Fast, rate-sensitive corticosteroid negative feedback during stress MASANORI Department
KANEKO AND TSUTOMU HIROSHIGE of Physiology, Hokkaido University School
rate-
of Medicine,
Supporo
060, Japan
sensitive corticosteruid negative feedback during stress. Am. ‘J. Physiol. 234(l): R39-R45, 1978 or Am. J. Physiol: Regulatory Integrative Comp. Physiol. 3(l): R39-R45, 1978. - Characteristics of the fast, rate-sensitive, negative-feedback regulation of adrenocorticotropin secretion during stress was quantitatively analyzed using rats anesthetized with pentobarbital sodium. Various levels of plasma corticosterone were achieved during morning hours by infusing corticosterone solutions of different concentrations. Blood was sampled serially from the carotid artery. An increase in plasma corticosterone concentration 15 min after intravenous, pulsed injection of histamine (230 pg) during saline intravenous infusion was defined as the “control response.” When plasma corticosterone was rising during corticosterone infusion, the response to histamine stimulus was distinctly inhibited (fast, rate-sensitive feedback inhibition), whereas such an inhibition was not observed when plasma corticosterone levels were not rising, regardless of the absolute level. The critical rate of rise of plasma corticosterone, at or above which the fast rate-sensitive feedback was manifested, was 4-6 pg/lOO ml per min. When three graded doses of histamine were injected while plasma corticosterone levels were increasing at a rate of 6 pg/lOO ml per min, the absolute value of the inhibition observed was independent of the administered dose of the stressor. A hypothetical model for the mechanism of this feedback inhibition, based on the assumption that the hormone effect was proportional to the rate of formation of hormone-receptor complex, satisfied the quantitative characteristics of the inhibition experimentally observed in this study.
times suppress the stress-induced activation of the pituitary-adrenal system in the rat (13). Having considered carefully the differences among the experimental designs of the workers cited, Dallman and Yates (2) then showed that two different kinds of inhibitory negative feedback exist: 1) a fast, rate-sensitive feedback and 2) a delayed, proportional, levelsensitive feedback. These findings not only explained the discrepancies among the experimental data from different laboratories, but also provided a new approach to the study of the negative-feedback regulation of ACTH secretion under stress. Subsequent studies from other laboratories (6, 18) confirmed the existence of the fast, rate-sensitive feedback inhibition in the rat under different experimental conditions. On the other hand, failure to confirm its existence was reported in the rat with laparotomy stress (14)In this paper, we aimed, first, to confirm the existence of this type of negative feedback under conditions similar to those described by Dallman and Yates (2), as well as under conditions with other noxious stimuli. Second, we proposed to analyze the characteristics of the fast, rate-sensitive negative feedback in order to disclose its operational features. Third, on the basis of our experimental observations, we attempted to construct a model that might explain the mode of action of this feedback inhibition under stress.
adrenocorticotropin;
MATERIALS
KANEKO,
MASANORI,
AND
TSUTOMU
HIROSHIGE.
Fast,
corticosterone
REGULATION of stress-induced adrenocorticotropin (ACTH) secretion by corticosteroids was clearly demonstrated long ago (E), but several quantitative and temporal aspects of the inhibition are still unclear. Yates and Urquhart (16) proposed an hypothesis that assumed the existence of a set point for the operation of the hypothalamo-pituitary-adrenal system at rest and under stress. Their theory claimed that the activation of the system under stress followed elevation of a set point probably located in the central nervous system (CNS). The difference between this set point and the actual blood steroid level activates the system; the operation of the system is controlled by a closed feedback loop even under stress. This hypothesis, met several dificulties. For though very attractive, example, maintenance of elevated blood steroid levels by continuous infusion of corticosteroid did not at all NEGATIVE-FEEDBACK
AND
METHODS
Female Wistar rats weighing 170-220 g were used. They were housed at constant temperature of 23 t 1°C under 12-h light-dark cycle with the light period beginning at 6:30 A.M. Rat chow (Oriental Yeast Co.) and water were available ad libitum. The experiments were performed between 8 A,M. and 11 A.M. when plasma corticosterone levels are low and stable. Corticosterone (50 mg) dissolved in 1.5 ml of acetone was added to 100 ml of physiological saline solution, which was then placed for 2 h in a water bath (37°C) under nitrogen, to evaporate acetone and to prevent oxidation of corticosterone. The completion of evaporation was estimated by the disappearance of the smell of acetone. Thus prepared, the solution was stored at 5°C until use. Immediately before use it was further diluted with saline solution to appropriate concentrations. Intravenous infusions. The rats were anesthetized with pentobarbital sodium (Nembutal, Abbott) (5.5 mg/ 100 g body wt). Forty minutes later, when plasma
R39
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R40 corticosterone levels were basal, an intravenous infusion was started through a lateral caudal vein at a rate of 28 pl/min. The infusion was either saline, or corticosterone in various concentrations. Stresses. Two stresses were used: histamine and laparotomy plus traction. Histamine (Wako Junyaku Co.) was injected (230, 460, and 1,150 pg dissolved in saline, with final volumes of 0.25-0.50 ml) through a lateral caudal vein under pentobarbital anesthesia. The injection was completed within 10-G s. Laparotomy stress consistedbof abdominal incision 2-3 cm long under pentobarbital anesthesia, followed by mild traction of the intestinal tract with forceps. In all the stress experiments, incremental responses in plasma corticosterone levels were examined 15 min after the start of stress, unless otherwise indicated. In the early stage of the experiment, trunk blood was collected by decapitation. Afterward, to study changes in plasma corticosterone levels in single rats, arterial blood was withdrawn serially through a plastic cannula that had been inserted into the left common carotid artery 2 days before the experiment. The amount of blood sampled at one time was 0.6 ml or less, and the total volume of blood removed in the longest experiment (135 min) was less than 3.0 ml per rat. Plasma corticosterone concentrations were determined by the fluorometric method of Zenker and Bernstein (17), with minor modification. To simulate the overall operation of the fast, ratesensitive negative feedback, an analog computer of Hitachi 200X type was used. Statistical analysis was performed with the Student f-test.
M.
I 0
KANEKO
AND
T. HIROSHIGE
1
I
15
30
Time(md FIG. 1. Response of pituitary-adrenal axis to histamine stress. At zero time, 230 pg histamine dissolved in 0.25 ml saline (standard stimulus) were injected through lateral caudal vein in rats under pentobarbital anesthesia. Incremental change in plasma corticosterone concentrations at 15 min was defined as “control response.” Numbers of rats are shown in parentheses. Vertical lines give standard errors.
RESULTS
Demonstration of a fast, rate-sensitive feedback inhibition under histamine stress. The time course of the response to 230 pg histamine (standard dose) is shown in Fig. 1. This dose of histamine intravenously caused a peak increment in plasma corticosterone of approximately 30 pg/100 ml, 15 min after the injection. Therefore, in the following experiments the change in plasma corticosterone level 15 min after the onset of stress was adopted as a measure of the responsiveness to stress. In Fig. 2, the time course of plasma corticosterone concentration is shown before and during a constant infusion of corticosterone-saline solution. The plasma corticosterone concentration showed a tendency to rise almost linearly during the first 5 min of infusion. The rate of rise then fell off, and 15 min after the onset of infusion the plasma steroid level attained a new plateau that was approximately 40 pg/100 ml higher than the initial basal level. Thereafter corticosterone concentration was maintained at the elevated level over the 120min period of infusion. Infusion of physiological saline alone at the same rate caused no elevation. Intravenous injections of the standard dose of histamine were made at various times after the start of infusion. At the 3rd min, the response was significantly reduced, but at the Eth, 20th, and 30th min the responses were as large as the control response obtained when the plasma corticosterone was at the lowest level in the morning. At the 120th min, the response was completely abolished.
I
I
I
0
30 Time
#/
1
IJ 90
L
120
(min>
2. Negative-feedback inhibition of stress response by plasma corticosterone. Plasma corticosterone was elevated by intravenous infusion of corticosterone-saline solution (2.52 pg/r&n) (vm), started at 0 min. Each point is mean value of data from 4-9 rats (2 one standard error (SE) indicated by vertical bars). Control group was infused with physiological saline solution under the sami conditions (v&Standard stress stimulus (iv injection of 230 pg histamine) was given in various phases of changes in plasma corticosterone concentration 3, 15, 20, 30, 90, and 120 min. Rats were decapitated 15 min after histamine injection to collect trunk blood for measurement of plasma corticosterone concentration. Plasma corticosterone levels attained (x) are shown at times of histamine injections, when responses were initiated. Numbers of ratts used are shown in parentheses. Responses were distinctly reduced in the phase of rapid increase of plasma corticosterone (P < 0.0011, and no response was observed 2 h after a high plasma corticosterone level had been attained, FIG.
Characteristics of the fast, rate-sensitive feedback inhibition. To facilitate quantitative analysis of the fast, rate-sensitive feedback inhibition, blood from individual rats was sampled serially during constant infusions of various amounts of corticosterone. As shown in Fig. $4, the basal level of plasma corticosterone was somewhat higher in these chronically cannulated rats, but was within a reasonable range for the present purposes. The rate of rise of plasma corticosterone level was ascertained in each rat by serial sampling,
by estimating the rate from the differencebetween the
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FAST
FEEDBACK
INHIBITION
R41
MODEL
*-v 1 0
1
1
1
5
10
15
Time
0 0 ff
1
JJ
30
of stress. Three different doses of histamine were injected 3 min after the start of corticosterone infusions, when plasma corticosterone levels were rising at the rate of 6 pg/lOO ml per min. To obtain this rate of rise, corticosterone infusions at the rate of 2.27-4.03 pglmin were used, The rate of rise differed to some extent in different rats, for a given infusion rate, Only the data from rats that showed approximately 6 pg/lOO ml per min rate of rise ape recorded in Fig. 5. The control responses show a clear dose-dependent relationship. The responses observed when plasma corticosterone
(mid
a l
l
8
r
l
l 1
1
1
0
15 Time
1
1
30
45
(mid
FIG. 3. Changes in plasma corticosterone levels of individual rats during continuous infusions of corticosterone-saline solutions. Arterial blood was repeatedly withdrawn through an indwelling catheter in the carotid artery. A: at zero time, infusions of corticosterone ranging from 0 to 4.03 kglmin were started (closed circles). Control group was infused with saline under the same conditions (open circles). B: corticosterone infusion was started at 0 min and stopped at 15 min.
basal level and the 5th min value. Then the magnitude of stress-induced response was measured in the same animal. By varying the rates of rise of plasma corticosterone concentration, we could estimate the relationship between the rate of rise and the degree of suppression of the response. The plasma steroid level, once elevated, fell sharply as soon as the infusion was stopped (Fig. 3B). Here in the same manner we can estimate the rate of decline of plasma corticosterone levels in relation to the magnitude of stress-induced responses. The results are summarized in Fig. 4. The control response obtained when the standard dose of histamine was given during steady, basal concentrations of plasma corticosterone (i.e., during a rate of change of 0 pg/lOO ml per min) was approximately 30 pg/lOO ml. As the rate of rise of plasma corticosterone was increased, the magnitude of response became progressively smaller: the inhibition of the response became progressively larger. At the rate of 4-6 pg/lOO ml per min, the stress response was almost completely abolished. In contrast, when plasma corticosterone levels were declining, no inhibition was observed. In the next series of experiments, the degree of inhibition was examined in relation to the mamitude
-6 Rate
1
1
-4 -2 of change
1
1
I
?
0 2 4 6 in plasma corticosterone(
1
I
8 10 pg/lmml/min)
FIG. 4. Relationship between rate of change in plasma corticosterone concentration and degree of fast, rate-sensitive inhibition. Fifteen-minute incremental response of plasma corticosterone levels to 230 pg histamine was measured at each rate of change in plasma corticosterone. Different rates of rise and fall of plasma corticosterr one levels were obtained by turning on and off graded infusions of corticosterone, as shown in Fig. 3, A and B. Histamine was injected 3 min after the start of infusion, in the rising phase of corticosterone levels and at various times in the declining phase after the infusion was stopped.
460 Histamine()rg)
1150
FIG. 5. Relationship between degree of fast, rate-sensitive inhibition and intensity of stressful stimulus. White coZumn in each group indicates control responses to histamine, 230, 460, and 1,150 pg, respectively, Shaded columns indicate responses obtained when plasma corticosterone was increasing at the rate of 6 pg/lOO ml per min. Numbers in parentheses indicate numbers of determinations. Lines at tops of columns are + 2 SE.
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R42
M.
was rising at a rate of 6 pg/lOO ml per min also appeared to show a dose-response relationship. Inhibition of the responses, given as the differences between white and shaded columns in respective pairs in Fig. 5, appeared to have a constant absolute value regardless of the intensity of stimulus applied, if the rate of rise of plasma corticosterone was fixed. With laparotomy plus traction stress the inhibitory effect was not clear-cut. As shown in Fig. CIA, the response to laparotomy plus traction during corticosterone infusions, producing rates of rise in corticosterone levels of 6 pg/lOO ml per min, was not significantly different from the control response in the absence of corticosterone infusion. It is, however, noteworthy that the effect of laparotomy stress on plasma corticosterone levels continued at least 1 h, in marked contrast to the effect of histamine, which disappeared during the l-h period of observation (Fig. 6.B). Model of the mode of action of the fast, rate-sensitive feedback inhibition. Unique features of this feedback inhibition are its rapidity and rate sensitivity, both of which are rather difficult to reconcile with the general
KANEKO
AND
T. HIROSHIGE
concept of the mode of action of steroid hormones on various target tissues (3). The initial event in a series of processes of hormone action in the fast, rate-sensitive feedback inhibition must be the recognition of the rate of change in hormone concentration by target cells. As a basis for this recognition, we adopted the following two assumptions: 1) the union of hormone and receptor obeys the law of mass action; and 2) the hormone effect is proportional to the rate of formation of hormonereceptor complex. These assumptions are represented by the following mathematical expressions where X: hormone concentration in the blood R: “concn” of unoccupied receptors located on the target cells XR: “concn” of occupied receptors (bound) K 1: association rate constant K,: dissociation rate constant P: ratio of bound receptors to total receptors, (XRI(R + XR)); fraction of receptors that is occupied Y: hormone effect, i.e., inhibition of the response to stress (lessening of the increase in hormone concentration provoked by stress) t: time m, n, a, /3, 4: constants from mass action (assumption 1): K X+RsXR K2 and therefore, from definition of P dP dt = K,X - (KJ
Lapar
lapar under
inf.
+ K,)P
(1)
and from rate hypothesis (assumption 2): Y = $K,X(l
- P)
(2)
The changes in the hormone concentration in the circulating blood produced by a constant intision of hormone, or constant secretion, with first-order kinetics of hormone removal, are given by X = m(1 - emu’)+ n
(3)
which corresponds to the phase of rise of hormone concentration, and bY 0 A
-5
A
1
0
15 Time
fM
(min)
FIG. 6. Response to laparotomy plus intestinal traction. In the rat anesthetized with pentobarbital sodium, an abdominal incision was made and the intestinal tract was mildly manipulated. A: failure to demonstrate fast, rate-sensitive inhibition to laparotomy+ White column indicates control response 15 min after stress. Shaded column indicates response when plasma corticosterone concentration was rising at rate of 6 pgflO0 ml per min. B: duration of effect of laparotomy. Rats were left untouched after laparotomy stress until decapitation at 60th min (0). Response pattern to histamine stress is also shown for comparison @---a>.
X
= me-@’
+ n
(4)
which gives the time course of the declining phase of hormone concentration, To simulate the feedback inhibition on the basis of these equations, the values of constants must be determined within the range where physiological criteria are well satisfied. ti and p represent coefficients of exponential functions and need not be equal to each other because of dynamic asymmetries in the system (2). 4 represents the constant by which the rate of formation of hormone-receptor complex is converted to the physiological inhibitory effect. These constants can therefore be arbitrarily determined so as to fit the
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FAST
FEEDBACK
appropriate
INHIBITION
R43
MODEL
data of Figs. 3, A and B, 4, and 5. Constant
n represents the morning basal level of plasma corticos-
terone (10 pg/lOO ml). The physiological range of the plasma corticosterone concentration (X) in female rats is from 10 to 100 pg/lOO ml, i.e., from 2.9 x 10e7to 2.9 x 10-” M. Therefore, if n takes the value of 10, m. varies between 0 and 90, as shown by Eq. 5 below. The hormone concentration in the steady state, during infusion of secretion is 1imX F lim {m(l - e-l*/) + n} = m + n I--+=
t-r:
(5)
We now assume an ideal response to the standard histamine stimulus when there is initially no corticosterone in the body fluid (when t = 0, p = 0). When X now adopts any positive value as a consequence of stimulation, from Eq. 1, we obtain, by integration, and by assuming that X is fully independent of P and may be treated as a parameter in the relation of P to t
p=-
X
(1 K x+2 Kl
Therefore, after substituting
e-(K,.‘t’
+ h’,,t
I
(6)
into
Time 7. Theoretical curves of fast, rate-sensitive inhibition based on our hypothesis. Hormonal effect, feedback inhibition of stress response, corresponding to the basal, steady level of plasma corticosterone was arbitrarily set to zero. Dashed curue indicates declining phase of plasma corticosterone after cessation of infusion, during which no hormonal inhibitory effect was found. Curves numbered 1-7 in upper figure correspond to curves numbered l-7 in lower figure, and represent responses to increasing rates of infusion of corticosterone. FIG.
(7) the rising phase of the plasma corticosterone response. and so, in the steady state, the hormone effect is expressed as lim Y = +K.,I t-3;
[
~--_---m+n
(m + n) +y
K2
1
(8)
In addition, the degree of inhibition is proportional to the rate of rise of the plasma corticosterone (dose response). This model describes the effects of corticosteroid feedback during an initial exposure. We do not know to what extent data from repeated stress exposures, repeated infusions, or adrenalectomy might require a modification of the model.
1
From Eqs. 6 and 8, it follows that in the steady state the hormonal inhibitory effect, Y, is simply proportional to the fraction of receptors that is occupied (P)
where the subscript ss denotes steady-state. However, the inhibitory effect, Y, actually appears to be independent of the levels of corticosterone, for several hours. (The magnitude of the corticosterone response to the standard histamine stimulus remains the same (about +30 pg/lOO ml) regardless of the initial steady levels of plasma corticosterone, which may be basal (Fig. 1) or elevated by infusions (Fig. Z).) Therefore, K,/K, must be enough smaller than (m + n) so Y remains relatively constant for various values of (m + n) in the range of 10-100. For this reason, the value of the equilibrium dissociation constant K,/K, was set in the range of 0,005-O. 15 by assigning 987 to K, and the range 5-150 to K,. Simulation on an analog computer on the basis of this model produced a set of curves (Fig. 7), predicting the time course of corticosterone concentration, and its inhibitory effects, during constant infusions. According to this simulation, the hormonal effect or degree of negative feedback inhibition of stress-induced response is apparently short-lived, and only manifested during
DISCUSSION
The present study has amply confirmed the finding of Dallman and Yates (2) that there are two different types of negative-feedback inhibition of stress-induced activation of ACTH secretion: 1) the fast, rate-sensitive and 2) the delayed, proportional feedback inhibition. We have also confirmed that the fast feedback component is unidirectional under conditions similar to those employed by Dallman and Yates. Recently, Papaikonomou and Smelik (9) presented evidence that suggested the existence of a derivative controller which is sensitive to rates of decrease in blood corticosterone levels in the rat. Their results, however, cannot be directly compared with ours, because their experiments were performed with acutely operated rats, and in the afternoon. It has been reported that the fast, rate-sensitive negative feedback is demonstrable with other stressors such as sham adrenalectomy (6, 11). We also observed that the same feedback mechanism effectively inhibited the response induced by adrenaline injection (unpublished observation). Thus it appears that the fast, ratesensitive feedback is not a limited mechanism, designed for a particular type of stressor, but is a more general characteristic applicable to a wide variety of stressors. In this connection, failure to demonstrate the inhibition
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R44 with laparotomy stress both in the li terature (14) and in th e present experiment, des lerves commen t. In our experiment, laparotomy was performed together with mild traction of the intestinal tract. Because the effect of this combined stress lasted at least 1 h (Fig. W), and because this type of stress is known to be resistant to corticosteroid feedback (11, our failure to demonstrate fast feedback inhibition of it may not be surprising. The critical rate of rise of plasma corticosterone, above which the response to histamine stress was virtually abolished, was 4-6 pg/lOO ml per min in our study, using female rats. On the other hand, Jones et al. (6) reported the critical rate to be 1.3 Fg/lOO ml per min with sham adrenalectomy in male rats. Such a difference in the critical rate seems to show a sex difference. However, since free and not bound corticosterone is the effective feedback signal (7) and the proportion of the bound form is significantly greater in female rats than in male rats (4), the difference is more likely apparent than real. In our attempts to construct a hypothetical model to explain the mode of action of the fast, rate-sensitive feedback, we adopted the assumptions employed in Paton’s rate theory of drug action (10): 1) the union of drug with receptor obeys the law of mass act ion and 2) the drug effect is proportional to th e rate of formation of drug-receptor complex. He performed an outstanding analysis on the mode of action of acetylcholine on the smooth muscle of the guinea pig ileum in v itro. The drug concentration in the incubati on med ium was kept constant in the experiment of Paton, whereas in our experiment the plasma corticosterone levels were changing during transient phases. Jones et al. (6) also proposed the Paton theory as a model for fast ratesensitive feedback mechanism in 1972. Essential features, theoretically derived, of the fast feedback inhibition based on our hypothesis are as follows. 1) As soon as the hormone concentration begins to increase, the hormone effect starts to appear and reaches a maximum rapidly. As the rate of rise in hormone concentration becomes smaller, the effect is progressively reduced and finally disappears before the hormone level reaches a ne W platea u. 2) The magnitude of the effect is proportional to the ra.te of rise of hormone concentration, but no effect is demonstrable when the hormone concentration is declining. 3) The magnitude of the effect is not essentially influenced by the intensity of stress, but depends on the rate of rise of hormone concentration. These predictions appear to agree well with the characteristics of the fast rate-sensitive feedback inhibition which were observed experimentally in this study. In the processes of this simulation, one of the crucial points was the choice of the value for the dissociation equilibrium constant, K,/K,. The dissociation equilibrium constant of corticosterone from site(s) which must be related to the fast rate-sensitive feedback is still unknown. According to Suyemitsu and Terayama (15), the dissociation equilibrium constant of cortisol from the hepatic cell membrane is 1.4 x 10mgto l-3 x lop8 M,values roughly one-tenth of the physiological level of free corticosterone concentration in the rat plasma (2.9 x lO+ to 2.9 x W7 M). Munck and Brinck-Johnsen (8)
M.
KANEKO
AND
T. HIROSHIGE
estimated the dissociation equilibrium constant of cortisol in thymus lymphocytes to be 4 x 10W8M. Provided that the dissociation equilibrium constant of corticosterone from the putative site(s) of the rate-sensitive feedback is comparable to the above cited constants, variations in the magnitude of the control response obtainable at different steady Ievels of plasma corticosterone (e.g., from 2.9 x 10e7 to 2.9 x lo-” M, with roughly one-tenth of it in the free form) can be calculated to be only 5% or less according to our model. These considerations are consistent with our experimental finding that the response to standard stress remains almost the same when steady levels of plasma corticosterone are either high or low. As will be noticed, the principal assumption behind our model is that the rate-sensitive feedback action is equi valent to the phenomenon of binding itself. The model is really a description of the binding process. The theoretical curves (Fig. 7) imply that the actual time course of the hormone effect may vary subtly according to different rates of rise of plasma steroid concentration, It is predicted that infusions of corticos&one at low rates lead to prolonged feedback effects, compared to infusions at high rates, despite the fact that the rising concentration of corticosterone continued longer at the high rate of infusion. But we have no evidence to confirm or deny this point. To clarify this point, changes in plasma ACTH concentration must be followed. In spite of the apparently successful simulation, the basic assumptions employed here are not necessarily valid, because we have no evidence in their favor at the cellular or subcellular level. Hallahan et al. (5) examined the time course of inhibition of glucose. uptake by glucocorticoid in the thymus lymphocytes in vitro. According to them, the essential step in this phenomenon is the attachment of corticoid-receptor complex to a certain specific site on the surface of the nuclear membrane. Furthermore, quite interestingly, the subsequent messenger RNA synthesis proceeds pari passu with the rate of union of the corticoid-receptor complex with the acceptor sites on the nuclear membrane. Thus, their finding seems to be in terpreta .ble by a mechanism similar to rate-sensitive feedback inhibition. However, in the case of the thymus lymphocytes nearly 30 min are required for cortisol to begin to inhibit glucose uptake, whereas in this feedback phenomenon the effect becomes manifested within several minutes. This astonishing rapidity of effect suggests that synthetic processes of a messenger RNA, or protein, are not involved in its operation. In the following paper we demonstrate that the fast, rate-sensitive feedback involves a catecholaminergic stage in the brain, whereas delayed, level-sensitive corticosteroid negative feedback involves a serotonergic stage (6a). The authors are indebted to Professor K. Furukawa, Dept. of Anesthesiology, Hokkaido University School of Medicine, for his help in the use of an analog computer of Hitachi 200 X type. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education of Japan Grants 911304 of Specified Category I974 and 010904 of Specified Category 1975). Received
for publication
8 March
1977.
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REFERENCES 1. DALLMAN, M. F., AND F. E. YATES. Anatomical and functional mapping of central neural input and feedback pathways of the adrenocortical system. Mem. Sot. Endocrinol. 17: 39-72, 1968. 2. DALLMAN, M. F., AND F. E. YATES. Dynamic asymmetries in the corticosteroid feedback path and distribution-metabolismbinding elements of the adrenocortical system. Ann. N. Y. Acad. Sci. 156: 696-721, 1969. 3. FEIGELSON, P., AND M. FEIGELSON. Studies on the mechanism of cortisone action. In: Actions of’ Hormones on Molecular Processes, edited by G. Litwick and D. Kritchevsky. New York: Wiley, 1964, p. 218-233. 4. GARA, R. R., AND U. WESTOPHAL. Corticosteroid-binding globulin in the rat: studies on the sex difference. Endocrinology 77: 841-851, 1965. 5. HALLAHAN, C., D. A. YOUNG, AND A. MUNCK. Time course of’ early events in the glucocorticoids on rat thymus cells in vitro. J. BioL. Chem. 248: 2922-2927, 1973. 6. JONES, M. T., F. FL BRUSH, AND P. L. B. NEAME. Characteristics of fast feedback control of corticotropin release by corticosteroids. J. Endocrinol. 55: 489-497, 1972. 6a. KANEKO, M., AND T. HIROSHIGE. Site of fast, rate-sensitive feedback inhibition of adrenocorticotropin secretion during stress. Am. J. Physiol. 234: R46-R51, 1978 or Am. J. Physiol.: Regulatory Integrative Comp. Physiul. 3: R46-R51, 1978. 7. KWAI, A., AND F.E. YATES. Interference with feedback inhibition of adrenocorticotropin release by protein binding of corticosterone. Endocrinology 79: 1040-1046, 1966. 8. MUNCK, A., AND T. BRINCK-JOHNSEN. Specific and nonspecific physicochemical interactions of glucocorticoids and related steroids with rat thymus cells in vitro. J. Biol. Chem. 243: 55565565, 1968.
9. PAPAIKONOMOU, E., AND P. G. SMELIK. Evidence for derivative control in the rat pituitary-adrenal system. Am. J. Physiol. 227: 137-143, 1974. 10. PATON, W. D. H. A theory of drug action based on the rate of drug-receptor combination. Proc. Roy. Sot. London, Ser. B 154: 21-69, 1961. 11. SATO, T., M, SATO, J. SHINSAKO, AND M.DALLMAN. Corticosterone-induced changes in hypothalamic corticotropin-releasing factor (CRF) content after stress. Endocrinology 97: 265-274, 1975. 12. SAYERS, G., AND M. SAYERS. Regulation of pituitary adrenocorticotropic activity during the response of the rat to acute stress. Endocrinology 40: 265-273, 1947. 13. SMELIK, P. G. Failure to inhibit corticotrophin secretion by experimentally induced increases in corticoid levels. Acta Endocrinol. 44: 36-46, 1963. 14. SMELIK, P. G. Failure to demonstrate a fast rate sensitive feedback action of corticosteroids during adrenocortical activation. Acta Endocrinol. Suppl. 177: 149, 1973. 15. SUYEMITSU, T., AND H. TERAYAMA. Specific binding sites for natural glucocorticoids in plasma membranes of rat liver. Endocrinology 96: 1499-1508, 1975. 16. YATES, F. E., AND J. URQUHART. Control of plasma concentrations of adrenocortical hormones. Physiol. Rev. 42: 359-443, 1962. 17. ZENKER, N., AND D, E. BERNSTEIN. The estimation of small amounts of corticosterone in rat plasma. J. Biol. Chem. 231: 695-701, 1958. 18. ZIMMERMAN, E., AND V. CRTTCHLOW. Short-latency suppression of pituitary-adrenal function with physiological plasma levels of corticosterone in the female rat. Neuroendocrinobgy 9: 235243, 1972.
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KANEKO AND TSUTOMU HIROSHIGE of Physiology, Hokkaido University School
rate-
of Medicine,
Supporo
060, Japan
sensitive corticosteruid negative feedback during stress. Am. ‘J. Physiol. 234(l): R39-R45, 1978 or Am. J. Physiol: Regulatory Integrative Comp. Physiol. 3(l): R39-R45, 1978. - Characteristics of the fast, rate-sensitive, negative-feedback regulation of adrenocorticotropin secretion during stress was quantitatively analyzed using rats anesthetized with pentobarbital sodium. Various levels of plasma corticosterone were achieved during morning hours by infusing corticosterone solutions of different concentrations. Blood was sampled serially from the carotid artery. An increase in plasma corticosterone concentration 15 min after intravenous, pulsed injection of histamine (230 pg) during saline intravenous infusion was defined as the “control response.” When plasma corticosterone was rising during corticosterone infusion, the response to histamine stimulus was distinctly inhibited (fast, rate-sensitive feedback inhibition), whereas such an inhibition was not observed when plasma corticosterone levels were not rising, regardless of the absolute level. The critical rate of rise of plasma corticosterone, at or above which the fast rate-sensitive feedback was manifested, was 4-6 pg/lOO ml per min. When three graded doses of histamine were injected while plasma corticosterone levels were increasing at a rate of 6 pg/lOO ml per min, the absolute value of the inhibition observed was independent of the administered dose of the stressor. A hypothetical model for the mechanism of this feedback inhibition, based on the assumption that the hormone effect was proportional to the rate of formation of hormone-receptor complex, satisfied the quantitative characteristics of the inhibition experimentally observed in this study.
times suppress the stress-induced activation of the pituitary-adrenal system in the rat (13). Having considered carefully the differences among the experimental designs of the workers cited, Dallman and Yates (2) then showed that two different kinds of inhibitory negative feedback exist: 1) a fast, rate-sensitive feedback and 2) a delayed, proportional, levelsensitive feedback. These findings not only explained the discrepancies among the experimental data from different laboratories, but also provided a new approach to the study of the negative-feedback regulation of ACTH secretion under stress. Subsequent studies from other laboratories (6, 18) confirmed the existence of the fast, rate-sensitive feedback inhibition in the rat under different experimental conditions. On the other hand, failure to confirm its existence was reported in the rat with laparotomy stress (14)In this paper, we aimed, first, to confirm the existence of this type of negative feedback under conditions similar to those described by Dallman and Yates (2), as well as under conditions with other noxious stimuli. Second, we proposed to analyze the characteristics of the fast, rate-sensitive negative feedback in order to disclose its operational features. Third, on the basis of our experimental observations, we attempted to construct a model that might explain the mode of action of this feedback inhibition under stress.
adrenocorticotropin;
MATERIALS
KANEKO,
MASANORI,
AND
TSUTOMU
HIROSHIGE.
Fast,
corticosterone
REGULATION of stress-induced adrenocorticotropin (ACTH) secretion by corticosteroids was clearly demonstrated long ago (E), but several quantitative and temporal aspects of the inhibition are still unclear. Yates and Urquhart (16) proposed an hypothesis that assumed the existence of a set point for the operation of the hypothalamo-pituitary-adrenal system at rest and under stress. Their theory claimed that the activation of the system under stress followed elevation of a set point probably located in the central nervous system (CNS). The difference between this set point and the actual blood steroid level activates the system; the operation of the system is controlled by a closed feedback loop even under stress. This hypothesis, met several dificulties. For though very attractive, example, maintenance of elevated blood steroid levels by continuous infusion of corticosteroid did not at all NEGATIVE-FEEDBACK
AND
METHODS
Female Wistar rats weighing 170-220 g were used. They were housed at constant temperature of 23 t 1°C under 12-h light-dark cycle with the light period beginning at 6:30 A.M. Rat chow (Oriental Yeast Co.) and water were available ad libitum. The experiments were performed between 8 A,M. and 11 A.M. when plasma corticosterone levels are low and stable. Corticosterone (50 mg) dissolved in 1.5 ml of acetone was added to 100 ml of physiological saline solution, which was then placed for 2 h in a water bath (37°C) under nitrogen, to evaporate acetone and to prevent oxidation of corticosterone. The completion of evaporation was estimated by the disappearance of the smell of acetone. Thus prepared, the solution was stored at 5°C until use. Immediately before use it was further diluted with saline solution to appropriate concentrations. Intravenous infusions. The rats were anesthetized with pentobarbital sodium (Nembutal, Abbott) (5.5 mg/ 100 g body wt). Forty minutes later, when plasma
R39
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R40 corticosterone levels were basal, an intravenous infusion was started through a lateral caudal vein at a rate of 28 pl/min. The infusion was either saline, or corticosterone in various concentrations. Stresses. Two stresses were used: histamine and laparotomy plus traction. Histamine (Wako Junyaku Co.) was injected (230, 460, and 1,150 pg dissolved in saline, with final volumes of 0.25-0.50 ml) through a lateral caudal vein under pentobarbital anesthesia. The injection was completed within 10-G s. Laparotomy stress consistedbof abdominal incision 2-3 cm long under pentobarbital anesthesia, followed by mild traction of the intestinal tract with forceps. In all the stress experiments, incremental responses in plasma corticosterone levels were examined 15 min after the start of stress, unless otherwise indicated. In the early stage of the experiment, trunk blood was collected by decapitation. Afterward, to study changes in plasma corticosterone levels in single rats, arterial blood was withdrawn serially through a plastic cannula that had been inserted into the left common carotid artery 2 days before the experiment. The amount of blood sampled at one time was 0.6 ml or less, and the total volume of blood removed in the longest experiment (135 min) was less than 3.0 ml per rat. Plasma corticosterone concentrations were determined by the fluorometric method of Zenker and Bernstein (17), with minor modification. To simulate the overall operation of the fast, ratesensitive negative feedback, an analog computer of Hitachi 200X type was used. Statistical analysis was performed with the Student f-test.
M.
I 0
KANEKO
AND
T. HIROSHIGE
1
I
15
30
Time(md FIG. 1. Response of pituitary-adrenal axis to histamine stress. At zero time, 230 pg histamine dissolved in 0.25 ml saline (standard stimulus) were injected through lateral caudal vein in rats under pentobarbital anesthesia. Incremental change in plasma corticosterone concentrations at 15 min was defined as “control response.” Numbers of rats are shown in parentheses. Vertical lines give standard errors.
RESULTS
Demonstration of a fast, rate-sensitive feedback inhibition under histamine stress. The time course of the response to 230 pg histamine (standard dose) is shown in Fig. 1. This dose of histamine intravenously caused a peak increment in plasma corticosterone of approximately 30 pg/100 ml, 15 min after the injection. Therefore, in the following experiments the change in plasma corticosterone level 15 min after the onset of stress was adopted as a measure of the responsiveness to stress. In Fig. 2, the time course of plasma corticosterone concentration is shown before and during a constant infusion of corticosterone-saline solution. The plasma corticosterone concentration showed a tendency to rise almost linearly during the first 5 min of infusion. The rate of rise then fell off, and 15 min after the onset of infusion the plasma steroid level attained a new plateau that was approximately 40 pg/100 ml higher than the initial basal level. Thereafter corticosterone concentration was maintained at the elevated level over the 120min period of infusion. Infusion of physiological saline alone at the same rate caused no elevation. Intravenous injections of the standard dose of histamine were made at various times after the start of infusion. At the 3rd min, the response was significantly reduced, but at the Eth, 20th, and 30th min the responses were as large as the control response obtained when the plasma corticosterone was at the lowest level in the morning. At the 120th min, the response was completely abolished.
I
I
I
0
30 Time
#/
1
IJ 90
L
120
(min>
2. Negative-feedback inhibition of stress response by plasma corticosterone. Plasma corticosterone was elevated by intravenous infusion of corticosterone-saline solution (2.52 pg/r&n) (vm), started at 0 min. Each point is mean value of data from 4-9 rats (2 one standard error (SE) indicated by vertical bars). Control group was infused with physiological saline solution under the sami conditions (v&Standard stress stimulus (iv injection of 230 pg histamine) was given in various phases of changes in plasma corticosterone concentration 3, 15, 20, 30, 90, and 120 min. Rats were decapitated 15 min after histamine injection to collect trunk blood for measurement of plasma corticosterone concentration. Plasma corticosterone levels attained (x) are shown at times of histamine injections, when responses were initiated. Numbers of ratts used are shown in parentheses. Responses were distinctly reduced in the phase of rapid increase of plasma corticosterone (P < 0.0011, and no response was observed 2 h after a high plasma corticosterone level had been attained, FIG.
Characteristics of the fast, rate-sensitive feedback inhibition. To facilitate quantitative analysis of the fast, rate-sensitive feedback inhibition, blood from individual rats was sampled serially during constant infusions of various amounts of corticosterone. As shown in Fig. $4, the basal level of plasma corticosterone was somewhat higher in these chronically cannulated rats, but was within a reasonable range for the present purposes. The rate of rise of plasma corticosterone level was ascertained in each rat by serial sampling,
by estimating the rate from the differencebetween the
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FAST
FEEDBACK
INHIBITION
R41
MODEL
*-v 1 0
1
1
1
5
10
15
Time
0 0 ff
1
JJ
30
of stress. Three different doses of histamine were injected 3 min after the start of corticosterone infusions, when plasma corticosterone levels were rising at the rate of 6 pg/lOO ml per min. To obtain this rate of rise, corticosterone infusions at the rate of 2.27-4.03 pglmin were used, The rate of rise differed to some extent in different rats, for a given infusion rate, Only the data from rats that showed approximately 6 pg/lOO ml per min rate of rise ape recorded in Fig. 5. The control responses show a clear dose-dependent relationship. The responses observed when plasma corticosterone
(mid
a l
l
8
r
l
l 1
1
1
0
15 Time
1
1
30
45
(mid
FIG. 3. Changes in plasma corticosterone levels of individual rats during continuous infusions of corticosterone-saline solutions. Arterial blood was repeatedly withdrawn through an indwelling catheter in the carotid artery. A: at zero time, infusions of corticosterone ranging from 0 to 4.03 kglmin were started (closed circles). Control group was infused with saline under the same conditions (open circles). B: corticosterone infusion was started at 0 min and stopped at 15 min.
basal level and the 5th min value. Then the magnitude of stress-induced response was measured in the same animal. By varying the rates of rise of plasma corticosterone concentration, we could estimate the relationship between the rate of rise and the degree of suppression of the response. The plasma steroid level, once elevated, fell sharply as soon as the infusion was stopped (Fig. 3B). Here in the same manner we can estimate the rate of decline of plasma corticosterone levels in relation to the magnitude of stress-induced responses. The results are summarized in Fig. 4. The control response obtained when the standard dose of histamine was given during steady, basal concentrations of plasma corticosterone (i.e., during a rate of change of 0 pg/lOO ml per min) was approximately 30 pg/lOO ml. As the rate of rise of plasma corticosterone was increased, the magnitude of response became progressively smaller: the inhibition of the response became progressively larger. At the rate of 4-6 pg/lOO ml per min, the stress response was almost completely abolished. In contrast, when plasma corticosterone levels were declining, no inhibition was observed. In the next series of experiments, the degree of inhibition was examined in relation to the mamitude
-6 Rate
1
1
-4 -2 of change
1
1
I
?
0 2 4 6 in plasma corticosterone(
1
I
8 10 pg/lmml/min)
FIG. 4. Relationship between rate of change in plasma corticosterone concentration and degree of fast, rate-sensitive inhibition. Fifteen-minute incremental response of plasma corticosterone levels to 230 pg histamine was measured at each rate of change in plasma corticosterone. Different rates of rise and fall of plasma corticosterr one levels were obtained by turning on and off graded infusions of corticosterone, as shown in Fig. 3, A and B. Histamine was injected 3 min after the start of infusion, in the rising phase of corticosterone levels and at various times in the declining phase after the infusion was stopped.
460 Histamine()rg)
1150
FIG. 5. Relationship between degree of fast, rate-sensitive inhibition and intensity of stressful stimulus. White coZumn in each group indicates control responses to histamine, 230, 460, and 1,150 pg, respectively, Shaded columns indicate responses obtained when plasma corticosterone was increasing at the rate of 6 pg/lOO ml per min. Numbers in parentheses indicate numbers of determinations. Lines at tops of columns are + 2 SE.
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R42
M.
was rising at a rate of 6 pg/lOO ml per min also appeared to show a dose-response relationship. Inhibition of the responses, given as the differences between white and shaded columns in respective pairs in Fig. 5, appeared to have a constant absolute value regardless of the intensity of stimulus applied, if the rate of rise of plasma corticosterone was fixed. With laparotomy plus traction stress the inhibitory effect was not clear-cut. As shown in Fig. CIA, the response to laparotomy plus traction during corticosterone infusions, producing rates of rise in corticosterone levels of 6 pg/lOO ml per min, was not significantly different from the control response in the absence of corticosterone infusion. It is, however, noteworthy that the effect of laparotomy stress on plasma corticosterone levels continued at least 1 h, in marked contrast to the effect of histamine, which disappeared during the l-h period of observation (Fig. 6.B). Model of the mode of action of the fast, rate-sensitive feedback inhibition. Unique features of this feedback inhibition are its rapidity and rate sensitivity, both of which are rather difficult to reconcile with the general
KANEKO
AND
T. HIROSHIGE
concept of the mode of action of steroid hormones on various target tissues (3). The initial event in a series of processes of hormone action in the fast, rate-sensitive feedback inhibition must be the recognition of the rate of change in hormone concentration by target cells. As a basis for this recognition, we adopted the following two assumptions: 1) the union of hormone and receptor obeys the law of mass action; and 2) the hormone effect is proportional to the rate of formation of hormonereceptor complex. These assumptions are represented by the following mathematical expressions where X: hormone concentration in the blood R: “concn” of unoccupied receptors located on the target cells XR: “concn” of occupied receptors (bound) K 1: association rate constant K,: dissociation rate constant P: ratio of bound receptors to total receptors, (XRI(R + XR)); fraction of receptors that is occupied Y: hormone effect, i.e., inhibition of the response to stress (lessening of the increase in hormone concentration provoked by stress) t: time m, n, a, /3, 4: constants from mass action (assumption 1): K X+RsXR K2 and therefore, from definition of P dP dt = K,X - (KJ
Lapar
lapar under
inf.
+ K,)P
(1)
and from rate hypothesis (assumption 2): Y = $K,X(l
- P)
(2)
The changes in the hormone concentration in the circulating blood produced by a constant intision of hormone, or constant secretion, with first-order kinetics of hormone removal, are given by X = m(1 - emu’)+ n
(3)
which corresponds to the phase of rise of hormone concentration, and bY 0 A
-5
A
1
0
15 Time
fM
(min)
FIG. 6. Response to laparotomy plus intestinal traction. In the rat anesthetized with pentobarbital sodium, an abdominal incision was made and the intestinal tract was mildly manipulated. A: failure to demonstrate fast, rate-sensitive inhibition to laparotomy+ White column indicates control response 15 min after stress. Shaded column indicates response when plasma corticosterone concentration was rising at rate of 6 pgflO0 ml per min. B: duration of effect of laparotomy. Rats were left untouched after laparotomy stress until decapitation at 60th min (0). Response pattern to histamine stress is also shown for comparison @---a>.
X
= me-@’
+ n
(4)
which gives the time course of the declining phase of hormone concentration, To simulate the feedback inhibition on the basis of these equations, the values of constants must be determined within the range where physiological criteria are well satisfied. ti and p represent coefficients of exponential functions and need not be equal to each other because of dynamic asymmetries in the system (2). 4 represents the constant by which the rate of formation of hormone-receptor complex is converted to the physiological inhibitory effect. These constants can therefore be arbitrarily determined so as to fit the
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FAST
FEEDBACK
appropriate
INHIBITION
R43
MODEL
data of Figs. 3, A and B, 4, and 5. Constant
n represents the morning basal level of plasma corticos-
terone (10 pg/lOO ml). The physiological range of the plasma corticosterone concentration (X) in female rats is from 10 to 100 pg/lOO ml, i.e., from 2.9 x 10e7to 2.9 x 10-” M. Therefore, if n takes the value of 10, m. varies between 0 and 90, as shown by Eq. 5 below. The hormone concentration in the steady state, during infusion of secretion is 1imX F lim {m(l - e-l*/) + n} = m + n I--+=
t-r:
(5)
We now assume an ideal response to the standard histamine stimulus when there is initially no corticosterone in the body fluid (when t = 0, p = 0). When X now adopts any positive value as a consequence of stimulation, from Eq. 1, we obtain, by integration, and by assuming that X is fully independent of P and may be treated as a parameter in the relation of P to t
p=-
X
(1 K x+2 Kl
Therefore, after substituting
e-(K,.‘t’
+ h’,,t
I
(6)
into
Time 7. Theoretical curves of fast, rate-sensitive inhibition based on our hypothesis. Hormonal effect, feedback inhibition of stress response, corresponding to the basal, steady level of plasma corticosterone was arbitrarily set to zero. Dashed curue indicates declining phase of plasma corticosterone after cessation of infusion, during which no hormonal inhibitory effect was found. Curves numbered 1-7 in upper figure correspond to curves numbered l-7 in lower figure, and represent responses to increasing rates of infusion of corticosterone. FIG.
(7) the rising phase of the plasma corticosterone response. and so, in the steady state, the hormone effect is expressed as lim Y = +K.,I t-3;
[
~--_---m+n
(m + n) +y
K2
1
(8)
In addition, the degree of inhibition is proportional to the rate of rise of the plasma corticosterone (dose response). This model describes the effects of corticosteroid feedback during an initial exposure. We do not know to what extent data from repeated stress exposures, repeated infusions, or adrenalectomy might require a modification of the model.
1
From Eqs. 6 and 8, it follows that in the steady state the hormonal inhibitory effect, Y, is simply proportional to the fraction of receptors that is occupied (P)
where the subscript ss denotes steady-state. However, the inhibitory effect, Y, actually appears to be independent of the levels of corticosterone, for several hours. (The magnitude of the corticosterone response to the standard histamine stimulus remains the same (about +30 pg/lOO ml) regardless of the initial steady levels of plasma corticosterone, which may be basal (Fig. 1) or elevated by infusions (Fig. Z).) Therefore, K,/K, must be enough smaller than (m + n) so Y remains relatively constant for various values of (m + n) in the range of 10-100. For this reason, the value of the equilibrium dissociation constant K,/K, was set in the range of 0,005-O. 15 by assigning 987 to K, and the range 5-150 to K,. Simulation on an analog computer on the basis of this model produced a set of curves (Fig. 7), predicting the time course of corticosterone concentration, and its inhibitory effects, during constant infusions. According to this simulation, the hormonal effect or degree of negative feedback inhibition of stress-induced response is apparently short-lived, and only manifested during
DISCUSSION
The present study has amply confirmed the finding of Dallman and Yates (2) that there are two different types of negative-feedback inhibition of stress-induced activation of ACTH secretion: 1) the fast, rate-sensitive and 2) the delayed, proportional feedback inhibition. We have also confirmed that the fast feedback component is unidirectional under conditions similar to those employed by Dallman and Yates. Recently, Papaikonomou and Smelik (9) presented evidence that suggested the existence of a derivative controller which is sensitive to rates of decrease in blood corticosterone levels in the rat. Their results, however, cannot be directly compared with ours, because their experiments were performed with acutely operated rats, and in the afternoon. It has been reported that the fast, rate-sensitive negative feedback is demonstrable with other stressors such as sham adrenalectomy (6, 11). We also observed that the same feedback mechanism effectively inhibited the response induced by adrenaline injection (unpublished observation). Thus it appears that the fast, ratesensitive feedback is not a limited mechanism, designed for a particular type of stressor, but is a more general characteristic applicable to a wide variety of stressors. In this connection, failure to demonstrate the inhibition
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R44 with laparotomy stress both in the li terature (14) and in th e present experiment, des lerves commen t. In our experiment, laparotomy was performed together with mild traction of the intestinal tract. Because the effect of this combined stress lasted at least 1 h (Fig. W), and because this type of stress is known to be resistant to corticosteroid feedback (11, our failure to demonstrate fast feedback inhibition of it may not be surprising. The critical rate of rise of plasma corticosterone, above which the response to histamine stress was virtually abolished, was 4-6 pg/lOO ml per min in our study, using female rats. On the other hand, Jones et al. (6) reported the critical rate to be 1.3 Fg/lOO ml per min with sham adrenalectomy in male rats. Such a difference in the critical rate seems to show a sex difference. However, since free and not bound corticosterone is the effective feedback signal (7) and the proportion of the bound form is significantly greater in female rats than in male rats (4), the difference is more likely apparent than real. In our attempts to construct a hypothetical model to explain the mode of action of the fast, rate-sensitive feedback, we adopted the assumptions employed in Paton’s rate theory of drug action (10): 1) the union of drug with receptor obeys the law of mass act ion and 2) the drug effect is proportional to th e rate of formation of drug-receptor complex. He performed an outstanding analysis on the mode of action of acetylcholine on the smooth muscle of the guinea pig ileum in v itro. The drug concentration in the incubati on med ium was kept constant in the experiment of Paton, whereas in our experiment the plasma corticosterone levels were changing during transient phases. Jones et al. (6) also proposed the Paton theory as a model for fast ratesensitive feedback mechanism in 1972. Essential features, theoretically derived, of the fast feedback inhibition based on our hypothesis are as follows. 1) As soon as the hormone concentration begins to increase, the hormone effect starts to appear and reaches a maximum rapidly. As the rate of rise in hormone concentration becomes smaller, the effect is progressively reduced and finally disappears before the hormone level reaches a ne W platea u. 2) The magnitude of the effect is proportional to the ra.te of rise of hormone concentration, but no effect is demonstrable when the hormone concentration is declining. 3) The magnitude of the effect is not essentially influenced by the intensity of stress, but depends on the rate of rise of hormone concentration. These predictions appear to agree well with the characteristics of the fast rate-sensitive feedback inhibition which were observed experimentally in this study. In the processes of this simulation, one of the crucial points was the choice of the value for the dissociation equilibrium constant, K,/K,. The dissociation equilibrium constant of corticosterone from site(s) which must be related to the fast rate-sensitive feedback is still unknown. According to Suyemitsu and Terayama (15), the dissociation equilibrium constant of cortisol from the hepatic cell membrane is 1.4 x 10mgto l-3 x lop8 M,values roughly one-tenth of the physiological level of free corticosterone concentration in the rat plasma (2.9 x lO+ to 2.9 x W7 M). Munck and Brinck-Johnsen (8)
M.
KANEKO
AND
T. HIROSHIGE
estimated the dissociation equilibrium constant of cortisol in thymus lymphocytes to be 4 x 10W8M. Provided that the dissociation equilibrium constant of corticosterone from the putative site(s) of the rate-sensitive feedback is comparable to the above cited constants, variations in the magnitude of the control response obtainable at different steady Ievels of plasma corticosterone (e.g., from 2.9 x 10e7 to 2.9 x lo-” M, with roughly one-tenth of it in the free form) can be calculated to be only 5% or less according to our model. These considerations are consistent with our experimental finding that the response to standard stress remains almost the same when steady levels of plasma corticosterone are either high or low. As will be noticed, the principal assumption behind our model is that the rate-sensitive feedback action is equi valent to the phenomenon of binding itself. The model is really a description of the binding process. The theoretical curves (Fig. 7) imply that the actual time course of the hormone effect may vary subtly according to different rates of rise of plasma steroid concentration, It is predicted that infusions of corticos&one at low rates lead to prolonged feedback effects, compared to infusions at high rates, despite the fact that the rising concentration of corticosterone continued longer at the high rate of infusion. But we have no evidence to confirm or deny this point. To clarify this point, changes in plasma ACTH concentration must be followed. In spite of the apparently successful simulation, the basic assumptions employed here are not necessarily valid, because we have no evidence in their favor at the cellular or subcellular level. Hallahan et al. (5) examined the time course of inhibition of glucose. uptake by glucocorticoid in the thymus lymphocytes in vitro. According to them, the essential step in this phenomenon is the attachment of corticoid-receptor complex to a certain specific site on the surface of the nuclear membrane. Furthermore, quite interestingly, the subsequent messenger RNA synthesis proceeds pari passu with the rate of union of the corticoid-receptor complex with the acceptor sites on the nuclear membrane. Thus, their finding seems to be in terpreta .ble by a mechanism similar to rate-sensitive feedback inhibition. However, in the case of the thymus lymphocytes nearly 30 min are required for cortisol to begin to inhibit glucose uptake, whereas in this feedback phenomenon the effect becomes manifested within several minutes. This astonishing rapidity of effect suggests that synthetic processes of a messenger RNA, or protein, are not involved in its operation. In the following paper we demonstrate that the fast, rate-sensitive feedback involves a catecholaminergic stage in the brain, whereas delayed, level-sensitive corticosteroid negative feedback involves a serotonergic stage (6a). The authors are indebted to Professor K. Furukawa, Dept. of Anesthesiology, Hokkaido University School of Medicine, for his help in the use of an analog computer of Hitachi 200 X type. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education of Japan Grants 911304 of Specified Category I974 and 010904 of Specified Category 1975). Received
for publication
8 March
1977.
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REFERENCES 1. DALLMAN, M. F., AND F. E. YATES. Anatomical and functional mapping of central neural input and feedback pathways of the adrenocortical system. Mem. Sot. Endocrinol. 17: 39-72, 1968. 2. DALLMAN, M. F., AND F. E. YATES. Dynamic asymmetries in the corticosteroid feedback path and distribution-metabolismbinding elements of the adrenocortical system. Ann. N. Y. Acad. Sci. 156: 696-721, 1969. 3. FEIGELSON, P., AND M. FEIGELSON. Studies on the mechanism of cortisone action. In: Actions of’ Hormones on Molecular Processes, edited by G. Litwick and D. Kritchevsky. New York: Wiley, 1964, p. 218-233. 4. GARA, R. R., AND U. WESTOPHAL. Corticosteroid-binding globulin in the rat: studies on the sex difference. Endocrinology 77: 841-851, 1965. 5. HALLAHAN, C., D. A. YOUNG, AND A. MUNCK. Time course of’ early events in the glucocorticoids on rat thymus cells in vitro. J. BioL. Chem. 248: 2922-2927, 1973. 6. JONES, M. T., F. FL BRUSH, AND P. L. B. NEAME. Characteristics of fast feedback control of corticotropin release by corticosteroids. J. Endocrinol. 55: 489-497, 1972. 6a. KANEKO, M., AND T. HIROSHIGE. Site of fast, rate-sensitive feedback inhibition of adrenocorticotropin secretion during stress. Am. J. Physiol. 234: R46-R51, 1978 or Am. J. Physiol.: Regulatory Integrative Comp. Physiul. 3: R46-R51, 1978. 7. KWAI, A., AND F.E. YATES. Interference with feedback inhibition of adrenocorticotropin release by protein binding of corticosterone. Endocrinology 79: 1040-1046, 1966. 8. MUNCK, A., AND T. BRINCK-JOHNSEN. Specific and nonspecific physicochemical interactions of glucocorticoids and related steroids with rat thymus cells in vitro. J. Biol. Chem. 243: 55565565, 1968.
9. PAPAIKONOMOU, E., AND P. G. SMELIK. Evidence for derivative control in the rat pituitary-adrenal system. Am. J. Physiol. 227: 137-143, 1974. 10. PATON, W. D. H. A theory of drug action based on the rate of drug-receptor combination. Proc. Roy. Sot. London, Ser. B 154: 21-69, 1961. 11. SATO, T., M, SATO, J. SHINSAKO, AND M.DALLMAN. Corticosterone-induced changes in hypothalamic corticotropin-releasing factor (CRF) content after stress. Endocrinology 97: 265-274, 1975. 12. SAYERS, G., AND M. SAYERS. Regulation of pituitary adrenocorticotropic activity during the response of the rat to acute stress. Endocrinology 40: 265-273, 1947. 13. SMELIK, P. G. Failure to inhibit corticotrophin secretion by experimentally induced increases in corticoid levels. Acta Endocrinol. 44: 36-46, 1963. 14. SMELIK, P. G. Failure to demonstrate a fast rate sensitive feedback action of corticosteroids during adrenocortical activation. Acta Endocrinol. Suppl. 177: 149, 1973. 15. SUYEMITSU, T., AND H. TERAYAMA. Specific binding sites for natural glucocorticoids in plasma membranes of rat liver. Endocrinology 96: 1499-1508, 1975. 16. YATES, F. E., AND J. URQUHART. Control of plasma concentrations of adrenocortical hormones. Physiol. Rev. 42: 359-443, 1962. 17. ZENKER, N., AND D, E. BERNSTEIN. The estimation of small amounts of corticosterone in rat plasma. J. Biol. Chem. 231: 695-701, 1958. 18. ZIMMERMAN, E., AND V. CRTTCHLOW. Short-latency suppression of pituitary-adrenal function with physiological plasma levels of corticosterone in the female rat. Neuroendocrinobgy 9: 235243, 1972.
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