basis of the regulatory processes. Cholinergic effects on tissue are connected with the synthesis and hydrolysis of
120
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acetylcholine and the reactivity of choline-sensitive receptors. We investigated agelinked changes in the metabolism of acetylcholine in the heart, skeletal muscles, vegetative ganglia, intestine and endocrine glands. In old age the concentration of acetylcholine in the heart falls (Fig. 6) because of the increased rate of hydrolysis and diminished rate of synthesis. The latter is largely due to the reduced activity of the choline acetylase which activates the transfer of the acetyl group from coenzyme A to choline. But Cholinesterase activity falls simultaneously, and the overall rate of acetylcholine hydrolysis is reduced. The reduction in the rate of hydrolysis of the enzyme combined with diminished rate of its synthesis helps to maintain the cholinergic control of the cell. At an earlier stage of our investigation we demonstrated age-linked changes in the choline-sensitive receptors themselves. With advancing age the number of active groups in the receptor diminishes but their sensitivity to the mediator increases. A connection between these changes in the receptor and alterations in the reactive capacity of sulphydryl groups has been experimentally demonstrated.
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121
REGULATORY PROCESS IN THE MECHANISM OF AGEING
These data suggest the following mechanism of the age-linked changes of cholinergic impulses. Loss of nerve terminals and reduced rates of synthesis of acetylcholine weaken the nervous influences on tissues. The reduced intensity of hydrolysis of the mediator and changes in the choline-sensitive receptors enhance the sensitivity of the ceils to cholinergic humoral factors. Data on age-linked changes in the metabolism of catecholamines are presented in Fig. 7. The reduction in the amount of protein-bound noradrenaline and of the potential capacity of the tissues prolongs the action of exogenous adrenaline and thus enhances its effect in aged animals. This is associated with rearrangements in the various metabolic pathways of catecholamines. So the activity of monoaminooxidase increases with age in spite of the fact that activities of other metabolic pathways are reduced. Hormones play an important role in energy metabolism. Changes in the latter lead to functional alterations in the cell. Smaller amounts of mediators or hormones are required to induce changes of the cell energy metabolism in the aged animals compared with the mature ones. The increases in the rates of phosphorylase activity, glycolysis and glycogenolysis are more pronounced after injections of small doses of adrenaline in aged rats, and after large doses-in mature animals. How prominent the action of adrenaline is will depend to a large extent on the effect of the hormone on the mitochondrial processes of oxidative phosphorylation. A dissociation of oxidation from phosphorylation, with an increased rate of free oxidation, are responsible for the so-called "caloric" effect of adrenaline. Figure 9 shows that in aged rats an injection of adrenaline produces a considerable dissociation between oxidation and phosphorylation processes in the mitochondria of the cells of the heart. Thyroxine is an important factor in the regulation of cell energy metabolism. Our experiments have shown that small doses of thyroid hormone produce more prominent Laclate % 300 28O 26O 240 P in rn~/g Activity of tissue 220 5 of '~' phosphonilase 200
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glycogenolysis intensity is more pronounced in the hearts of old rats but the effect of high doses is more pronounced in adult rats.
122
v . v . FROL'KIS 8-12 months old rats
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FIG. 9. Uncoupling adrenaline effect at oxidative phosphorylation was more pronounced in the old rats heart. changes in the gaseous exchanges, in the intensity of tissue respiration of the liver and myocardium, more evident dissociation of oxidation from phosphorylation and more marked enhancement of glycolysis in aged rats. When large doses are given the effects are more prominent in mature animals. It appears therefore that in aged animals smaller amounts of hormones or mediators are required to produce metabolic changes. However, with higher doses the changes are more marked in mature animals. This explains the age-linked increase of tissue sensitivity to humoral factors and the depression of their reactivity. In an aged animal we are dealing therefore with a qualitatively different pattern of neurohormonal regulation. The enhanced tissue sensitivity to hormones along with depressed endocrine function, the increased tissue sensitivity to humoral factors along with depressed neural influences, the decrease of excitability in some of the neural structures and its increase in others, changes in the feed-back information from the receptors, etc. can be considered as specific instances of adaptation. Thanks to these allometric changes in the various control mechanisms, homeostasis is still maintained into old age. In the aged in various pathological situations the regulatory mechanisms are upset relatively easily by strong stimuli with consequent collapse of homeostasis. The results of our investigation show that in different periods of life different mechanisms are responsible for maintaining the indices of homeostasis at constant levels. For instance, different values for peripheral resistance and cardiac output concur in the maintenance of the blood pressure at the same level in the young and in the elderly. The maintenance of constant blood sugar level is achieved, as already indicated, by increasing the tissue sensitivity to insulin, adrenaline and cortisone,
REGULATORY PROCESS IN THE MECHANISM OF AGEING
123
by increasing the sensitivity of the receptors to variations in the level of blood sugar and by enhancing cellular glycolysis. In this way the outward immutability of a number of homeostatic indices in the aged masks quantitative and qualitative changes in the regulatory mechanisms involving complex and coordinated depression of some, and emergence of other adaptative reactions. Acknowledgements--Data presented in this paper were received due to research work of my collaborators : V. Bezrukov, L. Bogatskaya, T . Duplenko, E. Yenis, A. Murashina, N. Verkhratsky, N. Verzhykovskaya, V. Shevchuk, V. Sinitsky, S. T a n i n and J. Oschogoleva.