Propranolol-induced alterations in the pathophysiology of spontaneously hypertensive rats

Life Sciences, Vol. 29, pp. 1409-1416 Printed in the U.S.A.

Pergamon Press

PROPR#~OLOL-II~UCED ALTEILATIONS IN THE PATHOPI~SIOLOGY OF SPONTANEOUSLY ~[YPERTENSIVE RATS Bernard C. Wexler and John P. McMurtry May Institute for Medical Research of Jewish Hospital and Departments of Medicine and Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229 (Received in final form July 24, 1981) Summary Male and female, spontaneously hypertensive rats (SHR) were treated with propranolol (14 weeks) when they became I, 4 and 8 months old prior to, during the steep ascent, and when severe high blood pressure becomes ~stablished. Propranolol prevented the usual increase in blood pressure at an early age but did not effectively lower blood pressure at all age levels~ Propranololtreated SHR manifested marked alterations in pituitary~ adrenal, thymus, heart and gonadal weights. Despite progressively increasing hyperlipidemia and hyperglycemia, there were no significant differences between lipid, glucose, BUN, or serum enzyme levels in treated vs nontreated SHR. Circulating aldosterone and corticosterone levels were reduced in propranolol-treated SHR. Male SHR, treated or untreated, developed severe cardiovascular-renal lesions when they became B months old; none of the female Sh~ manifested any pathologic changes. It is suggested that the anti-hypertensive effects of propranolol were partially mediated by hormonal as well as by hemodynamic mechanisms. The pathophysiology of high blood pressure in the spontaneously hypertensive rat (SHR) mimics essential hypertension in man (1-3). The SHR develops left ventricular hypertrophy, increased cardiac output, expanded blood volume, and increased peripheral vasoconstriction. We (4-6) and others (1-3) have shown that nutritional and hormonal factors also play a role in the pathogenesis of SHR hypertension. For example, hypophysectomy, adrenalectomy, and gonadectomy will interfere with the development of naturally-occurring hypertension (2-5). SHR are unusually sensitive to handling, sudden noise, and stress (7,8). This hypersensitivity to stress becomes exacerbated with age and parallels the acutely rising blood pressure (7). We have shown that under strictly quiescent conditions, the diurnal secretion of the adrenal stress hormones, corticosterone and aldosterone, are similar in SHR to normotensive rats; under conditions of comparatively mild stress, SHR secrete copious quantities of corticosterone, deoxycorticosterone, aldosterone (7-9), and catecholamines (i0) Propranolol, a potent beta-adrenergic blocking agent, has been used widely as a anti-hypertensive drug. In addition to slowing the heart rate and decreasing cardiac output, propranolol has a sedative effect (ii). Preliminary trials with propranolol demonstrated that it had a remarkably tranquilizing effect on our hyperkinetic SHR. Therefore, we elected to treat male and female SHR with propranolol at various stages of their developing hypertension, i.e., prior to the appearance of hypertension, at the mid-stage of developing hypertension, 0024-3205/81/141409-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

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and when hypertension had become well established. Our purpose was to determine: a) whether SHR hypertension could be ameliorated irrespective of its temporal development, b) what hormonal changes occur concomitant with the progressively increasing spontaneous hypertension or reduction of high blood pressure (with propranolol), and c) what pathophysiologic changes occur with increasing or decreasing blood pressure. Methods All of the animals used in this experiment were obtained from our SHR Breeding Colony by brother:sister matings of original stock provided by the NIH (Bethesda, Maryland) through the courtesy of Dr. Carl T. Hansen. This breeder stock was derived from the original Kyoto-Wistar hypertensive strain developed by Okamoto and Aoki (i). These SHR manifest progressively increasing blood pressure shortly after weaning, and stable high blood pressure (180 • 5 mmHg) is established and maintained by 8-10 weeks. The rats were kept in our Research Animal Colony where temperature (26 • 1 ° C), humidity (45-50%), and lighting were controlled. The lighting in the Animal Research Colony was regulated on a 14 h "on" (0700-2100) and i0 h "off" cycle. The rats were fed a commercial rat chow (Purina) which has a relatively low fat content (4%) and were given tap water to drink ad libitum, Propranolol-d, i, (l-Isopropylamino)-3-(l-naphthyloxy)-2-propranolol hydrochloride, Ayerst Laboratories, Inderal R, was suspended in sterile water and 0.33 mg/100 g bw injected sc, three times per week for 14 weeks. A large number of untreated male and female SHR served as baseline controls from the time they were 1 month old until 11.5 months of age. Three groups (24 males and 24 females per group) were selected at random to receive propranolol RX started at: i) 1 month of age, just prior to the characteristic rise in blood pressure in SHR at 5 weeks of age, 2) 4 months of age when high blood pressure is rising rapidly in SHR, and 3) 8 months when high blood pressure has reached a zenith and has become chrcnic. Blood pressures were recorded at weekly intervals using light secobarbital (Seconal) anesthesia and the Friedman:Freed microphonic manometer indirect tail cuff method which measures systolic blood pressure only. The blood pressures were taken under quiescent conditions and without restraint. The rats were killed by decapitation at a site removed from the sight, smell, or sound of the remaining animals (within 1 min after removal from the cage). Blood was collected from the severed neck vessels, centrifuged, and stored at -20°C until ready for biochemical analyses. All animals were fasted 18 h prior to autopsy in anticipation of lipid analyses. Because adrenocortical steroidogenesis is affected by circadian rhythm, all animals were killed at a set time in the morning (0900-1000 h). The following biochemical parameters for each animal were measured using the Auto-analyzer (Technicon): serum enzymes-creatine phosphokinase (CPK), glutamic oxalo and pyruvic transaminases (SGOT, SGPT), and lactic dehydrogenase (LDH), lipids - triglycerides, free fatty acids, total cholesterol, glucose, and blood urea nitrogen (BUN). All of the above automated procedures are detailed in the manual published by the Technicon Co., "Automation in Analytical Chemistry," Technicon, Mediad, Inc., New York. Aldosterone and corticosterone were measured as an index of adrenocortical activity by radioimmunoassay (7). Antisera and standards for the immunoassay for aldosterone were kindly provided by Dr. A. Parlow and the Hormone Distribution Officer, NIAMDD, NIH (Bethesda, Maryland). Final body weight as well as the weight of pertinent organs were Gross and microscopic observations were made of the heart, aortae, for salient pathological features. Analysis of variance and other analyses followed the procedures and tables cited by Snedecor (12)

recorded. and kidneys biostatistical and using

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Student's t test. Results Systolic Blood Pressure. At five weeks of age, the blood pressure of the untreated SHR began to rise steeply. By the 4th month, blood pressures were severely elevated, (190 • 5 mmHg) and by 8 months, high blood pressure was well established (220 i 8 mmHg)(Fig, i). The blood pressures of male and female SHR followed a similar pattern of rise, reaching an ultimate plateau at severely elevated levels. Treatment with propranolol one week prior to the inception of spontaneously rising blood pressure did not prevent hypertension but it effectively inhibited the intensity of the increase. (A systolic blood pressure above 135 mmHg in the rat is considered to be abnormally elevated.) Propranolol was also effective in retarding the steeply rising blood pressure of older SHR.(Fig i)

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FIG. i Course of the spontaneously developing systolic blood pressure of male SHR from 30 days to 8 months of age. Each point depicted is the mean ± SE; n = i00 SHR per point. Some of the animals began receiving propranolol when they were 1 month old, 4 months old, or 8 months old. The blood pressure of the treated and non-treated SHR were recorded weekly and the changes 2,4,6,8,10,12 and 14 weeks later are shown, n = 24 for each point. The blood pressures for female SHR follow the same pattern of change as shown for males, treated and nontreated, except that female SHR blood pressure averaged i0 mmHg less than male SHR. The same protocol was followed for Figs. 2 and 3.

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Changes in organ and body weight. There were no statistically significant differences in the final body weight between treated and untreated male or female SHR. The weight of the pituitary gland of control males and females manifested dichotomous changes with age, i.e., the pituitary glands tended to become lighter in males but heavier in females (P~ 0.001)(Table I). Propranolol treatment caused a reverse trend in male SHR, i.e., pituitary glands increased in weight with time and treatment. Pituitary glands of propranolol-treated female SHR were significantly (P~0.001) reduced in weight but the trend toward increased pitditary gland weight with age obtained. Adrenal glandular weight was two to three times greater in female vs male SHR concomitant with a progressive increase in adrenal weight with age (Table I). Analysis of variance demonstrated that there was an even greater and significant (P<0.001) increase in adrenal weight witN time in male and female SHR'given propranolol. Propranoloi treatment appeared to accelerate the rate of thymus gland involution (Table I). The progressively worsening high blood pressure was accompanied by progressively increasing heart weight (P~0.001). Heart weights were less (P~0.001) in male and female SI~ in which the usual high blood pressure rise was inhibited by propranolol treatment (Table I). The kidneys of these SHR were comparatively small in size and there were no significant differences between the kidney weights of treated vs non-treated SHR. The testes and ovaries of the untreated SHR manifested progressive, premature involution and loss in weight with time and worsening blood pressure (Table I). Propranolol treatment appeared to inhibit this premature loss of gonadal size or weight. TABLE I Gravimetric Changes in Male vs Female SHR Killed at 4.2, 7.2 and 11.2 Months of Age and Treated With Propranolol or Saline for 14 Weeks Commencing at i a, 4 b, and 8 c Months of Age, Respectively

Rx

Final b.w. (g)

Pit

asaline Prop. bSaline Prop. CSaline Prop.

33755 323~6 346~6 352~7 345~7 340~9

9.7±0.8 i0.9~0.5 9.7~i.0 ii.3~0.3 9.5~0.6 ii.9-1.5

18.650.8 20.9~0.3~ 20.8~0.6 23.0~0.5 f 24.2~2.0 25.2~2.0f

17658 138~4" 125~8 100~5"r 75~8 75~4

1511~18 1347~9" 1699~12 1361~13" 1921~21 1788~14"

i121~18 i140~12 1270~24 1241~21 1590~16 1575~36

FemaleaSaline Prop. bSaline Prop. CSaline Prop.

21654 21615 222,3 213~4 217~7 220~3

12.3,0.5 9.1~0.6" 13.5~0.5 i0.i~0.4" 14.2-0.4 ii.0~0.6"

29.3~i.0 29.850.5 29.6~I.0 31.2~i.0 30.25~i.0 34.3"1.5

163~4 158~7 152~7 125~4" 86~4 86~6

i021,16 I001~i0 i132~15 i021~Ii* 1311~14 1198513*

719~18 718~16 875+22 839~17 900~26 918~21

Male

Adr

Thy

Heart

Kid

Testis/ ovary

(mgs) 1601~12 1502~16" 1576+28 1584~26 1386~30 1536~18

39~2 35~2" 36~3 35~2 31~3 35~2

Values are expressed as mean • SE; n = 24. * P~ 0.001 P ~ 0.05 Because the body weights were essentially the same for all animals, all of the above changes in organ weights remained significant whether the data was compared on an absolute organ weight basis or on the basis of organ weight/body weight X i00 ratio.

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Propranolol and SHR Hypertension

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Bloodbiochemistry. There were no statistically significant differences in circulating CPK, SGOT, SGPT or LDH levels between treated and non-treated SHR. Except for the characteristic hyperlipidemia and hyperglycemia which accompanies aging and increasing blood pressure in SHR, there were no significant difference~ between the circulating lipid, i.e., triglycerides, free fatty acids, and total cholesterol, or glucose levels of treated vs non-treated SHR. Despite their severely elevated blood pressure, the BUN levels of these animals were not abnormally elevated, i.e., ranging from 21 to 34 mg%. There were no significant differences in the BUN levels of treated vs non-treated SHR. (Above data omitted, available on request) Changes in circulating aldosterone and corticosterone. Circulating aldosterone, deoxycorticosterone, corticosterone, and prolactin increase with age and increasing blood pressure in male and female SHR (7). A similar trend was observed in the SHR in this experiment (Figs. 2 and 3). There was a significant ( P < 0 . 0 0 1 ) reduction in the aldosterone levels of the propranolol-treated SHR in which the characteristic rise in blood pressure was inhibited (Fig. 2). In these SHR, the corticosterone levels (taken under quiescent conditions) appeared to become progressively lower with age. The circulating corticosterone levels were significantly (P< 0.001) reduced in all propranolol-treated vs non-treated SHR (Fig. 3). Histopathology. Except for increasing fatty infiltration of the liver, hyperplasia and increasing lipid staining of the zona glomerulosa of the adrenal gland, there were no discernible pathologic changes in any of these SHR up to 7.5 months of age. Between 8 and 11.5 months, a wide spectrum of degenerative histopathologic changes appeared exclusively in male SIR. Despite the advanced nature of these pathophysiologic changes in male SHR, no discernible pathologic changes, gross or microscopic, could be found in the female SHR of comparable age. Details of this sex dichotomy in histopathology will be published (13). Despite the reduction of blood pressure in propranolol-treated male SHR, the severity of lesions appeared to be the same in treated vs nontreated male SHR. Many of the older male SHR exhibited stroke-like muscle paralysis (unilateral). Only advanced cerebral edema was found, i.e., no evidence of cerebral necrosis. Pituitary glands manifested a high population of basophil cells and frequent hemorrhage. The majority of the hearts in this group exhibited extensive foci of myocardial fibrosis often attended by atrial thromboses. The kidneys manifested advanced glomerulosclerosis and arterial intimal fibrinohyalin deposits and muscular swelling associated with severe reduction of the lumen. The testes were aspermatic and severely involuted with an unusually high incidence of intimal fibrinohyalin occlusion of the medium-sized testicular arteries. Discussion SHR are hyper-reactive but become docile when treated with propranolol. This tranquilizing effect could account for the inhibition of rising blood pressure. Our success in retarding the ascent of high blood pressure with propranolol vs little or no effectiveness claimed by others could be due to the fact that we administered propranolol subcutaneously, whereas most investigators give propranolol by mouth (14-16). The dynamic changes in pituitary gland weight observed in this experiment are remarkable because pituitary gland weight is not usually amenable to experimental maneuvers. The dichotomous decrease in male pituitary gland weight vs increased glandular weight in females, both of which were altered by treatment with propranolol, is suggestive of possible aberrant hypothalamic-pituitary function in SHR. It has been our consistent observation in this and in previous experiments that although SHR have comparatively small adrenal glands, their pituitary glands manifest increasing basophilia. Concomitantly, they also manifest adrenal hyperplasia, extra responsiveness to stress,

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and thymus gland involution, in parallel with their rising blood pressure(7,8, 16-18). The fact that the hypertension-retarding effect of propranolol was accompanied by aggregate changes in adrenal, thymus, pituitary, and gonadal weights is additional putative evidence of the occurrence of hormonal alterations in the treated animals. Despite severe high blood pressure, increased heart weight, and left ventricular hypertrophy, it is remarkable that the kidneys of SHR remained comparatively small and free of pathologic change with normal BUN levels. Despite myocardial fibrosis and hepatic lipidosis, the circulating enzymes, e.g., CPK, SGOT, SGPT and LDH, were essentially normal in treated and untreated animals. SHR develop progressively worsening hyperlipidemia and hyperglycemia in parallel with their rising blood pressure (7,8,16-18). Propranolol will cause hyperlipidemia, i.e., mobilization of free fatty acids, and hypoglycemia (19,20) through it's inhibition of catecholamine secretion, but no such effect was observed in these SHR. Particularly significant is the reduction of circulating aldosterone and corticosterone levels in SHR in which rising blood pressure was inhibited. Propranolol is effective in blocking juxtaglomerular release of renin and reducing angiotensin-induced stimulation of aldosterone (21). The reduction in circulating corticosterone levels by propranolol is of interest but we are not convinced that corticosterone plays a key role in the pathogenesis of hypertension in SHR. Increased secretion of aldosterone appears to be more directly related to the high blood pressure of SHR° The most salient feature of this investigation was the total absence of histopathology in these severely hypertensive animals until the sudden appearance of severe pathologic changes at 8 months of age in male SHR exclusively and that despite the retardation of rising blood pressure by propranolol, the presence of equally severe histopathology in propranolol-treated male SHR indicates that once the lesions associated with high blood pressure in SHR are established, they do not regress. Acknowledgements The authors wish to commend the technical expertise of A. Wayne, P. Levitas, D. Burton, R. Brand, G. Williamson, E. Domingo and D. Conatser. The research was supported in part by grants from the National Institute on Aging (AG-585) and the National Heart, Lung and Blood Diseases Institute (HL-21418). References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14.

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