63
Atherosclerosis, 24 (1976) 63-73 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
ALDOSTERONE, DEOXYCORTICOSTERONE AND CORTICOSTERONE DIFFERENCES BETWEEN ARTERIOSCLEROTIC BREEDER VS NONARTERIOSCLEROTIC VIRGIN RATS
SAMUEL G. IAMS and BERNARD
C. WEXLER
May Institute for Medical Research, The Jewish Hospital, and the Departments of Physiology, Medicine and Pathology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45229 (U.S.A.) (Received
23rd June, 1975)
(Accepted
12th January, 1976)
Summary The circulating levels of the major adrenal steroids, corticosterone (Cmpd. B), deoxycorticosterone (DOC), and aldosterone were compared in male and female, arteriosclerotic, breeder and non-arteriosclerotic virgin, SpragueDawley rats under resting conditons, i.e., quiescence, and after exposure to a mild stress, i.e., movement from one room to another just prior to autopsy. Under resting conditions, the arteriosclerotic animals had significantly greater circulating levels of Cmpd. B, DOC and aldosterone than the non-arteriosclerotic animals. Both the arteriosclerotic and non-arteriosclerotic animals were able to respond adequately to the mild stress stimulus. However, the female breeder rats which manifest the most severe aortic sclerosis, showed the greatest increase in Cmpd. B and aldosterone in response to the mild stress. Although no statistically significant differences could be found between female breeders with grossly visible aortic sclerosis of clear, minimal, moderate, or severe degree, it was apparent that adrenal steroid responsiveness becomes progressively compromised with increasing severity of arteriosclerosis, e.g., unusually high or low levels of Cmpd. B and aldosterone under both quiescent and mild stress conditions. It is suggested that there may be some connection between abnormal hypothalamic-pituitary-adrenal-gonadal function in repeatedly-bred and the pathogenesis of their naturally-occurring ateriosclerosis. -This work was supported by grants from the Eastern and Southwestern Ohio Heart Associations and the National Heart and Lung Institute (HL-15, 304) N.I.H. Dr. lams was supported by a Research Fellowship from the Southwestern Ohio Heart Association. Please address all correspondence to: Dr. Samuel G. lams, May Institute for Medical Research. 421 Ridgeway Avenue, Cincinnati, Ohio 45229, U.S.A.
64
Key words:
ACTH
-
Catecholamines
Factor
-
Mineralocortocoids
gland
-
Cortico-medullary us glucocorticoids
axis -
Corticotrophin
Hypothalamus
Releasing -
Pituitary
- Steroidogenesis
Introduction Spontaneous hypertension, hyperglycemia, hyperlipidemia and arteriosclerosis appear in repeatedly-bred male and female rats. The incidence and severity of the arteriosclerosis and related pathophysiologic changes parallels the frequency of the reproductive effort [l--8]. Although it is not known whether the changes in the hypothalamic-pituitary-adrenal-gonadal axis are primary or secondary relative to the pathogenesis of the arterial disease, we have definite evidence that endogenous pituitary, adrenal and gonadal hormones do play a prominent role in the pathogenesis of this naturally-occurring vascular disease. In earlier investigations, we found that arteriosclerotic, breeder rats manifested dramatic adrenocortical histopathology, e.g., hypertrophy, hyperplasia, zonal lipid alterations, thromboses and infarction, as well as adrenomedullary histopathologic alterations, e.g., pheochromocytomas [ 91. Total steroidogenesis and individual corticosteroids as well as the ratio of norepinephrine : epinephrine become progressively altered commensurate with the number and frequency of breedings as well as the severity of the arterial disease. Despite superabundant stores of cholesterol, i.e., steroid precursor material, arteriosclerotic, breeder rats are less capable of responding to exogenous ACTH or stress than virgin rats with no arterial disease [lo]. In vitro steroid analyses of arteriosclerotic, breeder rat adrenal glands demonstrates that in parallel with the development of their arterial disease, their adrenal glands lose the capacity to convert steroid intermediates, e.g., pregnenolone, into more definitive steroids, e.g., corticosterone, and the entire spectrum becomes altered, e.g., ratio of aldosterone : 18-hydroxydeoxycorticosterone : deoxycorticosterone : corticosterone, etc. [lo]. That is, the change in the normal spectrum of adrenal steroids appears to be due to some derangement of the adrenocortical enzymes which regulate steroidogenesis [ 111. Subsequent in vivo investigations confirmed these earlier in vitro findings, and, in additon, demonstrated that arteriosclerotic breeder rats had deranged metabolic clearance rates as well as abnormal hepatic steroid metabolism [ 121. In more recent studies we have found that pituitary production and release of ACTH is progressively impaired in breeder rats in parallel with their progressively worsening arteriosclerosis, and that this defect in adrenocorticotrophic hormone may be due to abnormal hypothalamic production and release of Corticotrophin Releasing Factor (CRF) [13]. There is an interesting sex dichotomy between male and female breeder rats in the pathogenesis of their arterial disease. The female breeders develop grossly visible arterial lesions of the aorta; male breeders develop microscopic aortic lesions only [l-5]. Further, despite their seemingly more innocuous lesions, male breeder rats die significantly earlier than their female breeder counterparts, usually due to myocardial infarction. ECG studies demonstrated
65
definitely abnormal tracings as well as fundamental differences between arteriosclerotic and non-arteriosclerotic animals and between males and females [14, 151. When we compared various hemodynamic parameters indicative of the cardiac index or cardiac output of arteriosclerotic, breeder (male vs female) vs non-arteriosclerotic, virgin (male vs female) rats, we uncovered another seemingly paradoxical situation wherein the female breeders with severe, grossly visible aortic sclerosis had a significantly higher cardiac output compared to the other groups with less severe arteriosclerosis [16,17] . Mineralocorticoids, e.g., aldosterone, deoxycorticosterone, etc., can increase cardiac output, condition the arterial wall toward hypertension, as well as other cardiovascular effects. We elected to compare the relative circulating levels of aldosterone, deoxycorticosterone and corticosterone (the major circulating glucocorticoid produced by the rat’s adrenal cortex) in arteriosclerotic, breeder vs non-arteriosclerotic, virgin rats, in order to ascertain whether the deranged adrenal glandular function has any direct bearing on the cardiovascular degenerative changes in repeatedly-bred rats. Further, we compared the circulating levels of these dynamic mineralocorticoids vs glucocorticoids in male vs female rats to determine whether there are any adrenocortical steroid differences between the sexes, between arteriosclerotic vs non-arteriosclerotic males and females or between breeder rats with varying degrees of arteriosclerosis, i.e., male breeders with microscopic aortic sclerosis vs female breeders with clear, minimal, moderate, or severe aortic sclerosis. Materials and methods Mature, non-arteriosclerotic, virgin and arteriosclerotic, breeder rats (Sprague-Dawley strain) were divided into 4 groups: virgin, non-arteriosclerotic males (15) and females (26), and arteriosclerotic, breeder males (17) and females (93). All of these animals were 6 to 8 months of age. The female breeder rats which are prone to develop grossly-visible aortic sclerosis [l-5] were classified, at autopsy, as having grossly-visible aortic sclerosis of either clear, minimal, moderate, or severe degree aa described previously [l].The arteriosclerotic, male breeders were found to have microscopic aortic sclerosis only, and like the female breeders, to have coronary, carotid, mesenteric, renal and peripheral arterial disease [l-5] (confirmed at autopsy and histopathologicallY)* The animals were housed in our Animal Research Colony where temperature, humidity and light are carefully controlled. All of the animals were fed a commercial rat chow (Purina Rat Chow, St. Louis, Missouri), which is relatively low in fat (4%) and tap water ad libitum. Because adrenocortical steroidogenesis is affected by circadian rhythm, the lighting in the Animal Research Colony was regulated on a 14 hours “On” : 10 hours “Off” cycle and the animals were killed at a set time in the morning (2 to 4 h after “Lights On”). Because the stress of movement of animals from one laboratory to another is sufficient to cause an increase in adrenocortical steroid secretion, one group of animals of each of the varieties described was autopsied within the confines of our Animal Research Colony without movement, i.e., the quiescent or nonstressed group. A similar group was moved to our Research Laboratories prior
66
to autopsy, i.e., mild stress. All of the animals were killed by instant decapitation with a minimum of handling to avoid the relatively strong stress of anesthesia. Blood was collected from the severed neck vessels, centrifuged, and stored at -20°C until ready for steroid analyses. Steroid determinations Aldosterone. Aldosterone was extracted from the serum samples using 30X volumes of methylene chloride (MeC&) and separated from other interfering steroids by means of a Sephadex LH-20 Column [using MeClz : MeOH (98 : 2, v/v) as a mobile phase]. The extracted aldosterone was measured using the radioimmunoassay method of Ito et al. [18]. Anti-serum for assay of aldosterone was provided through the courtesy of the Hormone Distribution Officer (Dr. R.W. Bates), National Institutes of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. Corticosterone. Each of the serum samples was extracted with a mixture of hexane : benzene (80 : 20, v/v) to partially purify the sample, e.g., to remove lipids. The serum samples were extracted a second time with the hexane : benzene (20 : 80, v/v) mixture to separate the corticosterone for the assay. Corticosterone was determined by a modified radioimmunoassay method of Mayes et al. [19] . Antiserum for corticosterone was added to a 3 ml centrifuge tube containing 5,000 cpm tritiated corticosterone and an aliquot of serum extract. The solution was incubated in a water bath at 40°C for 5 min and then chilled at 4°C overnight. A Dextran-80 (250 mg) and a Norit A charcoal mixture (250 mg) in 100 ml of borate buffer (pH 8.0) was added to the solution to terminate
Fig. 1. Comparison of the circulating corticosterone levels of male. non-arteriosclerotic, virgin and art&osclerotic, breeder vs female, non-arteriosclerotic virgin and arteriosclerotic, breeder, Sprague-Dawley rats under resting or quiescent conditions vs after a mild stress such as moving the animals from one room to another just prior to autopsy. The height of each column indicates the Mean f Standard Error; the numbers in brackets indicate the number of samples assayed. Figures 2 and 3 follow the same protocol.
61
the reaction and to bind the free steroid. The solution was then centrifuged (2,000 rpm) for 10 min and an aliquot taken to determine the percent of steroid bound to the antibody. Deoxycorticosterone. Serum samples were extracted with petroleum ether to remove the major quantities of progesterone and lipid. Each sample was extracted a second time with a hexane : benzene (20 : 80, v/v) mixture to recover the remaining deoxycorticosterone. The extract was then passed through a 20 cm Sephadex LH-20 column using a MeCl, : benzene : MeOH (70 : 26 : 4, v/v/v) mixture to separate the progesterone from the deoxycorticosterone (progesterone has a 100% cross reactivity with the antiserum). Antiserum for the radioimmunoassay of deoxycorticosterone and corticosterone was obtained from Endocrine Science, 18418 Oxnard St., Tarzana, California 91356, U.S.A.. The radioimmunoassay procedure was the same as that used for corticosterone, i.e., the method of Mayes et al. [19]. The immunoassay data was subjected to logit-log transform and analyzed by the method of Midgeley et al. [20] , and Rodbard et al. [21] . Covariance analysis was also used as a test of inter-assay correlation. Statistical analyses of the differences in serum steroid values between the experimental and control groups were performed using Student’s t-test as described by Snedecor and Cochran [22]. Results Under quiescent conditions, the circulating levels of corticosterone (Cmpd. B), deoxycorticosterone (DOC), and aldosterone were much higher (P < 0.001) in the arteriosclerotic, breeder vs the non-arteriosclerotic, virgin rats (Figs. l3). Cmpd. B levels were twice as high in the female breeders with grossly-visible
I 0
Oulescent
Fig. 2. Comparison of circulating deoxycorticosterone
levels. (0 = Data lost due to technical problems.)
68
arteriosclerosis compared to female virgins with no arteriosclerosis, i.e., 18.09 + 1.20 vs 8.15 f 0.95 pug/100 ml (Fig. 1); DOC levels were four times higher in the arteriosclerotic female breeders compared to the non-arteriosclerotic, virgin females, i.e., 7.56 f 0.48 vs 1.63 + 0.30 ng/ml (Fig. 2); aldosterone levels were three times higher in the arteriosclerotic female breeders compared to the non-arteriosclerotic, virgin females, i.e., 21.6 f 1.93 vs 9.16 f 1.65 ng/lOO ml. The Cmpd. B level for the arteriosclerotic, male breeders were three times higher than those for the nonarteriosclerotic, virgin males (Fig. l), i.e., 12.00 k 1.30 vs 4.98 + 0.82 pug/100 ml. The samples for DOC and aldosterone for quiescent, male breeder rats were irretrievably lost. (Data from other experiments indicate that the DOC and aldosterone levels for male breeders with microscopic aortic sclerosis are considerably higher than those for the non-arteriosclerotic, male virgins.) The adrenal cortices of the non-arteriosclerotic, virgin and arteriosclerotic, breeder rats, males and females, were all capable of responding to the relatively mild stress of moving the animals from one room to another just prior to autopsy. This capacity to respond to a mild stress was reflected in each of the major steroids measured, i.e., Cmpd. B (Fig. l), DOC (Fig. 2) and aldosterone (Fig. 3). This increase in the major steroids in response to a mild stress was statistically highly significant (P < 0.001) with the exception of the increase in aldosterone in the arteriosclerotic, female breeders. It should be borne in mind that in most animals, the adrenal gland of the female is much heavier than that of the male and in most animals, especially the rat, the female has much higher circulating steroid levels. This characteristic sex difference of higher circulating levels of steroids in females vs males was
I ,-. 0
Quiescent
m
Mild
Stress
-113 (36
(9)
Fig. 3. Comparison of circulating aldosterone levels. (o = Data lost due to technical problems.)
69
also found in these animals pertaining to both mineralocorticoids as well as glucocorticoids, i.e., aldosterone, DOC and Cmpd. B (Figs. l-3). When a comparison was made of the steroidogenic responsiveness of female breeders on the basis of their varying degrees of arteriosclerosis, i.e., clear, minimal, moderate and severe, compared with non-arteriosclerotic, virgins, it was apparent that the serum of arteriosclerotic animals contained much more of the major glucocorticoid, Cmpd. B and the mineralocorticoid, aldosterone (Fig. 4). However, there were no statistically significant differences in circulating steroid levels between each of the various categories of grossly-visible arteriosclerosis, i.e., clear to severe. When the circulating aldosterone levels of arteriosclerotic vs non-arteriosclerotic, females were compared under conditions of quiescence and mild stress (Fig. 5), no statistically significant differences could be found between those female breeders having clear, minimal, moderate or severe arteriosclerosis. A definite trend toward progressively greater divergence of aldosterone responsiveness to the stimulus of a mild stress, i.e., high (responsive) and low (non-responsive) values could be discerned concomitant with progressively increasing severity of grossly-visible arteriosclerosis, especially in those female breeders having severe aortic sclerosis (Fig. 5).
0 Corticosterone 0
G
0
Aldosterone
No
Cleor
Minimal
Microscopic ARTf
i,
Moderate
Severe
Grossly-visible 2IOSCLEROSIS
VIRGINS
Fig. 4. Scattergram depicting individual serum aldosterone (closed circles) and corticosterone (open circles) levels of non-arteriosclerotic. virgin, female rats compared with arteriosclerotic, female breeder rats separated according to the appearance of their grossly-visible aortic sclerosis observed at autopsy, i.e., clear, minimal, moderate and severe degree. Both the corticosterone and aldosterone levels were taken under resting or quiescent conditions.
IO
??Mild 0
Stress
0
Quiescent
:
.
.
8
:
i
5
1
0%
0 0
@ d %
1
Clear
Minima
Microscopic I
L
. VIRGINS
1
L ...
.
??
No
.
ARTERIOSCLER BREEDER!
??
I
Moderate
(
Severe
xsly-visible SIS I
Fig. 5. Scattergram of individual aldosterone levels of non-arteriosclerotic, virgin, female rats compared with arteriosclerotic. female breeder rats, separated according to the severity of their grossly-visible aortic sclerosis observed at autopsy, i.e., clear, minimal, moderate and severe. under conditions of quiescence (open circles) and mild stress (closed circles).
Discussion We cannot determine, at this time, whether the deranged adrenocortical function in repeatedly-bred rats is primary or secondary to their spontaneous cardiovascular degenerative changes. However, the present observations reconfirm our earlier findings that there is a definite difference in the adrenal steroid levels between non-arteriosclerotic virgin rats and arteriosclerotic breeder rats. When considering all of the accumulated evidence we have concerning the possible role of hormones in the pathogenesis of breeder rat arteriosclerosis, we believe that there may be some connection between the cardiovascular degenerative changes with accompany repeated breeding and derangement of the hypothalamic-pituitary adrenal-gonadal axis. For example, in one of our most recent reports, we showed that the circulating level of luteinizing hormone (LH) was progressively decreased and that circulating prolactin was greatly increased, commensurate with the increasing severity of grossly-visible arteriosclerosis in female breeder rats [23] . These findings would suggest that hypothalamic releasing factors which regulate pituitary gland release of LH and prolactin, e.g., Prolactin Inhibiting Factor (PIF), become altered during the continuous hypothalamic stimulation of repeated breeding. Similarly, we have also observed increasing pituitary basophilia, adenomatosis, and alterations in pituitary gland content of
71
ACTH in concert with the increasing severity of arteriosclerosis in repeatedlybred rats [13] . In the present investigation, the definitely abnormal Cmpd. B, DOC, and aldosterone levels in arteriosclerotic, breeder rats is further confirmation and extension of our earlier in vitro [lO,ll] and in vivo [12] steroid studies and is in keeping with our findings of extensive histopathologic alterations in the adrenal cortices of arteriosclerotic breeder rats, e.g., extensive zonal lipid alterations, thromboses and infarction [9] . It would be pertinent to emphasize that repeatedly-bred rats also develop progressively changing catecholamine biosynthesis, i.e., adrenaline, noradrenaline and phenyl-ethanolamine-Nmethyl transferase (PNMT) [24], as well as pheochromocytomas [9], in parallel with their changing cortical steroidogenesis and worsening arteriosclerosis. That is, there may be an adrenocortical-medullary axis or inter-play involved in the pathogenesis of the abnormal metabolic and vascular degenerative changes in repeatedly-bred rats, since adrenal steroids, particularly the glucocorticoids, have been shown to condition the activity of the adrenomedullary enzyme PNMT in converting noradrenaline into definitive adrenaline [25] . The combined changes in adrenal corticosteroids and catecholamines in repeatedlybred rats could well account for their hypertension, hyperglycemia, hyperlipidemia, arteriosclerosis, frequent myocardial infarction, and premature aging. In view of the sex dichotomy between male vs female breeders, i.e., relatively mild aortic sclerosis in male breeders vs advanced, grossly-visible aortic sclerosis in female breeders, it is noteworthy that not only do female rats have larger adrenal glands and produce greater quantities of Cmpd. B under both quiescent and stressful conditions, but also, repeated pregnancies (or arteriosclerosis) changes their adrenal steroidogenic capacity toward even greater Cmpd. B, DOC, and aldosterone production, particularly Cmpd. B and aldosterone. This greater capacity to produce adrenocorticoids in female rats may play some role in conditioning the various morphologic components of the aortic wall toward a different kind of pathologic change in females vs males. In order to obtain an overall purview of the temporal adrenal glandular changes associated with repeated breeding and arteriosclerosis, we have combined all of the information from our in vitro and in vivo investigations with the result that it would appear that repeatedly-bred rats become progressively hyperadrenocorticoid during the early phases of their reproductive activity; at this time, the arterial lesions are microscopic or just beginning to become grossly-visible in the abdominal aortic segment. With continued breeding, some of the animals manifest extensive adrenocortical and medullary pathology, their pituitary glands become adenomatous, ACTH release is impaired, and/or the adrenal cortices become unresponsive to ACTH stimulation and steroidogenesis becomes altered; at this time, grossly-visible arteriosclerosis is usually rampant throughout the length of the aorta. We believe that the increased circulating levels of Cmpd. B, DOC, and aldosterone, as demonstrated in these animals, may account for the repeatedly-bred rat’s propensity toward increasing blood pressure, myocardial failure, and perhaps, arteriosclerosis. Our finding of increased serum levels of Cmpd. B, DOC, and aldosterone in the arteriosclerotic breeder rats under quiescent conditions indicates that under conditions free of extra demand for pituitary gland release of ACTH, the arteriosclerotic, breeder rats are definitely hyperadrenocorticoid. The two,
72
three and four-fold increases in Cmpd. B, aldosterone and DOC in the arteriosclerotic vs non-arteriosclerotic rats, are quite considerable and could have farreaching pathophysiologic effects. It is of particular interest that under conditions of mild stress, i.e., mild ACTH release and mild adrenocortical stimulation, circulating levels of Cmpd. B, DOC, and aldosterone increased, approximately in equal proportions in both the arteriosclerotic and non-arteriosclerotic animals. On the other hand, in all of our previous investigations, when a stress of greater proportion was used, e.g., confinement within an ether-filled chamber, the non-arteriosclerotic, virgin rats were capable of releasing maximal quantities of adrenal steroids, i.e., Cmpd. B. However, arteriosclerotic, breeder rats are completely incapable of increasing their steroidogenic potential beyond a certain level, a level which is much below that of non-arteriosclerotic, virgin rats [10,12,13]. In our original in vitro investigations [lo] and in one of our most recent in vivo studies [13] , we found that as the dose of exogenous ACTH was increased in graded increments, the ability of adrenal glands of arteriosclerotic animals to secrete Cmpd. B on demand, increased up to a given dose of ACTH and then were no longer able to respond to any greater increments of ACTH stimulation. The same doses of ACTH in non-arteriosclerotic, virgins, elicited dynamic and increasing serum levels of Cmpd. B. That is, the non-arteriosclerotic, virgin rats retained their ability to continue to respond to increasing increments of exogenous ACTH and their dose : response did not plateau until a level of ACTH was reached well above that to which the arteriosclerotic breeder could no longer respond [lo]. Since this suggested that there may be a progressively developing refractoriness to endogenous (and exogenous) ACTH in repeatedly-bred rats, we transplanted the pituitary glands taken from arteriosclerotic, breeder rats beneath the kidney capsule of hypophysectomized, non-arteriosclerotic rats. We found that the transplanted breeder rat pituitary glands secreted ACTH and gonadotrophic hormones which were able to maintain the adrenal glands and gonads of the hypophysectomized recipients in superior fashion than those animals in which pituitary glands, removed from non-arteriosclerotic, virgin rats, had been transplanted in a similar manner [26]. Subsequently, bio-assay of the actual pituitary gland content of ACTH of arteriosclerotic vs non-arteriosclerotic animals, demonstrated that although the pituitary glands became progressively hyperplastic and heavier in breeders with clear vs minimal vs moderate vs severe arteriosclerosis, their ACTH content becomes progressively less [ 131. In conclusion, despite the fact that there were no statistically significant differences in the circulating steroid levels between female breeders with varying degrees of severity of arteriosclerosis, i.e., clear, minimal, moderate and severe arteriosclerosis, there was, nonetheless, a definite difference in the amounts of circulating Cmpd. B, DOC and aldosterone between non-arteriosclerotic, virgin rats and arteriosclerotic breeder rats. The findings reported here, taken in context with our ancillary published findings of alterations in pituitary trophic hormones [13,23] , changes in pituitary histopathology and ACTH content [13,26], and adrenal cortical and medullary malfunction [g-13,23,24,26], suggest that there may be some connection between abnormal hypothalamicpituitary-adrenal-gonadal function in repeatedly-bred rats and the pathogenesis of their naturally-occurring arteriosclerosis.
73
Acknowledgement The authors are grateful for the expert natser, E. Domingo, G. Heap and J. Wexler.
assistance
of D. Schaffner,
D. Co-
References 1 Wexler, B.C., Spontaneous arteriosclerosis in repeatedly-bred male and female rats. J. Atheroscler. Res., 4 (1964) 57-80. 2 Wexler, B.C. and True. C.W.. Carotid and cerebral arteriosclerosis, Circ. Res., 12 (1963) 659466. 3 Wexler, B.C., Spontaneous coronary arteriosclerosis in repeatedly bred male and female rats, Circ. Res., 14 (1964) 32-43. 4 Wexler, B.C., Spontaneous arteriosclerosis of the mesenteric. renal and peripheral arteries of repeatedly bred rats. Circ. Res.. 15 (1964) 485-496. 5 Wexler, B.C., Arteriosclerosis of the renal artery of repeatedly bred male and female rats, Atherosclerosis, 11 (1970) 383-400. 6 Judd, J.T. and Wexler. B.C., The role of lactation and weaning in the pathogenesis of arteriosclerosis in female breeder rats, J. Atheroscler. Res., 10 (1969) 153-1’72. 7 Wexler. B.C. and Kittinger, G.W.. Spontaneous arteriosclerosis in male and female breeder rats - Effects of a high fat diet and hyperadrenocorticism on repeatedly bred rats, J. Path. Bact. 94 (1967) 231-246. 8 Lutmer. R.F., and Wexler. B.C.. Pathophysiologic changes associated with the development of arteriosclerosis during various phases of the reproductive cycles of repeatedly bred rats, Camp. Biochem. Physic& 37 (1970) 445-466. 9 Wexler, B.C., Correlation of adrenocortical histopathology with arteriosclerosis in breeder rats, Acta endocrinol., 46 (1964) 613-631. 10 Wexler. B.C. and Kittinger, G.W.. Adrenocortical function in arteriosclerotic female breeder rats, J. Atheroscler. Res.. 5 (1965) 317-329. and corticosteroid production in 11 Kittinger, G.W. and Wexler, B.C., Adrenal gland dehydrogenases normal and arteriosclerotic breeder rats, Proc. Sot. EXP. Biol. Med., 118 (1965) 365-367. 12 Saroff, J. and Wexler, B.C.. Metabolic clearance and production rates of corticosterone in male and female virgin and breeder rats, Acta endocrinol.. 62 (1969) 411-424. 13 Wexler. B.C. and Lutmer, R.F.. ACTH content in pituitary glands of arteriosclerotic breeder vs nonarteriosclerotic, virgin rats. Atherosclerosis, 22 (1975) 199-214. 14 Wexler. B.C.. Willen, D. and Greenberg, B.P., Electrocardiographic differences between non-arteriosclerotic and arteriosclerotic rats, Atherosclerosis, 18 (1973) 129-140. 15 Wexler. B.C., Willen, D. and Greenberg, B.P., Progressive electrocardiographic changes in male and female arteriosclerotic and non-arteriosclerotic rats during the course of isoproterenol-induced myocardial infarction, Cardiovasc. Res.. 8 (1974) 460-468. 16 Colfer. H.T., Iams. S.G. and Wexler, B.C., Cardiovascular function in non-arteriosclerotic vs arterio17 18 19 20
21 22 23 24 25 26
sclerotic rats. Amer. J. Physlol., 229 (1975) 18-22. Golfer. H.T.. Iams. S.G. and Wexler. B.C., Hemodynamic changes in arteriosclerotic YS non-arteriosclerotic rats during the acute stages of myocardial infarction, Angiology. 27 (1976) 32-41. Ito. T., Woo. J.. Haning. R. and Horton, R.. A radioimmunoassay for aldosterone in human peripheral plasma including a comparison of alterate techniques, J. Clin. Endocr. Metab., 34 (1972) 106-112. Mayes, D., Furuyama, S., Kern, D.C. and Nugent, C.A., A radioimmunoassay for plasma aldosterone, J. Clin. Endow. Metab., 30 (1970) 682-685. Midgley. A.R., Jr., Niswender, G.D. and Rebar, R.W.. Principles for the assessment of the reliability of radioimmunoassay methods (Karolinska Symposium on Immunoassay of Gonadotropins), Acta Endocr., Suppl., 142 (1969) 163-184. Rodbard, D., Rayford. P.L., Cooper, J.A. and Ross, G.T.. Statistical quality control of radioimmunoassays. J. Clin. Endocr. Metab., 28 (1968) 1412-1418. Snedecor. G.W. and Cochran. W.G., Statistical Methods, Iowa State University Press, Ames. Iowa. 1967. Lewis, B.K. and Wexler. B.C., Changes in LH and prolactin in arteriosclerotic female breeder rats, Atherosclerosis, 21 (1975) 301-314. Wexler. B.C. and Greenberg, B.P., Adrenal cortico-medullary function in arteriosclerotic (breeder) and non-arteriosclerotic (virgin) rats, Atherosclerosis, 20 (1974) 155-172. Wurtman. R.J. and Axelrod. J., Adrenaline synthesis - control by the pituitary gland and adrenal glucocorticoids, Science 150 (1965) 1464-1465. Wexler, B.C. and Saroff, J.. Effects of transplanted pituitary glands from non-arteriosclerotic virgin and arteriosclerotic breeder donors to hypophysectomized rats. Acta endow., 59 (1968) 249-260.
Atherosclerosis, 24 (1976) 63-73 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
ALDOSTERONE, DEOXYCORTICOSTERONE AND CORTICOSTERONE DIFFERENCES BETWEEN ARTERIOSCLEROTIC BREEDER VS NONARTERIOSCLEROTIC VIRGIN RATS
SAMUEL G. IAMS and BERNARD
C. WEXLER
May Institute for Medical Research, The Jewish Hospital, and the Departments of Physiology, Medicine and Pathology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45229 (U.S.A.) (Received
23rd June, 1975)
(Accepted
12th January, 1976)
Summary The circulating levels of the major adrenal steroids, corticosterone (Cmpd. B), deoxycorticosterone (DOC), and aldosterone were compared in male and female, arteriosclerotic, breeder and non-arteriosclerotic virgin, SpragueDawley rats under resting conditons, i.e., quiescence, and after exposure to a mild stress, i.e., movement from one room to another just prior to autopsy. Under resting conditions, the arteriosclerotic animals had significantly greater circulating levels of Cmpd. B, DOC and aldosterone than the non-arteriosclerotic animals. Both the arteriosclerotic and non-arteriosclerotic animals were able to respond adequately to the mild stress stimulus. However, the female breeder rats which manifest the most severe aortic sclerosis, showed the greatest increase in Cmpd. B and aldosterone in response to the mild stress. Although no statistically significant differences could be found between female breeders with grossly visible aortic sclerosis of clear, minimal, moderate, or severe degree, it was apparent that adrenal steroid responsiveness becomes progressively compromised with increasing severity of arteriosclerosis, e.g., unusually high or low levels of Cmpd. B and aldosterone under both quiescent and mild stress conditions. It is suggested that there may be some connection between abnormal hypothalamic-pituitary-adrenal-gonadal function in repeatedly-bred and the pathogenesis of their naturally-occurring ateriosclerosis. -This work was supported by grants from the Eastern and Southwestern Ohio Heart Associations and the National Heart and Lung Institute (HL-15, 304) N.I.H. Dr. lams was supported by a Research Fellowship from the Southwestern Ohio Heart Association. Please address all correspondence to: Dr. Samuel G. lams, May Institute for Medical Research. 421 Ridgeway Avenue, Cincinnati, Ohio 45229, U.S.A.
64
Key words:
ACTH
-
Catecholamines
Factor
-
Mineralocortocoids
gland
-
Cortico-medullary us glucocorticoids
axis -
Corticotrophin
Hypothalamus
Releasing -
Pituitary
- Steroidogenesis
Introduction Spontaneous hypertension, hyperglycemia, hyperlipidemia and arteriosclerosis appear in repeatedly-bred male and female rats. The incidence and severity of the arteriosclerosis and related pathophysiologic changes parallels the frequency of the reproductive effort [l--8]. Although it is not known whether the changes in the hypothalamic-pituitary-adrenal-gonadal axis are primary or secondary relative to the pathogenesis of the arterial disease, we have definite evidence that endogenous pituitary, adrenal and gonadal hormones do play a prominent role in the pathogenesis of this naturally-occurring vascular disease. In earlier investigations, we found that arteriosclerotic, breeder rats manifested dramatic adrenocortical histopathology, e.g., hypertrophy, hyperplasia, zonal lipid alterations, thromboses and infarction, as well as adrenomedullary histopathologic alterations, e.g., pheochromocytomas [ 91. Total steroidogenesis and individual corticosteroids as well as the ratio of norepinephrine : epinephrine become progressively altered commensurate with the number and frequency of breedings as well as the severity of the arterial disease. Despite superabundant stores of cholesterol, i.e., steroid precursor material, arteriosclerotic, breeder rats are less capable of responding to exogenous ACTH or stress than virgin rats with no arterial disease [lo]. In vitro steroid analyses of arteriosclerotic, breeder rat adrenal glands demonstrates that in parallel with the development of their arterial disease, their adrenal glands lose the capacity to convert steroid intermediates, e.g., pregnenolone, into more definitive steroids, e.g., corticosterone, and the entire spectrum becomes altered, e.g., ratio of aldosterone : 18-hydroxydeoxycorticosterone : deoxycorticosterone : corticosterone, etc. [lo]. That is, the change in the normal spectrum of adrenal steroids appears to be due to some derangement of the adrenocortical enzymes which regulate steroidogenesis [ 111. Subsequent in vivo investigations confirmed these earlier in vitro findings, and, in additon, demonstrated that arteriosclerotic breeder rats had deranged metabolic clearance rates as well as abnormal hepatic steroid metabolism [ 121. In more recent studies we have found that pituitary production and release of ACTH is progressively impaired in breeder rats in parallel with their progressively worsening arteriosclerosis, and that this defect in adrenocorticotrophic hormone may be due to abnormal hypothalamic production and release of Corticotrophin Releasing Factor (CRF) [13]. There is an interesting sex dichotomy between male and female breeder rats in the pathogenesis of their arterial disease. The female breeders develop grossly visible arterial lesions of the aorta; male breeders develop microscopic aortic lesions only [l-5]. Further, despite their seemingly more innocuous lesions, male breeder rats die significantly earlier than their female breeder counterparts, usually due to myocardial infarction. ECG studies demonstrated
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definitely abnormal tracings as well as fundamental differences between arteriosclerotic and non-arteriosclerotic animals and between males and females [14, 151. When we compared various hemodynamic parameters indicative of the cardiac index or cardiac output of arteriosclerotic, breeder (male vs female) vs non-arteriosclerotic, virgin (male vs female) rats, we uncovered another seemingly paradoxical situation wherein the female breeders with severe, grossly visible aortic sclerosis had a significantly higher cardiac output compared to the other groups with less severe arteriosclerosis [16,17] . Mineralocorticoids, e.g., aldosterone, deoxycorticosterone, etc., can increase cardiac output, condition the arterial wall toward hypertension, as well as other cardiovascular effects. We elected to compare the relative circulating levels of aldosterone, deoxycorticosterone and corticosterone (the major circulating glucocorticoid produced by the rat’s adrenal cortex) in arteriosclerotic, breeder vs non-arteriosclerotic, virgin rats, in order to ascertain whether the deranged adrenal glandular function has any direct bearing on the cardiovascular degenerative changes in repeatedly-bred rats. Further, we compared the circulating levels of these dynamic mineralocorticoids vs glucocorticoids in male vs female rats to determine whether there are any adrenocortical steroid differences between the sexes, between arteriosclerotic vs non-arteriosclerotic males and females or between breeder rats with varying degrees of arteriosclerosis, i.e., male breeders with microscopic aortic sclerosis vs female breeders with clear, minimal, moderate, or severe aortic sclerosis. Materials and methods Mature, non-arteriosclerotic, virgin and arteriosclerotic, breeder rats (Sprague-Dawley strain) were divided into 4 groups: virgin, non-arteriosclerotic males (15) and females (26), and arteriosclerotic, breeder males (17) and females (93). All of these animals were 6 to 8 months of age. The female breeder rats which are prone to develop grossly-visible aortic sclerosis [l-5] were classified, at autopsy, as having grossly-visible aortic sclerosis of either clear, minimal, moderate, or severe degree aa described previously [l].The arteriosclerotic, male breeders were found to have microscopic aortic sclerosis only, and like the female breeders, to have coronary, carotid, mesenteric, renal and peripheral arterial disease [l-5] (confirmed at autopsy and histopathologicallY)* The animals were housed in our Animal Research Colony where temperature, humidity and light are carefully controlled. All of the animals were fed a commercial rat chow (Purina Rat Chow, St. Louis, Missouri), which is relatively low in fat (4%) and tap water ad libitum. Because adrenocortical steroidogenesis is affected by circadian rhythm, the lighting in the Animal Research Colony was regulated on a 14 hours “On” : 10 hours “Off” cycle and the animals were killed at a set time in the morning (2 to 4 h after “Lights On”). Because the stress of movement of animals from one laboratory to another is sufficient to cause an increase in adrenocortical steroid secretion, one group of animals of each of the varieties described was autopsied within the confines of our Animal Research Colony without movement, i.e., the quiescent or nonstressed group. A similar group was moved to our Research Laboratories prior
66
to autopsy, i.e., mild stress. All of the animals were killed by instant decapitation with a minimum of handling to avoid the relatively strong stress of anesthesia. Blood was collected from the severed neck vessels, centrifuged, and stored at -20°C until ready for steroid analyses. Steroid determinations Aldosterone. Aldosterone was extracted from the serum samples using 30X volumes of methylene chloride (MeC&) and separated from other interfering steroids by means of a Sephadex LH-20 Column [using MeClz : MeOH (98 : 2, v/v) as a mobile phase]. The extracted aldosterone was measured using the radioimmunoassay method of Ito et al. [18]. Anti-serum for assay of aldosterone was provided through the courtesy of the Hormone Distribution Officer (Dr. R.W. Bates), National Institutes of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. Corticosterone. Each of the serum samples was extracted with a mixture of hexane : benzene (80 : 20, v/v) to partially purify the sample, e.g., to remove lipids. The serum samples were extracted a second time with the hexane : benzene (20 : 80, v/v) mixture to separate the corticosterone for the assay. Corticosterone was determined by a modified radioimmunoassay method of Mayes et al. [19] . Antiserum for corticosterone was added to a 3 ml centrifuge tube containing 5,000 cpm tritiated corticosterone and an aliquot of serum extract. The solution was incubated in a water bath at 40°C for 5 min and then chilled at 4°C overnight. A Dextran-80 (250 mg) and a Norit A charcoal mixture (250 mg) in 100 ml of borate buffer (pH 8.0) was added to the solution to terminate
Fig. 1. Comparison of the circulating corticosterone levels of male. non-arteriosclerotic, virgin and art&osclerotic, breeder vs female, non-arteriosclerotic virgin and arteriosclerotic, breeder, Sprague-Dawley rats under resting or quiescent conditions vs after a mild stress such as moving the animals from one room to another just prior to autopsy. The height of each column indicates the Mean f Standard Error; the numbers in brackets indicate the number of samples assayed. Figures 2 and 3 follow the same protocol.
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the reaction and to bind the free steroid. The solution was then centrifuged (2,000 rpm) for 10 min and an aliquot taken to determine the percent of steroid bound to the antibody. Deoxycorticosterone. Serum samples were extracted with petroleum ether to remove the major quantities of progesterone and lipid. Each sample was extracted a second time with a hexane : benzene (20 : 80, v/v) mixture to recover the remaining deoxycorticosterone. The extract was then passed through a 20 cm Sephadex LH-20 column using a MeCl, : benzene : MeOH (70 : 26 : 4, v/v/v) mixture to separate the progesterone from the deoxycorticosterone (progesterone has a 100% cross reactivity with the antiserum). Antiserum for the radioimmunoassay of deoxycorticosterone and corticosterone was obtained from Endocrine Science, 18418 Oxnard St., Tarzana, California 91356, U.S.A.. The radioimmunoassay procedure was the same as that used for corticosterone, i.e., the method of Mayes et al. [19]. The immunoassay data was subjected to logit-log transform and analyzed by the method of Midgeley et al. [20] , and Rodbard et al. [21] . Covariance analysis was also used as a test of inter-assay correlation. Statistical analyses of the differences in serum steroid values between the experimental and control groups were performed using Student’s t-test as described by Snedecor and Cochran [22]. Results Under quiescent conditions, the circulating levels of corticosterone (Cmpd. B), deoxycorticosterone (DOC), and aldosterone were much higher (P < 0.001) in the arteriosclerotic, breeder vs the non-arteriosclerotic, virgin rats (Figs. l3). Cmpd. B levels were twice as high in the female breeders with grossly-visible
I 0
Oulescent
Fig. 2. Comparison of circulating deoxycorticosterone
levels. (0 = Data lost due to technical problems.)
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arteriosclerosis compared to female virgins with no arteriosclerosis, i.e., 18.09 + 1.20 vs 8.15 f 0.95 pug/100 ml (Fig. 1); DOC levels were four times higher in the arteriosclerotic female breeders compared to the non-arteriosclerotic, virgin females, i.e., 7.56 f 0.48 vs 1.63 + 0.30 ng/ml (Fig. 2); aldosterone levels were three times higher in the arteriosclerotic female breeders compared to the non-arteriosclerotic, virgin females, i.e., 21.6 f 1.93 vs 9.16 f 1.65 ng/lOO ml. The Cmpd. B level for the arteriosclerotic, male breeders were three times higher than those for the nonarteriosclerotic, virgin males (Fig. l), i.e., 12.00 k 1.30 vs 4.98 + 0.82 pug/100 ml. The samples for DOC and aldosterone for quiescent, male breeder rats were irretrievably lost. (Data from other experiments indicate that the DOC and aldosterone levels for male breeders with microscopic aortic sclerosis are considerably higher than those for the non-arteriosclerotic, male virgins.) The adrenal cortices of the non-arteriosclerotic, virgin and arteriosclerotic, breeder rats, males and females, were all capable of responding to the relatively mild stress of moving the animals from one room to another just prior to autopsy. This capacity to respond to a mild stress was reflected in each of the major steroids measured, i.e., Cmpd. B (Fig. l), DOC (Fig. 2) and aldosterone (Fig. 3). This increase in the major steroids in response to a mild stress was statistically highly significant (P < 0.001) with the exception of the increase in aldosterone in the arteriosclerotic, female breeders. It should be borne in mind that in most animals, the adrenal gland of the female is much heavier than that of the male and in most animals, especially the rat, the female has much higher circulating steroid levels. This characteristic sex difference of higher circulating levels of steroids in females vs males was
I ,-. 0
Quiescent
m
Mild
Stress
-113 (36
(9)
Fig. 3. Comparison of circulating aldosterone levels. (o = Data lost due to technical problems.)
69
also found in these animals pertaining to both mineralocorticoids as well as glucocorticoids, i.e., aldosterone, DOC and Cmpd. B (Figs. l-3). When a comparison was made of the steroidogenic responsiveness of female breeders on the basis of their varying degrees of arteriosclerosis, i.e., clear, minimal, moderate and severe, compared with non-arteriosclerotic, virgins, it was apparent that the serum of arteriosclerotic animals contained much more of the major glucocorticoid, Cmpd. B and the mineralocorticoid, aldosterone (Fig. 4). However, there were no statistically significant differences in circulating steroid levels between each of the various categories of grossly-visible arteriosclerosis, i.e., clear to severe. When the circulating aldosterone levels of arteriosclerotic vs non-arteriosclerotic, females were compared under conditions of quiescence and mild stress (Fig. 5), no statistically significant differences could be found between those female breeders having clear, minimal, moderate or severe arteriosclerosis. A definite trend toward progressively greater divergence of aldosterone responsiveness to the stimulus of a mild stress, i.e., high (responsive) and low (non-responsive) values could be discerned concomitant with progressively increasing severity of grossly-visible arteriosclerosis, especially in those female breeders having severe aortic sclerosis (Fig. 5).
0 Corticosterone 0
G
0
Aldosterone
No
Cleor
Minimal
Microscopic ARTf
i,
Moderate
Severe
Grossly-visible 2IOSCLEROSIS
VIRGINS
Fig. 4. Scattergram depicting individual serum aldosterone (closed circles) and corticosterone (open circles) levels of non-arteriosclerotic. virgin, female rats compared with arteriosclerotic, female breeder rats separated according to the appearance of their grossly-visible aortic sclerosis observed at autopsy, i.e., clear, minimal, moderate and severe degree. Both the corticosterone and aldosterone levels were taken under resting or quiescent conditions.
IO
??Mild 0
Stress
0
Quiescent
:
.
.
8
:
i
5
1
0%
0 0
@ d %
1
Clear
Minima
Microscopic I
L
. VIRGINS
1
L ...
.
??
No
.
ARTERIOSCLER BREEDER!
??
I
Moderate
(
Severe
xsly-visible SIS I
Fig. 5. Scattergram of individual aldosterone levels of non-arteriosclerotic, virgin, female rats compared with arteriosclerotic. female breeder rats, separated according to the severity of their grossly-visible aortic sclerosis observed at autopsy, i.e., clear, minimal, moderate and severe. under conditions of quiescence (open circles) and mild stress (closed circles).
Discussion We cannot determine, at this time, whether the deranged adrenocortical function in repeatedly-bred rats is primary or secondary to their spontaneous cardiovascular degenerative changes. However, the present observations reconfirm our earlier findings that there is a definite difference in the adrenal steroid levels between non-arteriosclerotic virgin rats and arteriosclerotic breeder rats. When considering all of the accumulated evidence we have concerning the possible role of hormones in the pathogenesis of breeder rat arteriosclerosis, we believe that there may be some connection between the cardiovascular degenerative changes with accompany repeated breeding and derangement of the hypothalamic-pituitary adrenal-gonadal axis. For example, in one of our most recent reports, we showed that the circulating level of luteinizing hormone (LH) was progressively decreased and that circulating prolactin was greatly increased, commensurate with the increasing severity of grossly-visible arteriosclerosis in female breeder rats [23] . These findings would suggest that hypothalamic releasing factors which regulate pituitary gland release of LH and prolactin, e.g., Prolactin Inhibiting Factor (PIF), become altered during the continuous hypothalamic stimulation of repeated breeding. Similarly, we have also observed increasing pituitary basophilia, adenomatosis, and alterations in pituitary gland content of
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ACTH in concert with the increasing severity of arteriosclerosis in repeatedlybred rats [13] . In the present investigation, the definitely abnormal Cmpd. B, DOC, and aldosterone levels in arteriosclerotic, breeder rats is further confirmation and extension of our earlier in vitro [lO,ll] and in vivo [12] steroid studies and is in keeping with our findings of extensive histopathologic alterations in the adrenal cortices of arteriosclerotic breeder rats, e.g., extensive zonal lipid alterations, thromboses and infarction [9] . It would be pertinent to emphasize that repeatedly-bred rats also develop progressively changing catecholamine biosynthesis, i.e., adrenaline, noradrenaline and phenyl-ethanolamine-Nmethyl transferase (PNMT) [24], as well as pheochromocytomas [9], in parallel with their changing cortical steroidogenesis and worsening arteriosclerosis. That is, there may be an adrenocortical-medullary axis or inter-play involved in the pathogenesis of the abnormal metabolic and vascular degenerative changes in repeatedly-bred rats, since adrenal steroids, particularly the glucocorticoids, have been shown to condition the activity of the adrenomedullary enzyme PNMT in converting noradrenaline into definitive adrenaline [25] . The combined changes in adrenal corticosteroids and catecholamines in repeatedlybred rats could well account for their hypertension, hyperglycemia, hyperlipidemia, arteriosclerosis, frequent myocardial infarction, and premature aging. In view of the sex dichotomy between male vs female breeders, i.e., relatively mild aortic sclerosis in male breeders vs advanced, grossly-visible aortic sclerosis in female breeders, it is noteworthy that not only do female rats have larger adrenal glands and produce greater quantities of Cmpd. B under both quiescent and stressful conditions, but also, repeated pregnancies (or arteriosclerosis) changes their adrenal steroidogenic capacity toward even greater Cmpd. B, DOC, and aldosterone production, particularly Cmpd. B and aldosterone. This greater capacity to produce adrenocorticoids in female rats may play some role in conditioning the various morphologic components of the aortic wall toward a different kind of pathologic change in females vs males. In order to obtain an overall purview of the temporal adrenal glandular changes associated with repeated breeding and arteriosclerosis, we have combined all of the information from our in vitro and in vivo investigations with the result that it would appear that repeatedly-bred rats become progressively hyperadrenocorticoid during the early phases of their reproductive activity; at this time, the arterial lesions are microscopic or just beginning to become grossly-visible in the abdominal aortic segment. With continued breeding, some of the animals manifest extensive adrenocortical and medullary pathology, their pituitary glands become adenomatous, ACTH release is impaired, and/or the adrenal cortices become unresponsive to ACTH stimulation and steroidogenesis becomes altered; at this time, grossly-visible arteriosclerosis is usually rampant throughout the length of the aorta. We believe that the increased circulating levels of Cmpd. B, DOC, and aldosterone, as demonstrated in these animals, may account for the repeatedly-bred rat’s propensity toward increasing blood pressure, myocardial failure, and perhaps, arteriosclerosis. Our finding of increased serum levels of Cmpd. B, DOC, and aldosterone in the arteriosclerotic breeder rats under quiescent conditions indicates that under conditions free of extra demand for pituitary gland release of ACTH, the arteriosclerotic, breeder rats are definitely hyperadrenocorticoid. The two,
72
three and four-fold increases in Cmpd. B, aldosterone and DOC in the arteriosclerotic vs non-arteriosclerotic rats, are quite considerable and could have farreaching pathophysiologic effects. It is of particular interest that under conditions of mild stress, i.e., mild ACTH release and mild adrenocortical stimulation, circulating levels of Cmpd. B, DOC, and aldosterone increased, approximately in equal proportions in both the arteriosclerotic and non-arteriosclerotic animals. On the other hand, in all of our previous investigations, when a stress of greater proportion was used, e.g., confinement within an ether-filled chamber, the non-arteriosclerotic, virgin rats were capable of releasing maximal quantities of adrenal steroids, i.e., Cmpd. B. However, arteriosclerotic, breeder rats are completely incapable of increasing their steroidogenic potential beyond a certain level, a level which is much below that of non-arteriosclerotic, virgin rats [10,12,13]. In our original in vitro investigations [lo] and in one of our most recent in vivo studies [13] , we found that as the dose of exogenous ACTH was increased in graded increments, the ability of adrenal glands of arteriosclerotic animals to secrete Cmpd. B on demand, increased up to a given dose of ACTH and then were no longer able to respond to any greater increments of ACTH stimulation. The same doses of ACTH in non-arteriosclerotic, virgins, elicited dynamic and increasing serum levels of Cmpd. B. That is, the non-arteriosclerotic, virgin rats retained their ability to continue to respond to increasing increments of exogenous ACTH and their dose : response did not plateau until a level of ACTH was reached well above that to which the arteriosclerotic breeder could no longer respond [lo]. Since this suggested that there may be a progressively developing refractoriness to endogenous (and exogenous) ACTH in repeatedly-bred rats, we transplanted the pituitary glands taken from arteriosclerotic, breeder rats beneath the kidney capsule of hypophysectomized, non-arteriosclerotic rats. We found that the transplanted breeder rat pituitary glands secreted ACTH and gonadotrophic hormones which were able to maintain the adrenal glands and gonads of the hypophysectomized recipients in superior fashion than those animals in which pituitary glands, removed from non-arteriosclerotic, virgin rats, had been transplanted in a similar manner [26]. Subsequently, bio-assay of the actual pituitary gland content of ACTH of arteriosclerotic vs non-arteriosclerotic animals, demonstrated that although the pituitary glands became progressively hyperplastic and heavier in breeders with clear vs minimal vs moderate vs severe arteriosclerosis, their ACTH content becomes progressively less [ 131. In conclusion, despite the fact that there were no statistically significant differences in the circulating steroid levels between female breeders with varying degrees of severity of arteriosclerosis, i.e., clear, minimal, moderate and severe arteriosclerosis, there was, nonetheless, a definite difference in the amounts of circulating Cmpd. B, DOC and aldosterone between non-arteriosclerotic, virgin rats and arteriosclerotic breeder rats. The findings reported here, taken in context with our ancillary published findings of alterations in pituitary trophic hormones [13,23] , changes in pituitary histopathology and ACTH content [13,26], and adrenal cortical and medullary malfunction [g-13,23,24,26], suggest that there may be some connection between abnormal hypothalamicpituitary-adrenal-gonadal function in repeatedly-bred rats and the pathogenesis of their naturally-occurring arteriosclerosis.
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Acknowledgement The authors are grateful for the expert natser, E. Domingo, G. Heap and J. Wexler.
assistance
of D. Schaffner,
D. Co-
References 1 Wexler, B.C., Spontaneous arteriosclerosis in repeatedly-bred male and female rats. J. Atheroscler. Res., 4 (1964) 57-80. 2 Wexler, B.C. and True. C.W.. Carotid and cerebral arteriosclerosis, Circ. Res., 12 (1963) 659466. 3 Wexler, B.C., Spontaneous coronary arteriosclerosis in repeatedly bred male and female rats, Circ. Res., 14 (1964) 32-43. 4 Wexler, B.C., Spontaneous arteriosclerosis of the mesenteric. renal and peripheral arteries of repeatedly bred rats. Circ. Res.. 15 (1964) 485-496. 5 Wexler, B.C., Arteriosclerosis of the renal artery of repeatedly bred male and female rats, Atherosclerosis, 11 (1970) 383-400. 6 Judd, J.T. and Wexler. B.C., The role of lactation and weaning in the pathogenesis of arteriosclerosis in female breeder rats, J. Atheroscler. Res., 10 (1969) 153-1’72. 7 Wexler. B.C. and Kittinger, G.W.. Spontaneous arteriosclerosis in male and female breeder rats - Effects of a high fat diet and hyperadrenocorticism on repeatedly bred rats, J. Path. Bact. 94 (1967) 231-246. 8 Lutmer. R.F., and Wexler. B.C.. Pathophysiologic changes associated with the development of arteriosclerosis during various phases of the reproductive cycles of repeatedly bred rats, Camp. Biochem. Physic& 37 (1970) 445-466. 9 Wexler, B.C., Correlation of adrenocortical histopathology with arteriosclerosis in breeder rats, Acta endocrinol., 46 (1964) 613-631. 10 Wexler. B.C. and Kittinger, G.W.. Adrenocortical function in arteriosclerotic female breeder rats, J. Atheroscler. Res.. 5 (1965) 317-329. and corticosteroid production in 11 Kittinger, G.W. and Wexler, B.C., Adrenal gland dehydrogenases normal and arteriosclerotic breeder rats, Proc. Sot. EXP. Biol. Med., 118 (1965) 365-367. 12 Saroff, J. and Wexler, B.C.. Metabolic clearance and production rates of corticosterone in male and female virgin and breeder rats, Acta endocrinol.. 62 (1969) 411-424. 13 Wexler. B.C. and Lutmer, R.F.. ACTH content in pituitary glands of arteriosclerotic breeder vs nonarteriosclerotic, virgin rats. Atherosclerosis, 22 (1975) 199-214. 14 Wexler. B.C.. Willen, D. and Greenberg, B.P., Electrocardiographic differences between non-arteriosclerotic and arteriosclerotic rats, Atherosclerosis, 18 (1973) 129-140. 15 Wexler. B.C., Willen, D. and Greenberg, B.P., Progressive electrocardiographic changes in male and female arteriosclerotic and non-arteriosclerotic rats during the course of isoproterenol-induced myocardial infarction, Cardiovasc. Res.. 8 (1974) 460-468. 16 Colfer. H.T., Iams. S.G. and Wexler, B.C., Cardiovascular function in non-arteriosclerotic vs arterio17 18 19 20
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sclerotic rats. Amer. J. Physlol., 229 (1975) 18-22. Golfer. H.T.. Iams. S.G. and Wexler. B.C., Hemodynamic changes in arteriosclerotic YS non-arteriosclerotic rats during the acute stages of myocardial infarction, Angiology. 27 (1976) 32-41. Ito. T., Woo. J.. Haning. R. and Horton, R.. A radioimmunoassay for aldosterone in human peripheral plasma including a comparison of alterate techniques, J. Clin. Endocr. Metab., 34 (1972) 106-112. Mayes, D., Furuyama, S., Kern, D.C. and Nugent, C.A., A radioimmunoassay for plasma aldosterone, J. Clin. Endow. Metab., 30 (1970) 682-685. Midgley. A.R., Jr., Niswender, G.D. and Rebar, R.W.. Principles for the assessment of the reliability of radioimmunoassay methods (Karolinska Symposium on Immunoassay of Gonadotropins), Acta Endocr., Suppl., 142 (1969) 163-184. Rodbard, D., Rayford. P.L., Cooper, J.A. and Ross, G.T.. Statistical quality control of radioimmunoassays. J. Clin. Endocr. Metab., 28 (1968) 1412-1418. Snedecor. G.W. and Cochran. W.G., Statistical Methods, Iowa State University Press, Ames. Iowa. 1967. Lewis, B.K. and Wexler. B.C., Changes in LH and prolactin in arteriosclerotic female breeder rats, Atherosclerosis, 21 (1975) 301-314. Wexler. B.C. and Greenberg, B.P., Adrenal cortico-medullary function in arteriosclerotic (breeder) and non-arteriosclerotic (virgin) rats, Atherosclerosis, 20 (1974) 155-172. Wurtman. R.J. and Axelrod. J., Adrenaline synthesis - control by the pituitary gland and adrenal glucocorticoids, Science 150 (1965) 1464-1465. Wexler, B.C. and Saroff, J.. Effects of transplanted pituitary glands from non-arteriosclerotic virgin and arteriosclerotic breeder donors to hypophysectomized rats. Acta endow., 59 (1968) 249-260.
