Effect of high calcium diet on the developmenvt of high blood pressure in intact spontaneously hypertensive rats and in adrenalectomized spontaneously hypertensive rats treated with aldosterone W~sswa E. B. Semafuko and David J. Morris Department of Pathology and Laboratory Biology and Medicine, Brown University,
Medicine, Providence,
The Miriam Hospital, Rhode Island, USA
Division
of
T’he current investigation was designed to study the effect(s) of high calcium diet on the development of high blood pressure (BP) in both young intact spontaneously hypertensive ruts (SHRs) and in young adrenalectomized (ADX) male SHRs treated with aldosterone (ALDO). Weaned SHRs were fed either a control calcium diet (0.5% Ca as P04) (CCaDiet), a high calcium diet (2.5% Ca, 0.5% as PO4 and 2% as CO,) (HCaDiet), or Agway ProLab rat food containing 2.5% Ca (HCaPLDiet). The HCaDiet signl~cantly biunted the development of high BP in young intact SHRs fP < 0.001; n = 8 to IO). At 6 weeks of age, BP was Ii7 2 2 mm Hg (HCaDiet) compared with 135 2 3 mm Hg (CCaDiet); by 12.7 weeks of age, BP was 192 + 4 mm Hg (HCaD~et) compared with 233 i 3 mm Hg (CCaDiet). Similar results were observed in age-matched SHRs fed the HCaPLDiet. The results show that subcutaneous infusion of ALDO (1 .O ,ugId, osmotic pumps) for 2 weeks to young ADX male SHRs raised on the CCaDiet caused a significant increase in systolic BP when compared with SHRs implanted with Sham pumps (P < 0.001). High BP associated with ALDO infusion was attenuated by the HCaDiet (BP after 2 weeks was 138 +- 8 mm Hg for the HCaDiet group compared with 200 +- 5 mm Hg for the CCaDiet group, P < 0.001; n = 4 to 6). The results show that the HCaDiet blunts the development of high BP in intact SHRs and may protect against the development of ALDO hypertension in ADX young SHRs. (Steroids 56:131-135, 1991)
Keywords:steroids; high calcium diet; adreaalectomy; aldosterone; hypertension: spont~eo~sly
Introduction The spontaneous hypertensive rat (SHR)’ has proven to be a useful model for the study of human essential hypertension. Despite intensive study, the etiology of the disease remains obscure in both species. The early studies by Aoki’s* showed that the thyroid and adrenal glands were necessary for the development and maintenance of high blood pressure (BP) in SHRs. The role
Address reprint requests to Wasswa E. B. Semafuko, Ph.D., The Miriam Hospital, Department of Pathology and Laboratory Medicine, 164 Summit Avenue, Providence, RI 02906, USA. Received June 28, 1990; accepted September 21, 1990.
0 1991 Butterworth-Heinemann
hypertensive
rats
of the adrenal gland in the initiation of hypertension in young SHRs, however, is far from clear and has remained controversial. ‘-’ More recently, we reported that the inhibition of the early rise in BP in young male SHRs caused by adrenalectomy (ADX) can be reversed by infusions of low dosages of aldosterone (ALDO) and other mineraiocorticoids.“‘O It is known that ingestion of high calcium diets results in a decrease in BP in humans with essential hypertension’1-14 and in experimentai animals with high BP.“-” However, the mechanism(s) by which high dietary calcium lowers high BP is not known. It appears that high BP observed in SHRs or in humans with essential hypertension may be associated with changes
Steroids,
1991, vol. 56, March
131
Papers in calcium homeostasis. “.“‘The role of a factor(s) from another group of cells within the parathyroid glands of SHRs has also been recently suggested to be important
.zO.z’
.
The present investigation was designed to study the effect of a high calcium diet on the development and maintenance of high BP in intact SHRs, and the development of high BP in young ADX male SHRs following treatment with ALDO.
’ . f
4
Male SHRs were purchased from Charles River Breeding Laboratory (Wilmington, MA, USA) at 18 to 19 days of age. After weaning (21 days of age). rats were fed either a control calcium diet (AIN-76A Semipurified diet, ICN Biochemical Industries, Cleveland, OH, USA) containing 0.5% calcium as PO, (CCaDiet), a high calcium diet (control diet supplemented to contain 2.5% calcium, 2% as CO, and 0.5% as PO,) (HCaDiet), or Agway ProLab rat food (Agway Inc.. Syracuse, NY y USA) containing 2.5% calcium (enriched in Ca by adding CaCO,) (HCaPLDiet). All rats were housed in an animal facility with a 12-hour light-dark cycle control system and maintained at 22 C. The rats were handled daily to acclimatize them to the experimental conditions and given water to drink ad libitum.
qf’high blood pwssuw
Systolic BP and body weight (BW) were measured twice a week in all groups of intact SHRs (from 4 to 19 weeks of age). Blood pressure was measured between 9:00 AM and 12:OOnoon by the indirect tail-cuff method using a pneumatic pulse transducer (Narco Biosysterns, Houston, TX, USA), and the mean of three determinations was used as previously described.“’
Development of high blood pressure in uldosterone-treated ruts Young male SHRs exhibiting systolic BP of 100 to 110 mm Hg (about 5 to 6 weeks of age) underwent bilateral ADX under ether anesthesia and were immediately imwith miniosmotic pumps planted, subcutaneously, (Model 2002, Alza Corporation, Palo Alto, CA, USA) loaded with ALDO (1 .O pg/d for 2 weeks) dissolved in propylene gl ycol. ‘“Control ADX SHRs were implanted with Sham pumps containing propylene glycol only. Aldosterone was obtained from Andard Mount Chemicals (London, UK). Thereafter, all ADX SHRs were maintained with 0.9% NaCl solution as drinking water ad libitum. Systolic BP measurements were resumed 3 days post-ADX and were performed as described above.
analyis
The results are presented as mean values -+ SEM. The differences between groups were evaluated by analysis Steroids,
’
..
+
I
-*
i *
Animals
132
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.. 7’
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:,
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I, /i*
Development
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1991, vol. 56, March
6
e
10
AGE
‘Z
16
16
*e
20
(weeks)
Figure 1 Effect of HCaDiet on the development of high BP in intact SHRs. Weaned male SHRs were fed either CCaDiet (0.5% Ca as POJ, HCaDiet (control diet supplemented to contain 2.5% Ca, 0.5% as PO, and 2% as COJ, or HCaPLDiet (enriched in Ca by adding CaCO,). Values are expressed as means +- SEM of eight to 10 animals. Values were significantly different compared with CCaDiet: *P < 0.001.
of variance (ANOVA) and Newman-Keuls test. Differences between means were tested by the Student’s t test.
Results of II high cctlcir(m diet on the development of high blood pressure in intut spontaneously hypt>rtensive ruts
E&ct
Systolic BP recorded from SHRs (5 to 19 weeks of age) raised on HCaDiet was significantly lower when compared with BP measured from age-matched SHRs raised on CCaDiet (P < 0.001; n = 8 to 10; Figure I). At 6 weeks of age, systolic BP was 117 2 2 mm Hg for SHRs raised on HCaDiet compared with 135 + 3 mm Hg for SHRs raised on CCaDiet. At 12.7 weeks (89 days) of age, BP levels were 192 I+_4 mm Hg for the HCaDiet group compared with 233 ? 3 mm Hg for the CCaDiet group, and, by 19 weeks of age, BP levels were 2 17 ‘-’ 2 mm Hg for the HCaDiet group compared with 237 t 2 mm Hg for the CCaDiet group. In addition, adult SHRs raised on HCaPLDiet also showed lowered BP comparable to the levels observed in the HCaDiet group; these levels were significantly lower when compared with age-matched SHRs raised on CCaDiet (P < 0.001; n = 10 to II; Figure 1). However, the systolic BP levels of SHRs raised on either HCaPLDiet or CCaDiet were similar until the seventh week of age.
Efiect oj’a high cnlcium diet on body weight in intact spontaneously hypertensive ruts The HCaDiet decreased BW gain significantly in young and adult intact SHRs when compared with agematched SHRs raised on CCaDiet (P < 0.001; n = 8 to 10; Figure 2). Young SHRs (4 to 6 weeks of age) fed HCaPLDiet exhibited BWs similar to those observed in age-matched SHRs raised on CCaDiet. However,
Dietary calcium and aldosterone
hypertension:
Semafuko
and Morris
o CCoDiet HCaDiet
370
l
‘;;; 320
6 HCaPLDiet
y 270 _c .-o, 220 a, s 170 G. 0 120 m
z
70
:
20 2
4
6
8
10
12
14
16
18
20
Age (weeks) Figure 2 Effect of HCaDiet on BW gain in intact SHRs. Weaned male SHRs were fed one of the following: CCaDiet (0.5% Ca as POJ, HCaDiet (control diet supplemented to contain 2.5% Ca, 0.5% Ca as PO1 and 2% as CO&, or HCaPLDiet (enriched in Ca by adding CaCO,). Values are expressed as means * SEM of eight to 10 animals. Values were significantly different compared with CCaDiet: *P < 0.001.
BWs exhibited by adult SHRs (8 to 19 weeks of age) fed HCaPLDiet were similar to those observed in agematched SHRs raised on HCaDiet. Effect
100
3
of a high calcium diet on the
development of high blood pressure in young adrenalectomized spontaneously hypertensive rats Constant infusion of ALDO (I .O pgld subcutaneously) to young ADX male SHRs raised on CCaDiet resulted in the development of high BP (Figure 3A). After 3 days of infusion, systolic BP was 142 + 5 mm Hg for ALDO compared with I17 2 2 mm Hg for SHRs implanted with Sham pumps (P < 0.01; n = 4 to 5; Figure 3A). After 2 weeks of ALDO infusion, BP was 200 ? 5 mm Hg for ALDO compared with I29 + 4 mm Hg for controls (P < 0.001; n = 4 to 5; Figure 3A). Feeding young ADX male SHRs HCaDiet resulted in inhibition of the ALDO-induced development of high BP (Figure 3B). After a l-week infusion of ALDO, the systolic BP for SHRs fed CCaDiet was 167 ? 3 mm Hg compared with 113 + 3 mm Hg for SHRs fed HCaDiet and, by 2 weeks, BP was 200 + 5 mm Hg for the CCaDiet group compared with 138 2 8 mm Hg for the HCaDiet group (P < 0.001; n = 4 to 5; Figure 3B). Infusion of ALDO had no effect in SHRs raised on HCaDiet (BP levels were similar to those observed in SHRs implanted with Sham pumps; Figure 3B). Discussion Several studies have shown that high calcium diets are associated with a decrease of BP in humans with essential hypertension. “-14,22,23Other studies have shown that high dietary calcium also lowers high BP in SHRs ‘5-‘7 deoxycorticosterone-salt-treated rats 24 and Dahl’salt-sensitive rats.25 Furthermore. it is kno;n
t
ADX + pumps
/I
70
al m
Y 1
0
-4
I 4
8
12
16
220
o-o o-0
m 1go _O-O :
CCaDiet (ALDO, 1 yg/d) HCoDiet (ALOO, 1 @g/d) HCoDiet
TB
6/?
(Control)
/’
I 70 -4
0
TIME (days
4
after
8
12
16
adrenalectomy)
Figure 3 Effect of HCaDiet on ALDO-induced development of hypertension in young ADX male SHRs. Young male ADX SHRs (raised on either CCaDiet or HCaDiet) were infused (minipumps) with either ALDO (ALDO pumps, 1.0 pgid subcutaneously for 2 weeks) or vehicle (Sham pumps, controls). Values are expressed as means * SEM of four to six animals. (A) Aldosteroneinduced development of high BP. Values were significantly different compared with controls: *P < 0.01, l*P < 0.001. (B) The HCaDiet protects against ALDO hypertension. Values were significantly different compared with CCaDiet: *P < 0.01, l*P < 0.001.
that the adrenal gland is essential for the development of high BP in young SHRs, but its role in the maintenance of high BP in adult SHRs is less clear. Earlier studies in our laboratory confirmed the findings of Aoki’ that adrenalectomy of young SHRs (5 to 6 weeks of age) inhibits and that infusions of very low dosages of ALDO (as little as 0.5 and 1.O pg/d) permits the full restoration of the development of high BP to levels usually attained in intact SHRs of the same age.” Young ADX male SHRs are extremely sensitive to infusions of very small quantities of ALDO administered at rates similar in magnitude to estimated daily secretory output from adrenal glands of both SHRs and normotensive rats.26,27 The present study was designed to assess the effects of a high calcium diet on both the development of high BP in young SHRs and maintenance of high BP in adult male SHRs. In addition, we wished to study the effects of high dietary calcium on the development of high BP following subcutaneous infusion of low levels of ALDO in young ADX male SHRs. The results of this investigation show that feeding young intact SHRs a high calSteroids,
1991, vol. 56, March
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Papers
cium diet results in a decrease in the development ot high BP in young SHRs and lowers the BP level\ usually attained by adult SHRs. These observations are in agreement with those first reported by Ayachi.” which have since been confirmed by several investigators.‘3.‘6.‘7 In addition, we now report for the first time that high dietary calcium markedly blunts the development of high BP usually observed following infusions of ALDO (I .O pug/d) to young ADX male SHRs. These latter data suggest that the hypotensive activity of high dietary calcium may be associated, in part, with altered bioactivity and/or metabolism of adrenal corticosteroids in SHRs. The results also suggest that high dietary calcium may protect against the development of aldosterone hypertension in ADX SHRs. However, care should be taken when interpreting and comparing the results from the experiments reported here. since it is possible that the effects of high calcium diet on the development of high BP in intact SHRs may occur by a different mechanism(s). The results also showed that high dietary calcium was associated with low BW gain, as previously reported by other investigators in SHRs” and in Dahl salt-sensitive rats.‘5 However, analysis of the data suggested that there was no consistent relationship between systolic BP and BW gain that could account for the hypotensive activity of high dietary calcium in either the intact or ADX young male SHRs. These observations are in partial agreement with other investigators who came to similar conclusions in intact SHRS.“.?~ Furthermore, our preliminary observations, like those reported by Stern et al.,” found no differences in daily food consumption among the different dietary groups. The mechanism(s) by which high calcium diets decreases high BP is not known at this time. Numerous investigations concerning the role played by parathyroid hormone, calcitonin, I ,25-dihydroxyvitamin D,, calcium, and magnesium homeostasis are presently being undertaken.” Additionally, the effect of high calcium diets on the secretion of a recently discovered factor, parathyroid hypertensive factor,‘“.” elaborated in the parathyroid glands of SHRs may also play a major role. From the present studies, the actions of high dietary calcium on both development of high BP in young intact SHRs and in young ADX SHRs treated with ALDO are very interesting and raise new questions. Further studies are required to determine whether the alterations in serum and tissue electrolyte composition associated with high dietary calcium may affect the biologic functions of adrenal mineralocorticoids and/or possibly glucocorticoids, and the hypothalamic-pituitary-adrenal axis: ‘u’ in their control of BP and the cardiovascular system. Additional experiments are required to determine the effect of such changes caused
by high dietary calcium on the pharmacodynamics and pharmacokinetics of ALDO and/or other adrenal corticosteroids to help better understand their physiologic role in the development and maintenance of hypertension. 134
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1991, vol. 56, March
Acknowledgments This project ~a\ and administered Dairy Council.
funded by the National in cooperation with
Dairy Bourcl the National
References I.
2.
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Aoki K (1963). Experimental studies on the relationshtp hctween endocrine organs and hypertension in spontaneously hypertensive rats. I. Effects of hypophysectomy. adrenalectomy. thyroidectomy. nephrectomy and sympathectomy on blood pressure. Jl~n Hc,c~rr J 4:443-461. Aoki K (1963). Experimental studies on the relationship between endogenous organ and hypertensive rats. II. Effects of various hormones on blood pressure. JLJ~~Hczclrr .I 4:561569. Sowers J. Tuck M, A5p ND, Sollars E (1981). Plasma aldoster-one response to adrenocorticotropin. angiotensin. potassium and stress in spontaneously hypertensive rats. EndocGto/o~> 108:1216-1’21. lams SC. McMurtry JP. Wexler BC tlY7Yt. Aldosterone. deoxyc”rtico~terone. corticosterone. and prolactin changes during the life span ofchronically and spontaneously hypertensive rats. E&oc~rino/~~~ IOY: 1X411x45. Gorsline J. Morris DJ (1985). The hypertensinogenic acttvity of l9-nor-deoxycorticosterone in the adrenalectomiLed spontaneously hypertensive rat. .I Stctwid Bioc~llc,tn 23:535536. Gorsline J. Harnik M. Tre\co PA. Morris DJ (1986). Hypertensinogenic activities of ring A-reduced metabolites of aldostetone. Hyparrrmiorr 8:1187-I I90 (suppl. I ). McCarron DA. Morris CD. Cole C (1982). Dietary calcium in human hypertension. .ScirnccI 217:267-X9. Resnick LM. Laragh JH. Sealey JE. Alderman MH (1983). Divalent cations in essential hypertension: relations between serum ionized calcium. magnesium and plasma renin activity. N Elr,q/ J Med 309:88X-891. Belilan JM. Villar J. Pineda 0. Gonzalez AL’. Sainze E. Gerrera G. Sibrian R (1983). Reduction of blood pressure with calcium supplementation in young adults. JAMA 249:1161-1165. Zemel MB. Guadoni SM. Walsh M. Komanicky I’. Stanley P. Johnson D. Fitter W. Sowers JR (1986). Effect of sodium and calcium on calcium metabolism and blood pressure regulation in hypertensive black adults. J Hyprrtcns 4:S364-S366 (suppl. 5). Ayachi S (1979). Increased dietary calcium lowers blood pressure in spontaneously hypertensive rats. Mc~/clho/im 28: 1234-1238. McCarron DA, Lucas PA, Shneidman RJ. LaCour B, Drueke T (1985). Blood pressure development of the spontaneously hypertensive rat after concurrent manipulations of dietary Ca?’ and Na-. J Chin Invest 76:1147-l 1.54. Stern N. Lee DB. Sillis V, Beck FWJ, Deftos L. Manolagds SC. Sowers JR (1984). Effect of high calcium intake on blood
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19. 20. 21.
pressure and calcium metabolism in young SHR. Hypertension 6:639-646. McCarron DA, Pingree PA, Rubin RJ, Gaucher SM, Mohtch M, Krutziks S (1980). Enhanced parathyroid function in essential hypertension: a homeostasis response to a urinary calcium leak. Hypertension 2:162-168. McCarron DA (1982). Low serum concentration of ionized calcium in patients with hypertension. N EngI J Med 307:
23. 24. 25.
and Morris
reduces blood pressure in Dahl salt-sensitive rats by neuronal mechanisms. ~ypertensjon 9~159-165 (suppl. 111). Moll D. Dale SL, Melby JC (1975). Adrenal steroidogenesis in the spontaneously hypertensive rat (SHR). Endocrinology Milne CM, Balment RJ, Henderson IW, Mosley W, Jones IC (1982). Adrenocortical function in the Brattleboro rat. Ann N Y Acad Sci 394~230-240.
28.
Pang PKT, Lewanczuk RZ (1989). Parathyroid origin of a new circulating hypertensive factor in spontaneously hypertensive rats. Am J HvDertens 2~898-902. Lewanczuk RZ, Chen A, Pang PKT (1990). The effect of dietary calcium on blood pressure in spontaneously hypertensive rats may be mediated by parathyroid hypertensive factor.
29.
Muntzel MS, Hatton DC, Metz JA, McCarron DA (1989). Dietary calcium alters blood pressure in neonatal spontaneously hypertensive rats. Am J Hypertens 2:158-162. McCarron DA (1989). Calcium metabolism and hypertension. Kidney Int 35~717-736.
30.
3:349-353.
Grobbee DE, Hofman A (1986). Effect of calcium supplementation on diastolic blood pressure in young people with mild hypertension. Lancer 2~703-707. Saito K, Sano H, Furata Y, Fukuzaki H (1989). Effect of oral calcium on blood pressure in salt-loaded borderline hvperten.. sive patients. Hydertension 13:219-226. Kurtz TW. Morris RC (1986). Attenuation of deoxvcorticosterone-induced hypertension by supplemental dietary calcium. J Hypertens 4:S182-S131 (suppl. 5). Peuler JD, Morgan DA, Mark AL (1987). High calcium diet
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27.
226-228.
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22.
26.
hypertension:
31.
32.
Sowers J, Tuck M, Asp ND, Sollars E (1981). Plasmaaldosterone and corticosterone responses to adrenocorticotropin, angiotensin, potassium and stress in spontaneously hypertensive rats. Endocrinology 1~~1216-1221. McMurtry JP, Wexler LA (1981). Hypersensitivity of spontaneously hypertensive rats (SHR) to heat, ether and immobilization. Endocrinology 108: 1730-1736. Hashimoto K. Makino S, Hirasawa R, Takao T, Sugawara M, Murakami K, Ono K, Ota Z (1989). Abnormalities in the hypothalamo-pituitary-adrenal axis in spontaneously hypertensive rats during development of hypertension. Endocrinology 125:1161-l
167.
Steroids,
1991, vol. 56, March
135
Medicine, Providence,
The Miriam Hospital, Rhode Island, USA
Division
of
T’he current investigation was designed to study the effect(s) of high calcium diet on the development of high blood pressure (BP) in both young intact spontaneously hypertensive ruts (SHRs) and in young adrenalectomized (ADX) male SHRs treated with aldosterone (ALDO). Weaned SHRs were fed either a control calcium diet (0.5% Ca as P04) (CCaDiet), a high calcium diet (2.5% Ca, 0.5% as PO4 and 2% as CO,) (HCaDiet), or Agway ProLab rat food containing 2.5% Ca (HCaPLDiet). The HCaDiet signl~cantly biunted the development of high BP in young intact SHRs fP < 0.001; n = 8 to IO). At 6 weeks of age, BP was Ii7 2 2 mm Hg (HCaDiet) compared with 135 2 3 mm Hg (CCaDiet); by 12.7 weeks of age, BP was 192 + 4 mm Hg (HCaD~et) compared with 233 i 3 mm Hg (CCaDiet). Similar results were observed in age-matched SHRs fed the HCaPLDiet. The results show that subcutaneous infusion of ALDO (1 .O ,ugId, osmotic pumps) for 2 weeks to young ADX male SHRs raised on the CCaDiet caused a significant increase in systolic BP when compared with SHRs implanted with Sham pumps (P < 0.001). High BP associated with ALDO infusion was attenuated by the HCaDiet (BP after 2 weeks was 138 +- 8 mm Hg for the HCaDiet group compared with 200 +- 5 mm Hg for the CCaDiet group, P < 0.001; n = 4 to 6). The results show that the HCaDiet blunts the development of high BP in intact SHRs and may protect against the development of ALDO hypertension in ADX young SHRs. (Steroids 56:131-135, 1991)
Keywords:steroids; high calcium diet; adreaalectomy; aldosterone; hypertension: spont~eo~sly
Introduction The spontaneous hypertensive rat (SHR)’ has proven to be a useful model for the study of human essential hypertension. Despite intensive study, the etiology of the disease remains obscure in both species. The early studies by Aoki’s* showed that the thyroid and adrenal glands were necessary for the development and maintenance of high blood pressure (BP) in SHRs. The role
Address reprint requests to Wasswa E. B. Semafuko, Ph.D., The Miriam Hospital, Department of Pathology and Laboratory Medicine, 164 Summit Avenue, Providence, RI 02906, USA. Received June 28, 1990; accepted September 21, 1990.
0 1991 Butterworth-Heinemann
hypertensive
rats
of the adrenal gland in the initiation of hypertension in young SHRs, however, is far from clear and has remained controversial. ‘-’ More recently, we reported that the inhibition of the early rise in BP in young male SHRs caused by adrenalectomy (ADX) can be reversed by infusions of low dosages of aldosterone (ALDO) and other mineraiocorticoids.“‘O It is known that ingestion of high calcium diets results in a decrease in BP in humans with essential hypertension’1-14 and in experimentai animals with high BP.“-” However, the mechanism(s) by which high dietary calcium lowers high BP is not known. It appears that high BP observed in SHRs or in humans with essential hypertension may be associated with changes
Steroids,
1991, vol. 56, March
131
Papers in calcium homeostasis. “.“‘The role of a factor(s) from another group of cells within the parathyroid glands of SHRs has also been recently suggested to be important
.zO.z’
.
The present investigation was designed to study the effect of a high calcium diet on the development and maintenance of high BP in intact SHRs, and the development of high BP in young ADX male SHRs following treatment with ALDO.
’ . f
4
Male SHRs were purchased from Charles River Breeding Laboratory (Wilmington, MA, USA) at 18 to 19 days of age. After weaning (21 days of age). rats were fed either a control calcium diet (AIN-76A Semipurified diet, ICN Biochemical Industries, Cleveland, OH, USA) containing 0.5% calcium as PO, (CCaDiet), a high calcium diet (control diet supplemented to contain 2.5% calcium, 2% as CO, and 0.5% as PO,) (HCaDiet), or Agway ProLab rat food (Agway Inc.. Syracuse, NY y USA) containing 2.5% calcium (enriched in Ca by adding CaCO,) (HCaPLDiet). All rats were housed in an animal facility with a 12-hour light-dark cycle control system and maintained at 22 C. The rats were handled daily to acclimatize them to the experimental conditions and given water to drink ad libitum.
qf’high blood pwssuw
Systolic BP and body weight (BW) were measured twice a week in all groups of intact SHRs (from 4 to 19 weeks of age). Blood pressure was measured between 9:00 AM and 12:OOnoon by the indirect tail-cuff method using a pneumatic pulse transducer (Narco Biosysterns, Houston, TX, USA), and the mean of three determinations was used as previously described.“’
Development of high blood pressure in uldosterone-treated ruts Young male SHRs exhibiting systolic BP of 100 to 110 mm Hg (about 5 to 6 weeks of age) underwent bilateral ADX under ether anesthesia and were immediately imwith miniosmotic pumps planted, subcutaneously, (Model 2002, Alza Corporation, Palo Alto, CA, USA) loaded with ALDO (1 .O pg/d for 2 weeks) dissolved in propylene gl ycol. ‘“Control ADX SHRs were implanted with Sham pumps containing propylene glycol only. Aldosterone was obtained from Andard Mount Chemicals (London, UK). Thereafter, all ADX SHRs were maintained with 0.9% NaCl solution as drinking water ad libitum. Systolic BP measurements were resumed 3 days post-ADX and were performed as described above.
analyis
The results are presented as mean values -+ SEM. The differences between groups were evaluated by analysis Steroids,
’
..
+
I
-*
i *
Animals
132
*
.. 7’
Experimental
Statistical
:,
.
I, /i*
Development
r *
1991, vol. 56, March
6
e
10
AGE
‘Z
16
16
*e
20
(weeks)
Figure 1 Effect of HCaDiet on the development of high BP in intact SHRs. Weaned male SHRs were fed either CCaDiet (0.5% Ca as POJ, HCaDiet (control diet supplemented to contain 2.5% Ca, 0.5% as PO, and 2% as COJ, or HCaPLDiet (enriched in Ca by adding CaCO,). Values are expressed as means +- SEM of eight to 10 animals. Values were significantly different compared with CCaDiet: *P < 0.001.
of variance (ANOVA) and Newman-Keuls test. Differences between means were tested by the Student’s t test.
Results of II high cctlcir(m diet on the development of high blood pressure in intut spontaneously hypt>rtensive ruts
E&ct
Systolic BP recorded from SHRs (5 to 19 weeks of age) raised on HCaDiet was significantly lower when compared with BP measured from age-matched SHRs raised on CCaDiet (P < 0.001; n = 8 to 10; Figure I). At 6 weeks of age, systolic BP was 117 2 2 mm Hg for SHRs raised on HCaDiet compared with 135 + 3 mm Hg for SHRs raised on CCaDiet. At 12.7 weeks (89 days) of age, BP levels were 192 I+_4 mm Hg for the HCaDiet group compared with 233 ? 3 mm Hg for the CCaDiet group, and, by 19 weeks of age, BP levels were 2 17 ‘-’ 2 mm Hg for the HCaDiet group compared with 237 t 2 mm Hg for the CCaDiet group. In addition, adult SHRs raised on HCaPLDiet also showed lowered BP comparable to the levels observed in the HCaDiet group; these levels were significantly lower when compared with age-matched SHRs raised on CCaDiet (P < 0.001; n = 10 to II; Figure 1). However, the systolic BP levels of SHRs raised on either HCaPLDiet or CCaDiet were similar until the seventh week of age.
Efiect oj’a high cnlcium diet on body weight in intact spontaneously hypertensive ruts The HCaDiet decreased BW gain significantly in young and adult intact SHRs when compared with agematched SHRs raised on CCaDiet (P < 0.001; n = 8 to 10; Figure 2). Young SHRs (4 to 6 weeks of age) fed HCaPLDiet exhibited BWs similar to those observed in age-matched SHRs raised on CCaDiet. However,
Dietary calcium and aldosterone
hypertension:
Semafuko
and Morris
o CCoDiet HCaDiet
370
l
‘;;; 320
6 HCaPLDiet
y 270 _c .-o, 220 a, s 170 G. 0 120 m
z
70
:
20 2
4
6
8
10
12
14
16
18
20
Age (weeks) Figure 2 Effect of HCaDiet on BW gain in intact SHRs. Weaned male SHRs were fed one of the following: CCaDiet (0.5% Ca as POJ, HCaDiet (control diet supplemented to contain 2.5% Ca, 0.5% Ca as PO1 and 2% as CO&, or HCaPLDiet (enriched in Ca by adding CaCO,). Values are expressed as means * SEM of eight to 10 animals. Values were significantly different compared with CCaDiet: *P < 0.001.
BWs exhibited by adult SHRs (8 to 19 weeks of age) fed HCaPLDiet were similar to those observed in agematched SHRs raised on HCaDiet. Effect
100
3
of a high calcium diet on the
development of high blood pressure in young adrenalectomized spontaneously hypertensive rats Constant infusion of ALDO (I .O pgld subcutaneously) to young ADX male SHRs raised on CCaDiet resulted in the development of high BP (Figure 3A). After 3 days of infusion, systolic BP was 142 + 5 mm Hg for ALDO compared with I17 2 2 mm Hg for SHRs implanted with Sham pumps (P < 0.01; n = 4 to 5; Figure 3A). After 2 weeks of ALDO infusion, BP was 200 ? 5 mm Hg for ALDO compared with I29 + 4 mm Hg for controls (P < 0.001; n = 4 to 5; Figure 3A). Feeding young ADX male SHRs HCaDiet resulted in inhibition of the ALDO-induced development of high BP (Figure 3B). After a l-week infusion of ALDO, the systolic BP for SHRs fed CCaDiet was 167 ? 3 mm Hg compared with 113 + 3 mm Hg for SHRs fed HCaDiet and, by 2 weeks, BP was 200 + 5 mm Hg for the CCaDiet group compared with 138 2 8 mm Hg for the HCaDiet group (P < 0.001; n = 4 to 5; Figure 3B). Infusion of ALDO had no effect in SHRs raised on HCaDiet (BP levels were similar to those observed in SHRs implanted with Sham pumps; Figure 3B). Discussion Several studies have shown that high calcium diets are associated with a decrease of BP in humans with essential hypertension. “-14,22,23Other studies have shown that high dietary calcium also lowers high BP in SHRs ‘5-‘7 deoxycorticosterone-salt-treated rats 24 and Dahl’salt-sensitive rats.25 Furthermore. it is kno;n
t
ADX + pumps
/I
70
al m
Y 1
0
-4
I 4
8
12
16
220
o-o o-0
m 1go _O-O :
CCaDiet (ALDO, 1 yg/d) HCoDiet (ALOO, 1 @g/d) HCoDiet
TB
6/?
(Control)
/’
I 70 -4
0
TIME (days
4
after
8
12
16
adrenalectomy)
Figure 3 Effect of HCaDiet on ALDO-induced development of hypertension in young ADX male SHRs. Young male ADX SHRs (raised on either CCaDiet or HCaDiet) were infused (minipumps) with either ALDO (ALDO pumps, 1.0 pgid subcutaneously for 2 weeks) or vehicle (Sham pumps, controls). Values are expressed as means * SEM of four to six animals. (A) Aldosteroneinduced development of high BP. Values were significantly different compared with controls: *P < 0.01, l*P < 0.001. (B) The HCaDiet protects against ALDO hypertension. Values were significantly different compared with CCaDiet: *P < 0.01, l*P < 0.001.
that the adrenal gland is essential for the development of high BP in young SHRs, but its role in the maintenance of high BP in adult SHRs is less clear. Earlier studies in our laboratory confirmed the findings of Aoki’ that adrenalectomy of young SHRs (5 to 6 weeks of age) inhibits and that infusions of very low dosages of ALDO (as little as 0.5 and 1.O pg/d) permits the full restoration of the development of high BP to levels usually attained in intact SHRs of the same age.” Young ADX male SHRs are extremely sensitive to infusions of very small quantities of ALDO administered at rates similar in magnitude to estimated daily secretory output from adrenal glands of both SHRs and normotensive rats.26,27 The present study was designed to assess the effects of a high calcium diet on both the development of high BP in young SHRs and maintenance of high BP in adult male SHRs. In addition, we wished to study the effects of high dietary calcium on the development of high BP following subcutaneous infusion of low levels of ALDO in young ADX male SHRs. The results of this investigation show that feeding young intact SHRs a high calSteroids,
1991, vol. 56, March
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Papers
cium diet results in a decrease in the development ot high BP in young SHRs and lowers the BP level\ usually attained by adult SHRs. These observations are in agreement with those first reported by Ayachi.” which have since been confirmed by several investigators.‘3.‘6.‘7 In addition, we now report for the first time that high dietary calcium markedly blunts the development of high BP usually observed following infusions of ALDO (I .O pug/d) to young ADX male SHRs. These latter data suggest that the hypotensive activity of high dietary calcium may be associated, in part, with altered bioactivity and/or metabolism of adrenal corticosteroids in SHRs. The results also suggest that high dietary calcium may protect against the development of aldosterone hypertension in ADX SHRs. However, care should be taken when interpreting and comparing the results from the experiments reported here. since it is possible that the effects of high calcium diet on the development of high BP in intact SHRs may occur by a different mechanism(s). The results also showed that high dietary calcium was associated with low BW gain, as previously reported by other investigators in SHRs” and in Dahl salt-sensitive rats.‘5 However, analysis of the data suggested that there was no consistent relationship between systolic BP and BW gain that could account for the hypotensive activity of high dietary calcium in either the intact or ADX young male SHRs. These observations are in partial agreement with other investigators who came to similar conclusions in intact SHRS.“.?~ Furthermore, our preliminary observations, like those reported by Stern et al.,” found no differences in daily food consumption among the different dietary groups. The mechanism(s) by which high calcium diets decreases high BP is not known at this time. Numerous investigations concerning the role played by parathyroid hormone, calcitonin, I ,25-dihydroxyvitamin D,, calcium, and magnesium homeostasis are presently being undertaken.” Additionally, the effect of high calcium diets on the secretion of a recently discovered factor, parathyroid hypertensive factor,‘“.” elaborated in the parathyroid glands of SHRs may also play a major role. From the present studies, the actions of high dietary calcium on both development of high BP in young intact SHRs and in young ADX SHRs treated with ALDO are very interesting and raise new questions. Further studies are required to determine whether the alterations in serum and tissue electrolyte composition associated with high dietary calcium may affect the biologic functions of adrenal mineralocorticoids and/or possibly glucocorticoids, and the hypothalamic-pituitary-adrenal axis: ‘u’ in their control of BP and the cardiovascular system. Additional experiments are required to determine the effect of such changes caused
by high dietary calcium on the pharmacodynamics and pharmacokinetics of ALDO and/or other adrenal corticosteroids to help better understand their physiologic role in the development and maintenance of hypertension. 134
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Acknowledgments This project ~a\ and administered Dairy Council.
funded by the National in cooperation with
Dairy Bourcl the National
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