Relaxation responses of aortic rings from salt-loaded high calcium fed rats to potassium chloride, calcium chloride and magnesium sulphate

Pathophysiology 4 (1998) 275 280 Relaxation responses of aortic rings from salt-loaded high calcium fed rats to potassium chloride, calcium chloride and magnesium sulphate B.J. Adegunloye, O.A. Sofola * Department of Physiology, College of Medicine, Uni ersity of Lagos, Lagos, Nigeria Received 26 January 1996; received in revised form 24 July 1997; accepted 5 August 1997 Abstract The relaxation responses of aortic rings of rats fed either normal rat feed, 8% salt diet, 8% salt+2.5% Ca diet or 2.5% Ca diet for 6 8 weeks has been investigated. The mean arterial pressure of salt loaded rats (148.63 8.54 mmhg) was higher (P 0.05) than those of control (108.36 7.81 mmhg), salt-loaded Ca-fed (101.62 7.38) and Ca fed (98.54 8.72) rats. The maximum KCl-induced relaxation of the aortic rings was least (55.5 3.5%, P 0.05) in salt loaded rats when compared to the maximum KCl-induced relaxation of control (70.76 3.65%), salt+ca fed (73.12 3.46%) and Ca fed (79.3 3.68%) rats. Salt loading attenuated the maximum relaxation to CaCl 2 (P 0.05) when compared to the maximum relaxation responses of control rats, salt+ca fed and Ca fed rats. The Mg-induced relaxation was lower (P 0.05) in salt loaded rats than any other group. Increasing the extracellular fluid calcium concentration to 2.61 and 5.1 mmol l 1 significantly (P 0.05) increased the relaxation responses to MgSO 4 in aortic rings from all the groups. The result of this study indicates that salt-loading caused significant increase in the blood pressure of the rats and also reduced the relaxation responses of the aortic rings to KCl, MgSO 4 and CaCl 2 which suggest altered Na +,K + -ATPase activity and resistance of the vascular smooth muscle membrane to calcium-induced membrane stabilization. These defects were reversed by dietary calcium supplement. 1998 Elsevier Science B.V. Keywords: Dietary salt; Calcium; Vascular responses 1. Introduction Previous studies have shown that dietary salt-loading induced an increase in blood pressure of rats [1 4]. It has also been demonstrated that the salt-induced hypertension in Sprague-Dawley rats is associated with enhanced contractile responses of isolated aortic rings to norepinephrine [2]. Results from several studies including measurement of radioactive 45 Ca influx [5], isolated vessel contractility [6,7] and subcellular calcium handling [8,9] have suggested that vascular smooth muscle of experimental hypertensive animals exhibit abnormal cellular calcium metabolism. Many mechanisms have been proposed to explain the altered cellular calcium handling in hypertension. It has been suggested that * Corresponding author. there is a circulating factor with natriuretic [10] and Na +,K + -ATPase inhibiting activity [11 13] in hypertension. Magnesium calcium-atpase pump activity has also been reported to be decreased in vascular smooth muscle cells of spontaneously hypertensive rats [14]. Vesicles isolated from vascular smooth muscle cells of spontaneously hypertensive rats have also been shown to exhibit decreased calcium uptake [15,16] suggesting that there is abnormality in the calcium pump of vascular smooth muscle. It has long been observed that communities with hard drinking water containing calcium and magnesium salts have lower blood pressures and mortality from cardiovascular diseases, suggesting that dietary calcium and magnesium have a role in reducing the incidence of hypertension [17,18]. It has been reported that low dietary calcium is associated with hypertension while 0928-4680/98/$19.00 1998 Elsevier Science B.V. All rights reserved. PII S0928-4680(97)10004-9

276 B.J. Adegunloye, O.A. Sofola / Pathophysiology 4 (1998) 275 280 high calcium intake could reverse some existing hypertension or prevent the development of hypertension [19 21]. High dietary calcium has been shown to lower blood pressure in nearly all models of hypertensive rats as well as their normotensive counterparts [20,22,21]. The blood pressure lowering effect of high calcium intake appears to be more effective in sodium chloride-induced hypertension [23,24]. The mechanism by which dietary calcium lowers blood pressure has not been ascertained. The present study therefore examined the effects of raised dietary calcium on salt-loaded rats and the relaxation responses of aortic rings from these rats to potassium, calcium and magnesium ions with the view of providing explanations for the antihypertensive effect of dietary calcium. 2. Materials and methods Sprague-Dawley rats aged 12 weeks and weighing between 120 150 g were randomly divided into four groups. The first group which was used as control was fed on normal rat diet containing 0.3% NaCl and 0.9% Ca. The second group (salt-loaded) received diet containing 8% NaCl and 0.9% Ca. The third group (saltloaded, high Ca-fed) received diet containing 8% NaCl and 2.5% Ca (as calcium carbonate) while the fourth group (calcium-fed) received diet containing 0.3% NaCl and 2.5% Ca. All the rats were provided with their respective diet with tap water to drink ad libitum for 6 8 weeks. At the end of feeding period, the rats were first anaesthetized with ether before 0.25 ml 100 g 1 of body weight of a mixture of 25% urethane and 1% chloralose was given intraperitoneally. After this the right femoral artery was immediately cannulated with polyethylene catheter which was filled with heparinised saline solution. Mean arterial pressure (MAP) was determined by using a Statham P23ID pressure transducer which was connected to a Grass Polygraph (model 7D) after calibrating with a mercury manometer. 2.1. Preparation of aortic rings The rats were killed by cervical dislocation and the thoracic aorta was immediately removed and cut into 2 mm ring segments and put in a petri dish containing physiological salt solution (PSS). A few minutes later, each ring was suspended in a 20 ml organ bath containing (PSS) of the following composition (mmol l 1 ): NaCl, 119; KCl, 4.7; NaHCO 3, 14.9; KH 2 PO 4, 1.2; MgSO 4, 1.2; CaCl 2, 1.6; glucose, 11.5. The medium was bubbled with 95% O 2 5% CO 2 gas mixture. Each ring was connected to a force transducer (model FT03 Grass instrument) which was coupled to Grass Polygraph (model 7D) for the recording of isometric tension. A passive tension of 2 g was applied to each ring. The ring was allowed to equilibrate for 90 min during which it was stimulated three times with 10 7 mol l 1 norepinephrine (Sigma) for 5 min at 30 min interval. 2.2. Relaxation response to potassium chloride (KCl) The PSS used for this experiment was potassium free. This was done by substituting all the KCl and KH 2 PO 4 in the PSS with equimolar concentrations of NaCl and NaH 2 PO 4, respectively. The aortic rings were incubated in this medium for 15 min before being precontracted with 10 7 mol l 1 norepinephrine. When the contraction has reached a steady level KCl (0.05 10 mmol l 1 ) was added cumulatively [22]. 2.3. Relaxation response to magnesium sulphate (MgSO 4 ) The relaxation responses to MgSO 4 was carried out by incubating the aortic rings in Mg-free PSS containing 1.6 mmol l 1 CaCl 2 for 30 min before being precontracted with 10 7 mol l 1 norepinephrine. The norepinephrine-induced contraction was allowed to reach a plateau before relaxation responses to MgSO 4 (0.05 10 mmol l 1 ) were carried out in a stepwise manner. After this the Mg-free PSS was replaced with normal PSS for 45 60 min during which the rings were rinsed at every 15 min interval. Later the aortic rings were incubated in Mg-free PSS containing 2.61 mmol l 1 or 5.1 mmol l 1 CaCl 2 for 30 min before another set of cumulative relaxation responses to MgSO 4 were carried out. 2.4. Relaxation response to calcium chloride (CaCl 2 ) The PSS used for this experiment contained 6 mmol l 1 NaHCO 3 (low bicarbonate PSS) instead of 14.9 mmol l 1 that the normal PSS contained. The low bicarbonate solution was to prevent precipitation of CaCO 3 during the course of adding cumulative concentrations of CaCl 2. The aortic rings were first precontracted with 10 7 mol l 1 norepinephrine and when the contractions had reached a steady level CaCl 2 (2.6 25.1 mmol l 1 ) was added cumulatively. 2.5. Statistics and data analysis The concentration of either KCl, MgSO 4 or CaCl 2 producing 50% relaxation (IC 50 ) was calculated using program for logit transformation of concentration response curves. The data are presented as mean SEM. One way analysis of variance was used to asses for statistical difference among the groups and Student paired t-test was used for data obtained from the same tissue (P 0.05 was considered significant).

B.J. Adegunloye, O.A. Sofola / Pathophysiology 4 (1998) 275 280 277 Fig. 1. Relaxation response curves for potassium chloride in aortic rings from control (, n=7), salt-loaded (, n=7), salt+ca fed (, n=6) and Ca-fed (, n=6) rats. * P 0.05 as compared with the controls. 3. Results 3.1. Blood pressure and heart rate Salt-loading induced a significant increase in the mean arterial pressure of salt-loaded rats (148.63 8.54 mmhg, n=8, P 0.05) but not in salt-loaded Ca-fed rats (108.36 7.81 mmhg, n=9) when compared to control rats (101.62 7.38 mmhg, n=8). The MAP of Ca-fed rats was similar (98.54 8.72 mmhg, n=9) to that of control group. There was no significant difference in the heart rates of the different groups which were: 370 13.22 for the controls, 405 14.38 for the salt-loaded rats, 385 12.57 for the salt-loaded calcium-fed rats and 375 12.73 for the calcium-fed rats (beats min 1 ). 3.2. Relaxation response to potassium chloride Fig. 1 shows the relaxation response curves of the aortic rings to KCl in K + -free PSS. Aortic rings from salt-loaded rats showed attenuated relaxation to KCl when compared with those of control rats (Table 1). There was no significant difference in the KCl-induced relaxation response of aortic rings from salt-loaded Fig. 2. Relaxation response curves for magnesium sulphate (MgSO 4 ) in aortic rings from control (, n=7), salt-loaded (, n=7), salt+ Ca-fed (, n=6) and Ca-fed (, n=6) rats. * P 0.05 as compared with the controls. Ca-fed rats and Ca-fed rats when compared to the responses of aortic rings from control rats. The IC 50 for KCl was thus higher (P 0.05) in salt-loaded rats than control rats while the IC 50, values in salt-loaded Ca-fed rats were similar to that of control. 3.3. Relaxation responses to MgSO 4 and the effect of increasing the PSS CaCl 2 concentration on the responses Fig. 2 shows the relaxation response curves to MgSO 4 in aortic rings from the different groups. The maximum relaxation response to MgSO 4 was lower in aortic rings from salt-loaded rats than in aortic rings from either control, salt-loaded Ca-fed or Ca-fed rats. Increasing the concentration of CaCl 2 in the PSS to 2.61 and 5.1 mmol l 1 did not alter the pattern of contractile responses to 10 7 mol l 1 norepinephrine. However, it significantly increased the relaxation responses to MgSO 4 in the aortic rings from all the groups (Fig. 3). The results also indicate that the effect of raised PSS CaCl 2 on the Mg-induced relaxation was more apparent in aortic rings from salt-loaded rats than in aortic rings from control, salt-loaded Ca-fed or Ca-fed rats Table 2. Table 1 The IC 50, values and % change in maximum relaxation of aortic rings in response to potassium and calcium chlorides Rat groups Control Salt-loaded Salt-loaded Ca-fed Ca-fed KCl IC 50 3.86 0.19 6.34 0.33* 3.84 0.03 3.25 0.25 Maximum relaxation 70.76 3.65 55.50 3.51* 73.10 3.46 79.3 3.11 CaCl 2 IC 50 15.16 2.36 15.26 2.48 14.52 2.11 Maximum relaxation 62.01 2.90 48.26 3.26* 60.11 2.56 62.13 2.23 Values are presented as mean SEM. The maximum fall in 10 7 mol l 1 noradrenaline induced contraction that was produced by either KCl or CaCl 2 was taken as maximum relaxation. ( ) maximum relaxation was not up to 50%. * P 0.05 when compared with the control.

278 B.J. Adegunloye, O.A. Sofola / Pathophysiology 4 (1998) 275 280 Fig. 4. Relaxation response curves for calcium chloride in aortic rings from control (, n=7), salt-loaded (, n=7), salt+ca fed (, n=6) and Ca-fed (, n=6) rats. * P 0.05 as compared with the controls. Fig. 3. The effect of varying the PSS calcium chloride concentration on the relaxant effect of magnesium sulphate on aortic rings from normal rats (A), salt-loaded rats (B), salt-loaded Ca-fed rats (C) and Ca-fed rats (D). The relaxation response was carried out in PSS containing either 1.6 mmol l 1 CaCl 2 ( ), 2.51 mmol l 1 CaCl 2 ( ) or 5.1 mmol l 1 CaCl 2 ( ). * P 0.05 when compared to the responses in 1.6 mmol l 1 CaCl 2 PSS (n=6 in each group). 3.4. Relaxation responses to calcium chloride The relaxation response curves of the aortic rings to CaCl 2 in low-bicarbonate PSS is shown in Fig. 4. The maximum relaxation of aortic rings from salt-loaded rats was lower than the maximum relaxation of aortic rings from control rats (Table 1). The IC 50 for CaCl 2 was also higher (P 0.05) in salt-loaded rats when compared with the IC 50 from control rats. Aortic rings from salt-loaded Ca-fed rats have similar maximum relaxation responses and IC 50 when compared to those of control rats but they were however significantly different from those of salt-loaded rats (Fig. 4, Table 1). 4. Discussion The antihypertensive effect of high dietary calcium observed in this study is consistent with our previous reports [25,26] that high dietary calcium prevents saltinduced hypertension in Sprague-Dawley rats by attenuating salt-induced enhanced contractile responses of vascular smooth muscle to vasopressors. Calcium supplementation has also been shown in other studies to attenuate the development of hypertension in spontaneously hypertensive rats [20,27] and prevents DOCAinduced blood pressure increases in spontaneously hypertensive rats by reducing contractile responses to norepinephrine whilst augmenting relaxation responses to acetylcholine and nitroprusside [28]. Elevated intracellular calcium concentration has been reported in various forms of hypertension [20,21]. It has been documented that excess salt-loading is associated with increases in intracellular calcium ion concentration [29,13]. Many mechanisms have been proposed for this observation. One of such mechanism is altered Na +, K + -ATPase activity [29,2,30]. Moreover high intracellular calcium concentration has been reported to inhibit Na +,K + -ATPase or vice versa [21]. The magnitude of KCl-induced relaxation of isolated blood vessels exposed to K + -free PSS has been used as a functional indicator of Na +,K + -ATPase activity [31,2,22]. The attenuated KCl-induced relaxation of aortic rings from salt-loaded rats observed in this study is suggestive of altered Na +,K + -ATPase activity. Since the magnitude Table 2 IC 50 values and maximal relaxation response of aortic rings to MgSO 4 Rat groups (n) IC Max relaxation a 50 (mmol l 1 ) (%) Max relaxation b (%) Control (6) 4.36 0.64 72.03 3.48 20.00 2.16 Salt-loaded (6) 29.89 3.60* 29.00 5.18* 49.00 4.15 Salt-loaded Ca-fed (6) 5.27 3.12 63.72 4.48 20.85 2.76 Ca-fed (5) 4.67 1.37 68.90 3.30 14.25 3.42 Values are presented as mean SEM. Maximum relaxation was obtained by subtracting relaxation in 1.6 mmol l 1 CaCl 2 PSS from maximum relaxation in 5.10 mmol l 1 CaCl 2 PSS. n=number of experiments in the group. a Increase (%) in maximum relaxation response to MgSO 4 in 1.6 mmol l 1 CaCl 2 PSS and b to MgSO 4 in 5.10 mmol l 1 CaCl 2 PSS. * P 0.05 as compared to control.

B.J. Adegunloye, O.A. Sofola / Pathophysiology 4 (1998) 275 280 279 of KCl-induced relaxation of salt-loaded Ca-fed rats was similar to those of control, then dietary calcium must have reversed the altered Na +,K + -ATPase activity induced by salt-loading. Porsti et al. [32] reported that high calcium diet augments potassium induced relaxation of aorta from spontaneously hypertensive rat aorta which suggested an increase in the activity of vascular smooth muscle Na +,K + -ATPase. The result of this study shows that high dietary calcium can prevent the impaired relaxation responses of the vascular smooth muscle to magnesium ion. The cause of the attenuated relaxation responses of the salt-loaded aorta to Mg 2+ is speculative and the mechanism by which high dietary calcium prevents this is not clear but it is known that Mg 2+ -induced relaxation of vascular smooth muscle is dependent on vascular Na +,K + -ATPase, Ca 2+ -ATPase and movement of ions across the membrane [28,33,34]. Earlier studies have indicated that raised extracellular magnesium ion concentration reduces contractile responses to calcium ions while low extracellular magnesium ion concentration elevates the tone of vascular smooth muscle [35,36]. These previous studies have suggested that raised extracellular fluid magnesium ion interferes with the influx of calcium from extracellular fluid into the cytosol. However the result of the present study indicates that as the extracellular fluid calcium concentration increases the magnitude of relaxation response to magnesium increases. This probably implies that high extracellular fluid calcium concentration might be playing complementary role to magnesium-induced relaxation of vascular smooth muscle. This effect could be due to the reported membrane stabilizing effect of high extracellular fluid calcium [37]. This may also account for why the rings from calcium fed rats did not show as much changes as those from control or salt-loaded rats. Membrane stabilization was investigated in this study by exposing norepinephrine precontracted aortic rings to calcium chloride. The magnitude of relaxation responses following addition of calcium chloride to the physiological salt solution is a measure of calcium-induced membrane stabilization [22,37]. Thus the attenuated relaxation responses to calcium chloride in salt-loaded rats indicate that the membrane of the vascular smooth muscle cell from this group was more difficult to stabilize but that high dietary calcium enhances the calcium-induced membrane stabilization of vascular smooth muscle. This observation is in agreement with the finding of Furspan et al. [38] who demonstrated that dietary calcium supplement increased serum ionized calcium by 10% in stroke-prone spontaneously hypertensive rats and this increase in extracellular fluid ionized calcium was associated with decreased net potassium efflux and reduced intracellular calcium concentration in lymphocytes. The lack of effect of dietary calcium on the relaxation responses of rats that were not salt-loaded suggests that high calcium intake in normotensive rats may not have any adverse effect. The result shown in Fig. 3 indicates that the increase in relaxation response to Mg 2+ as a result of increases in extracellular fluid calcium concentration were not as significant as the salt-loaded and control rats. The reason for this is because the vessels from high dietary calcium fed rats were already exposed to high extracellular fluid calcium in vivo and they therefore did not require much increase in the extracellular fluid calcium to attain their maximum relaxation responses to magnesium. The results of this present study demonstrate that salt-loading produces significant increases in blood pressure of rat and this effect of salt on blood pressure was prevented by dietary calcium supplement. This study also suggests that dietary calcium supplement blunted the salt-induced impaired Na +,K + -ATPase activity as well as membrane stabilization. Salt-loading also resulted in attenuated relaxation responses to MgSO 4 an effect that was reversed by high dietary calcium supplement. High extracellular fluid calcium was also shown in this study to enhance the relaxation responses to MgSO 4. The lack of any significant effect of high dietary calcium on Ca-fed rats can be an indication of the protective effect of high calcium diet on blood pressure and vascular responses. Acknowledgements The authors are grateful to Prof. Roger Hainsworth of the Department of Cardiovascular Studies, University of Leeds for allowing us to make use of the facilities in his laboratory for the preparation of this manuscript and also for his useful suggestions. This study was supported by the University of Lagos central research grant CRC 92/03. References [1] C.I. Nwaigwe, O.A. Sofola, Potassium but not nifedipine reduces hypertension in anaesthetized salt-loaded rats, Med. Sci. Res. 17 (1989) 767 768. [2] P.C.M. Obiefuna, A.B. Ebeigbe, O.A. Sofola, C.P. Aloamaka, Altered responses of aortic smooth muscle from Sprague-Dawley rats with salt induced hypertension, Clin. Exp. Pharmacol. Physiol. 18 (1991) 813 818. [3] O.A. Sofola, P.C.M. Obiefuna, O.P. Adesanya, Chloroquine and captopril prevent the development of hypertension in rats fed a high salt diet, Med. Sci. Res. 19 (1991) 379 380. [4] O.A. Sofola, P.C.M. Obiefuna, B.J. Adegunloye, Contractile response of normotensive rat aorta to serum from salt-loaded Sprague-Dawley rats, Pflug. Arch. Eur. J. Physiol. 423 (1993) 161 163.