Mineral balance and bone histology in spontaneously hypertensive rats

Physiology&Behavior,Vol. 52, pp. 731-736, 1992

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Mineral Balance and Bone Histology in Spontaneously Hypertensive Rats M I T S U H I R O F U R U S E , .1 M I S A O O K U M U R A , i " T O H R U WATANABE:~ A N D J U N - I C H I O K U M U R A *

*Laboratory of Animal Nutrition, School of Agriculture, Nagoya University, Nagoya 464-01, Japan, ? Tokai Gakuen Women's College, Nagoya 468, Japan, and 7tLaboratory of Functional Anatomy, School of Agriculture, Nagoya University, Nagoya 464-01, Japan Received 17 J u n e 1991 FURUSE, M., M. OKUMURA, T. WATANABEAND J.-I. OKUMURA. Mineralbalanceand bonehistologyin spontaneously hypertensiverats. PHYSIOL BEHAV 52(4) 731-736, 1992.--SHRs (9 weeks old) were fed diets with or without extra NaCI (2%) and were given water or saline (1%) for 7 weeks. Food and liquid intakes were measured weekly. Blood pressure was determined at 9, 11, 13, 15, and 16 weeks of age. The values for body weight gain and food intake were not influenced by any treatment. Liquid intake greatly increased with the surplus of dietary NaCI. The blood pressure increased with age in all treatments. The increase in blood pressure was enhanced by the addition of NaC1 to diet and water. Urinary potassium and calcium excretions were enhanced by added dietary NaCI and saline. Calcium content in the femoral bone was not changed by any treatment, although the number of osteoclast and the area of bone marrow were increased by saline supplementation. Sodium chloride

SHR

Food intake

Liquid intake

Bloodpressure

Osteoclast

in the drinking water, although it was not related to decreased food (i.e., Ca) intake or increased egg weight or production. The purpose of the current study was, therefore, to determine whether long-term NaCI load in food or water changes the mineral balances of SHR. Bone histology was also investigated, because a fracture of a bone was observed in the course of the experiment.

THE spontaneously hypertensive rat (SHR) has a stronger preference for NaCl solution compared with the Wistar-Kyoto (WKY) rat used as a normotensive control in 24-h test (6,8,15). Differences between SHR and WKY were also found for responses to salted foods. The SHR showed higher NaCl food preference in liquid milk products and consequently consumed more NaCI compared with WKY rats (5). Not only a preference for NaCl but the response of blood pressure by NaC1 load was different between SHR and WKY. The mean arterial pressure of SHR, but not WKY rats, was further enhanced by NaCl loading (21). According to Dahl (7), NaCl intake of Japanese people is very high when compared with Western people. In Japan, furthermore, most people consume large amounts of NaC! from liquid, such as soup everyday. To the authors' knowledge, however, no one has directly compared the effects on the blood pressure of NaCl supplementation of food and water. It is well known that increased intake of NaC1 consistently elevates blood pressure of laboratory animals and humans. Recently, it has been recognized that other minerals, such as calcium, potassium, and magnesium, are also related to the hypertension. Dietary potassium and calcium were found to be protective against hypertension (4). Dietary magnesium intake may be an important factor in the pathogenesis of hypertension, because blood pressure was lowered by dietary supplementation of magnesium (9). In contrast, Zemel et al. (23) reported magnesium supplementation had no effects on blood pressure. Balnave and Yoselewitz (3) reported that the incidence of damaged egg shell increased linearly with the increase of sodium

METHOD

Subjects and Housing A total of 20 male SHR (8 weeks of age) was purchased from Hoshino Laboratory Animals, Yashio, Saitama, Japan. The animals were housed in individual wire-bottomed cages (21.5 X 21.5 × 23.5 cm) in a constant temperature room (22°C) with a light-dark cycle of 0600 h on, 2200 h off. All rats were initially fed a control diet ad lib with water. After 7 days (9 weeks of age), they were ranked by weight and divided into four experimental groups in order to distribute initial body weight as uniformly as possible. The experimental groups were: 1. 2. 3. 4.

control diet with water (CW); control diet with saline (CS); salted diet with water (SW); salted diet with saline (SS).

The numbers of replicates were five per treatment.

J Requests for reprints should be addressed to Dr. M. Furuse.

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FURUSE 1!I A1. TABLE 1

Diet and Liquid Preparation The compositions of experimental diets are shown in Table 1. The salted diet was made including 2% NaC1 at the expense of corn starch in the control diet. The saline was made by the addition of NaCI at a level of 1%. The diets and liquid were given ad lib for 7 weeks. The NaCI used in diets and liquid was purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. The contents (g/kg diet) of sodium, calcium, potassium, and magnesium in the control diet were 1.02, 5.20, 3.60, and 0.50, respectively.

Measurement o['Food and Liquid Intakes and Blood Pressure Food and liquid intakes were measured weekly. Systolic blood pressure in conscious, prewarmed, restrained rats was measured biweekly and at the last day of the experiment by the tail-cuff method using an electrosphygmomanometer and physiological recorder (KN-208, Natsume Seisakusyo, Tokyo, Japan).

Determination o#Mineral Content in Feces, Urine, and Bone The feces and urine were collected separately during the last 6 days of the experimental period. Feces were ashed with an electric muffle at 580°C for 3 h after overnight drying at 98°C. At the final day of the experiment, rats were sacrificed by bleeding under ether anesthetization. After killing the animals, the right femoral bone was removed. Bones were thoroughly cleaned of soft tissues and weighed. The bone samples were ashed with the electric muffle at 580°C for 12 h, after overnight drying at 98°C. Analyses of urine (calcium, magnesium, sodium, and potassium), feces (magnesium, sodium, and potassium), and bone (magnesium) were made by flame atomic absorption spectrophotometry. Bone and feces were diluted with 1% HCI and urine was diluted with distilled water, and aspirated directly. Calcium content of bone and feces was determined by the method of Gitelman (12) using a kit (Calcium-C-test Wako, Wako Co. Ltd., Osaka, Japan). Phosphorus in the bone was determined by the Fiske-Subbarow method (10),

Bone tfistomorphometo' Left femoral bones, cleaned of soft tissues and fractured with a bone cutter, were fixed in Bouin's fluid for about 24 h at 4°C. They were decalcified in Plank-Rychlo's fluid for 10 h and then processed routinely for embedding in paraffin and cross-section serially at 7 um in thickness. After deparaffinization and rehydration, the sections were stained with Mayer's hematoxylineosin, dehydrated in graded ethanols, cleared in xylene, and mounted. Three sections of every five sections from each group were selected; cross-sectional areas of the epiphysis of femur and its marrow cavity were measured with a Zeiss IBAS image analysis system. The number of osteoclasts in the marrow cavity were counted in the same sections under a light microscope.

Statistical Procedure Data for body weight, food intake, liquid intake, Na intake, and blood pressure were subjected to three-way (food, liquid, and age) analysis of variance (ANOVA), by taking the rats as main-plot and age as sub-plot. Two-way ANOVA was applied for the other data. The data were considered significant when p < 0.05. All statistical procedures were done using a commercially available statistical package (18). RESU LTS

A fracture of a femur was observed in two rats of the CS group during week 4 of the experiment and in a rat in the SS

COMPOSITION OF EXPERIMENTAl, DIEIS

Ingredient

Sodium chloride Corn starch Sucrose Casein DL-Methionine Cellulose* Corn oil AIN76 Mineral mixt AIN76A Vitamin mix:~ Choline bitartrate

Control (g/kg)

NaCI Excess (g/kg)

0 150 500

i 30 5()~

200 3 50 50 35 10 2

* Toyo Roshi Co. Ltd., Tokyo, Japan. t Report of the American Institute of Nutrition ad hoc committeeon standardsfor nutritional studies(17). ~tSecond report of the ad hoc committee on standards for nutritional studies (19).

group during week 7. One rat of the SS group was dead during week 6 of the experiment. The values for these four rats were omitted from the data for the calculation. Figure I gives changes in body weight and intakes of food and liquid of SHR given diets and water with or without extra NaCI. The growth over the experimental period in all groups appeared similar and no significant difference was detected among treatments. Over the experimental period, food intake was significantly increased by saline, F( 1, 91) = 8.61, p = 0.0042, but no significant effect was detected by added dietary NaCI. Liquid intake was significantly increased by added dietary NaCI, F(I, 91) = 207.82, p < 0.0001, but not by the addition of 1% NaCI in water, F(I, 91) = 0.10. A significant interaction between added dietary NaC1 and added NaCI in water was detected, F( 1, 91) = 17.63, p < 0.0001. This implied that on the control diet liquid intake was enhanced by additional NaCI in water, although the reverse was true for the salted diet. Figure 2 shows changes in sodium intake and blood pressure of SH R given diets and water with or without extra NaCI. Daily sodium intake in each treatment was almost constant during the experiment. The effects of diet, F(I, 91) = 1097.35, p < 0.0001, liquid, F(1, 91) = 940.01, p < 0.0001, and diet X liquid interaction, F(I, 91) = 61.85, p < 0.0001, were significant. The sodium intake in the CW group showed the lowest and that in the SS the highest. The values for the CS and SW were comparable and remained between the CW and SS group. The blood pressure in all treatments increased with age, F(4, 51) = 112.20, p < 0.0001. The CW group gave the lowest value and the SS group the highest. The effects of diet, F(I, 64) = 28.50, p < 0.0001, liquid, F(1, 64) = 20.56, p < 0.0001, and diet X liquid interaction, F(I, 64) = 5.45, p = 0.0227, were significant. Sodium, potassium, magnesium, and calcium balances of SHR given diets and water with or without extra NaCi during the last 6 days of the experiment are shown in Tables 2, 3, 4, and 5, respectively. Sodium intake was enhanced by added dietary NaCI, F(1, 12) = 195.60, p < 0.0001, or saline, F(1, 12) = 192.02, p < 0.0001, and there was a synergistic increase in the SS group. Fecal sodium excretion was significantly increased by saline, F(I, 12) = 6.88, p = 0.0223, but it was not changed by added dietary NaC1. Urinary sodium excretion was enhanced by added dietary NaCI,/7(1, 12) = 79.97, p < 0.0001, or saline,

M I N E R A L B A L A N C E A N D B O N E H I S T O L O G Y IN S H R

733

50

300

Wks

Wks

Wks

FIG. I. Changes in body weight (left) and intakes of food (center) and liquid (right) in SHR given diets and water with or without extra NaCI. Horizontal axes indicate the week after the beginning of the experiment. Treatments: ©, control diet with water; t , control diet with saline; A, salted diet with water; &, salted diet with saline. Salted diet and water were made by the addition of sodium chloride at concentrations of 2% and 1%, respectively. Vertical bars represent standard errors of means.

F(1, 12) = 107.85, p < 0.0001, a n d an additive effect was observed in the SS treatment. U r i n a r y potassium excretion was increased by added NaC1 in the diet, F(I, 12) = 30.05, p < 0.0001, a n d liquid, F(1, 12) = 55.86, p < 0.0001. In all parameters o f m a g n e s i u m balance, no significant changes were produced by any treatments. There were n o significant differences

--/

Na intake (mg/d)

50(3

in parameters o f calcium balance except for urinary excretion. U r i n a r y calcium excretion was significantly e n h a n c e d by added dietary NaCl, F ( l , 12) = 10.81, p = 0.0065, a n d saline, F(1, 12) = 9.72, p = 0.0089, t h o u g h the differences were very small. Table 6 gives weights a n d mineral contents o f femoral b o n e of S H R given diets a n d water with or without extra NaC1. N o significant differences were observed in wet a n d dry weight a n d

TABLE 2 SODIUM BALANCE OF SHR GIVEN DIETS AND WATER WITH OR WITHOUT EXTRA NaCI Liquid Water Intake (mg/day) Control Salted Fecal excretion (rag/day) Control Salted Urinary excretion (mg/day) Control Salted

20C

100 t

2O 10

Saline

15 + 0.3 310 + 9

306 + 46 809 + 61

2.43 _ 0.21 2.96 _ 0.21

3.33 + 0.30 3.29 _+ 0.17

15 _+ 0.7 122 _+ 3

146 + 28 297 + 29

Effect of ANOVA i

l

i

l

i

2

3

4

5

l

t

6 7 Wks

160

t(~ 0

i 2

i 4

I I 6 7 Wks

FIG. 2. Changes in sodium intake (left) and blood pressure (right) in SHR given diets and water with or without extra NaCl. Horizontal axes indicate the week after the beginning of the experiment. Treatments: ©, control diet with water; I , control diet with saline; A, salted diet with water; A, salted diet with saline. Salted diet and water were made by the addition of sodium chloride at concentrations of 2% and 1%, respectively. Vertical bars represent standard errors of means.

Intake Fecal excretion Urinary excretion

Diet

Liquid

Diet × Liquid

* NS *

* $ *

t NS NS

Values are mean _ SEM. *t$ Significant levels: *p < 0.001, tP < 0.01, ~tp < 0.05; NS, not significant.

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t.[',RI St. f I \ i

FABLE 3 POTASSIUM BALANCE OF SHR GIVFN [)lETS AN[) WATER WITH OR WITHOUT EXTRA NaC1

[AB[.I-. 4

MAGNESIUM BALANCE OF SHR (ilVI!N DIEIS AND V~A[f!R WITH OR WITHOUT EXTRA Na('l

l_.iquid Water Intake (rag/day) Control Salted Fecal excretion (rag/day) Control Salted Urinary excretion (rag/day) Control Salted

Liquid Saline

54.3 ± 1.1 53.0 ± 1.5

56.3 + 2.7 59.9 + 4.9

4.06 _+ 0.24 4.66 +_ 0.29

4.09 ± 0.86 4.96 ± 0.42

9.2 + 1.3 25.1 _+ 0.9

30.4 _+ 5.6 43.8 ± 3.7

Intake (rag/day) Control Salted Fecal excretion (rag/day) Control Salted Urinary excretion (rag/day) Control Salted

Water

Saline

7.55 + 0.16 7.37 + 0.20

7.83 + 11.37 ~;.32 + 0.68

3.57 + 0.10 3.33 + 0.11

3.66 + 0.09 3.55 _+_0.28

0.057 +_ 0.025 0.088 ± 0.057

0.116 ± 0.059 0.113 + (t.(140

E~ctofANOVA

Effect of ANOVA

Intake Fecal excretion Urinary excretion

Diet

Liquid

Diet × Liquid

NS NS *

NS NS *

NS NS NS

Values are mean _+ SEM. Significant levels: *p < 0.001: NS, not significant.

calcium content. A significant interaction, F( 1, 12) = 6.68, p = 0.0239, was observed in phosphorus content, indicating that with the control diet phosphorus c o n t e n t decreased by saline, but with the N a C M o a d e d diet it increased. M a g n e s i u m c o n t e n t was lowered by added dietary NaC1, F ( I , 12) = 7.67, p = 0.0170, a n d saline, F(1, 12) = 7.05, p = 0.0210. Figure 3 shows cross sections of the epiphysis of femoral bone in S H R given diets a n d water with or without extra NaCI. There were no significant differences between the experimental and control in cross-sectional areas of b o n e a n d m a r r o w cavity, though a difference was observed in the developmental degree of e n d o c h o n d r a l b o n e in the marrow cavity (Fig. 3a-d). The area of m a r r o w cavity per cross-sectional area of the f e m u r increased by saline (Fig. 3b,d), but not by the added dietary NaC1 alone (Fig. 3c). There was a significant m a r k e d increase in the n u m b e r of osteoclasts in the CS group ( m e a n + SE, 291 +_ 6) a n d its n u m b e r was significantly lowered in the SS group (213 + 12). No significant changes were observed in the SW group ( 109 +_ 2) compared with the C W group (110 _+ 3). The osteoclast n u m b e r per unit area of m a r r o w cavity was significantly enhanced by water with extra NaC1, i.e., it was highest in the CS group (38 + 5) followed by the SS group (23 _+ 2); those of the C W (12 _+ 2) a n d SW (14 +_ 3) were comparable a n d low. A significant difference was observed between two divisions. Osteoclasts showing the pyknosis in the SS group only increased in n u m b e r per unit area of m a r r o w cavity, but the r e m a i n d e r were comparable to each other.

Intake Fecal excretion Urinary excretion

Diet

Liquid

Diet × Liquid

NS NS NS

NS NS NS

NS NS NS

Values are mean ± SEM. Significant levels: NS, not significant.

not affected by the level of sodium in the diet. With the salted diet in the present study, however, a m o u n t of saline c o n s u m e d was less t h a n that of water with the control diet. This result indicated that the S H R adjusted sodium intake in water when a large a m o u n t of sodium was offered in a diet even if they prefer saline. However, a two-bottle choice t e c h n i q u e was not used in the present study: this should be examined. Bertino a n d Beauc h a m p (5) also reported that S H R preferred salted food, whereas

TABLE 5 CALCIUM BALANCE OF SHR GIVEN DIETS AND WATER WITH OR WITHOUT EXTRA NaCI Liquid

Intake (rag/day) Control Salted Fecal excretion (mg/day) Control Salted Urinary' excretion (mg/day) Control Salted

Water

Saline

78.5 ± 1.6 76.6 ± 2. l

81.4 ± 3.9 86.6 ± 7. l

43.0 ~_ 2.0 43.8 _+ 1.1

46.1 :+_ 1.5 46.9 ± 4.8

I).091 _+ 0.029 0.440 ± 0.129

0.417 ± 0.225 (I.980 ± 0.187

E~ctofANOVA DISCUSSION It has previously been recognized that in short-term experiments (24 h) the S H R prefers salted water (6,8,15). In the present study, the a m o u n t of saline c o n s u m e d t e n d e d to be higher t h a n that of water c o n s u m e d by rats on the n o r m a l control diet. It was suggested that not only preference but liquid intake was increased by the supply of NaC1 in drinking water for SHR. Fregly et al. ( 11 ) f o u n d that preference for the saline in rats was

Intake Fecal excretion Urinary excretion

Diet

Liquid

Diet X Liquid

NS NS *

NS NS *

NS NS NS

Values are mean _+ SEM. Significant levels: *p < 0.01, NS, not significant.

M I N E R A L BALANCE A N D BONE H I S T O L O G Y IN SHR

TABLE 6 EFFECT OF INCLUDING ADDED NaCI IN DIET AND WATER ON MINERAL CONTENT OF FEMORAL BONE IN SHR

735

3a

CW

Liquid

Wet weight (g) Control Salted Dry weight (g) Control Salted Calcium content (mg/bone) Control Salted Phosphorus content (mg/bone) Control Salted Magnesium content (mg/bone) Control Salted

Water

Saline

0.925 _+ 0.013 0.951 _+ 0.026

0.926 + 0.021 0.939 + 0.027

0.585 + 0.015 0.596 + 0.018

0.578 _+ 0.002 0.603 + 0.016

21.3 _+ 0.6 21.5 + 0.2

20.7 + 1.2 20.3 + 0.3

9.79 _+ 0.04 9.73 _+0.06

9.59 _+ 0.05 9.89 + 0.13

0.465 + 0.004 0.449 _+ 0.006

0.450 _+ 0.005 0.437 _+ 0.004

Effect of ANOVA

Wet weight Dry weight Calcium content Phosphorus content Magnesium content

Diet

Liquid

Diet × Liquid

NS NS NS NS *

NS NS NS NS *

NS NS NS * NS

Values are mean _+ SEM. Significant levels: *p < 0.05, NS, not significant.

the present study indicated that food intake of the salted diet was not always increased compared with the control diet. The physical state, i.e., powder, of the diet used in the present study might have led to the fall in the NaC1 preference of SHR, because Bertino and Beauchamp (5) reported that the NaC1 preference of the SHR was changed according to the texture of diets. Thornton et al. (21) reported blood pressure of SHR was further enhanced by NaCI loading. An effect of added dietary NaC1 and saline on the blood pressure was also found in the present study. A similar increase in the blood pressure was observed in the CS and SW groups. Because sodium intake in both groups was identical, it can be assumed that the source of sodium from the diet or liquid was not an important factor for enhancing blood pressure of SHR. Amounts of intake or retention of sodium would be more important factors, since the highest values in sodium intake and blood pressure were observed in S H R loaded by added dietary NaCI and saline. A number of dietary studies have been conducted on the effects on blood pressure of addition and deletion of nutrients other than sodium. The role of minerals, such as potassium, calcium, and magnesium, in the diet as agents is what commands attention in the field of nutrition and hypertension. Potassium is the abundant cation in the body, and it has been recognized that the correlation of potassium excretion with systolic blood pressure was significant (14). This p h e n o m e n o n was confirmed in the present study, since the higher the urinary potassium excretion the higher the blood pressure (Table 3). Calcium is an

FIG. 3. Photomicrographs of femoral bones. The grayish spicules represent endochondral bones including calcified cartilage matrix, and the dark parts are bone marrow containing developing blood cells, connective tissue elements, and osteoclasts. (a) Control diet with plain water (CW), trabeculae develop very well; (b) salted diet with saline (SS), trabeculae are under weak development compared with CW; (c) salted diet with plain water (SW); (d) control diet with saline (CS). (c) and (d) show the low development of trabeculae. Bar: 1 mm.

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FURI*SI-l!l

essential component in the normal function of vascular smooth muscle cells (13) and contributes to blood pressure regulation at several extravascular sites (16). When SHR was compared with its normotensive control W K Y rat, there was no consistent effect on calcium excretion and absorption (22). In the present study, SHR loaded by added dietary, NaC1 and saline slightly enhanced calcium excretion. This would be due to the higher sodium excretion in the NaCMoaded SHR, because increasing sodium excretion by the kidney causes a rise in urinary calcium excretion (20). Magnesium is vital to many vascular-tissue-related processes that are functionally important for normal blood pressure regulation (1). We expected that magnesium balance in SHR would also be influenced by the NaC1 load, but it was not altered in all treatments. Because a fracture of femoral bone was observed in the CS and SS groups, mineral composition and morphological changes in the femoral bone were investigated. Previously, it had been reported that bone calcium content in SHR was not different from WKY rats when young, but significantly decreased with age (22). In the present study, the weight and calcium content in the SHR were not changed by any of the treatments, However, the number of osteoclast was increased by the extra NaCI in water, implying that a large amount of calcium might be present in the marrow cavity of the femoral bone of SHR loaded by saline: that of the other group was retained in the bone. The

41

osteoclast number was more greatly enhanced m the CS group than in tile SS group, though NaCI intakes were greater in the SS group than in the CS group, it was also lbund that the SW group fell considerably below the CS group in terms of osteoclast number irrespective of equal NaCI intakes. The authors could not determine the reasons for these results, though not only total NaC1 intake but also NaC1 sources from diets or liquid might be an important factor lbr osteoclast numbers. Further experiment remains to be investigated. In any event, these results suggested that the strength of bone was not simply correlated v~ith calcium content, but with the developmental degree of endochondral bone, trabeculae in the marrow cavity. The reason for this phenomenon was unclear, but in the laying hen a similar observation was reported in relation to higher sodium content in drinking water. The incidence of damaged egg shell increased linearly with the increase of sodium in the drinking water (3 L Although there is the difference between calcium carbonate for the egg shell and calcium phosphate for the bone, it is an interesting thct. Thornton et al. (211 and the present study demonstrated that SHR on a high-NaCl diet have an enhanced pressure response. On the other hand, it was reported thal increased dietary calcium lowers blood pressure in the SHR (2). According to these findings it seems to be important to study the possibility that the water excess in NaCl may stimulate the calcium reabsorption from the bone by osteoclasts.

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13. Kuriyama, H.; Yushi, I.: Suzuki, H.: Kitamura, K.; ltoh, T. Factors modifying contraction-relaxation cycle in vascular smooth muscles. Am, J. Physiol. 243:H641-H662: 1982. 14. Langlbrd, H. G. Dietary potassium and hypertension: Epidemiologic data. Ann. Intern. Med. 98:770-772: 1983. 15. McConnell, S, D.; Henkin. R. 1. Increased preference for Na + and K ~ salts in spontaneously hypertensive (SH) rats. Proc. Soc. Exp. Biol. Med. 143:185-188: 1973. 6. Rasmussen, H. Calcium and cAMP in stimulus-response coupling. Ann. NY Acad. Sci. 356:346-353; 1980. 7. Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1(17:1340-1348: 1977. 8. SAS Institute Inc. SAS user's guide: Statistics. Ca~, NC: SAS Institute Inc.: 1985. 19. Second report of the ad hoc committee on standards for nutritional studies. J. Nutr. 110:1726: 1980. 20. Suki, W. N. Calcium transport in the nephron. Am. J. Physiol, 237: FI-F6: 1979. 21. Thornton, R. M.: Davidson, J. M.; Oparil, S. Enhanced cold pressor response in spontaneously hypertensive rats on high-NaCl diet. Am. J. Physiol. 255:HI018-HI023: 1988. 22. Young, E. C.; Bukoski, R. D.; McCarron, D. A. Calcium metabolism in experimental hypertension. Proc. Soc. Exp. Biol. Med. 187:123141: 1988. 23. Zemel, P. C,; Zemel, M. B.: Urberg, M.; Douglas, F. L.; Geiser, R.; Sowers, J. R. Metabolic and hemodynamic effects of magnesium supplementation in patients with essential hypertension. Am. J, Clin. Nutr. 51:665-669: 1990.