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(Circulation. 1996;93:1000-1008.)
© 1996 American Heart Association, Inc.

Articles

Endothelial Function in Deoxycorticosterone-NaCl Hypertension

Effect of Calcium Supplementation

Heikki Mäkynen, MD; Mika Kähönen, MD; Xiumin Wu, MSc; Pertti Arvola, MD; Ilkka Pörsti, MD

From the Medical School (H.M., X.W.), University of Tampere, and Departments of Clinical Physiology (M.K.) and Internal Medicine (P.A., I.P.), Tampere University Hospital, Tampere, Finland.

Correspondence to Heikki Mäkynen, MD, University of Tampere, Medical School, Department of Pharmacology, Clinical Pharmacology, and Toxicology, PO Box 607, FIN-33101 Tampere, Finland.

	Abstract

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Background Dietary calcium intake has been suggested to correlate inversely with blood pressure in humans and experimental animals. However, the effects of calcium supplementation on hypertensive disturbances of the endothelium have not been well characterized.

Methods and Results Wistar-Kyoto rats were made hypertensive by deoxycorticosterone (DOC)-NaCl treatment, but a concurrent increase in chow calcium content from 1.1% to 2.5% markedly attenuated the rise in blood pressure. The function of isolated mesenteric arterial rings in vitro was investigated at the close of the 10-week study. In norepinephrine-precontracted rings, the relaxations to acetylcholine (ACh) and ADP, as well as to nitroprusside, 3-morpholinosydnonimine, and isoproterenol were attenuated in hypertensive rats on 1.1% calcium, but these responses were improved by calcium supplementation. In the presence of N^G-nitro-L-arginine methyl ester (L-NAME), the relaxations to ACh in hypertensive animals on normal calcium were practically absent, whereas in normotensive rats and calcium-supplemented hypertensive rats, distinct relaxations to higher concentrations of ACh were still present. These responses were reduced by 30% to 50% with apamin, a blocker of Ca²⁺-activated K⁺ channels, and were further inhibited by blockade of ATP-dependent K⁺ channels with glyburide. Interestingly, relaxations elicited by ACh and ADP during precontraction with 60 mmol/L KCl (preventing endothelium-dependent hyperpolarization) were not impaired in hypertensive animals. The contractile sensitivity of endothelium-intact arterial rings to 5-hydroxytryptamine and norepinephrine was higher in hypertensive rats on either normal or high-calcium diet, whereas the increase in contractile sensitivity caused by L-NAME corresponded in all groups.

Conclusions High-calcium diet markedly opposed experimental DOC-NaCl hypertension, an effect associated with improved arterial relaxation, while abnormalities of vascular contractile properties remained unaffected. In particular, the hyperpolarization-related component of endothelium-dependent arterial relaxation, mediated via opening of arterial K⁺ channels, could be augmented by calcium supplementation in DOC-NaCl hypertension.

Key Words: endothelium • hypertension • calcium • arteries • diet

	Introduction

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Several epidemiological studies have proposed that dietary calcium gain and blood pressure are inversely correlated, although contradictory results have been published as well.^{1 2 3} Calcium supplementation has also been suggested to decrease blood pressure in human hypertension,^{4 5 6 7} and certain subsets of patients, including those with hypertensive disorders of pregnancy⁷ and salt-sensitive individuals,⁵ appear to be more susceptible to the blood pressure–lowering action.

In experimental animals, the antihypertensive effect of high-calcium diet has been very consistent.⁸ Studies in various experimental models have shown that calcium supplementation is especially effective in sodium volume–dependent hypertension.^{9 10} The mechanisms underlying the antihypertensive action of increased dietary calcium intake have not been fully defined, but probably both vascular and nonvascular actions are involved. Since Ca²⁺ is acknowledged to be the main determinant of arterial smooth muscle tone, many studies have explored the effects of calcium intake on functional properties of vascular tissue. Indeed, high-calcium diet has been shown to improve arterial relaxation and attenuate contractile activity in experimental hypertension.^{10 11}

Endothelial cells produce and release many vasoactive substances, both relaxing and constrictive, and thereby regulate the tone of underlying vascular smooth muscle.^{12 13 14} Hypertension is associated with impairment of endothelial function, which is likely to contribute to the increased arterial resistance characteristic of hypertensive states.^{15 16} Interestingly, various pharmacological treatments have been reported to protect or recover endothelium-dependent vasomotion in hypertension.^{17 18 19} However, the effects of calcium supplementation on endothelial function remain unknown.

Therefore, the aim of the present study was to investigate whether increased dietary calcium as a nonpharmacological treatment of elevated blood pressure could beneficially influence endothelial function in experimental mineralocorticoid-NaCl hypertension. The study design also allowed evaluation of the roles of different endothelium-derived mediators in the vascular responses.

	Methods

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Animals and Experimental Design
Sixty 7-week-old male WKY rats were divided into four groups (n=10 to 12) with equal mean systolic blood pressures. The DOC and Ca-DOC groups were treated with DOC 25 mg/kg SC once a week, and 0.7% NaCl was added to the drinking fluid, whereas the WKY and Ca-WKY groups received equal volumes of saline instead of DOC and drank normal tap water. The standard chow in the WKY and DOC groups contained 1.1% calcium, and the modified chow in the Ca-WKY and Ca-DOC groups contained 2.5% calcium, with the extra calcium supplied as the carbonate salt. The two pellet chows were identical except for the calcium contents (R3 rat chow, Ewos). The systolic blood pressures of the conscious animals were measured by the tail-cuff method at 28°C (model 129 blood pressure meter, IITC Inc). At the age of 17 weeks, the rats were decapitated and exsanguinated, and the hearts and mesenteric arteries were immediately excised. The experimental design was approved by the Animal Experimentation Committee of the University of Tampere, Finland.

Mesenteric Arterial Responses In Vitro
The mesenteric artery was carefully cleaned of connective tissue, and six consecutive standard sections (3 mm long) were cut beginning 1 cm distal to the junction of the mesenteric artery and aorta. The vascular endothelium was removed from some rings by gentle rubbing with a jagged injection needle. The rings were placed between small hooks (diameter, 0.3 mm) and suspended in an organ bath chamber (volume, 20 mL) in PSS (pH 7.4) of the following composition (mmol/L): NaCl 119.0, NaHCO₃ 25.0, glucose 11.1, CaCl₂ 1.6, KCl 4.7, KH₂PO₄ 1.2, and MgSO₄ 1.2, aerated with 95% O₂/5% CO₂. The rings were initially equilibrated for 1 hour at 37°C with a resting tension of 1.5 g, which was determined to render maximal contractile force development to 125 mmol/L KCl in the study groups.^{10 11} The force of contraction was measured with an isometric force–displacement transducer and registered on a polygraph (FT 03 transducers and model 7E polygraph, Grass Instrument Co). The presence of functionally intact endothelium in vascular preparations was confirmed by the observation of at least 70% relaxation to 1 µmol/L ACh in rings precontracted with 1 µmol/L norepinephrine, and the absence of endothelium by the lack of this response. All rings were allowed a further 30-minute stabilization period in PSS after these test responses.

Endothelium-Dependent Relaxation After Receptor-Mediated Precontraction
Relaxations to two endothelium-dependent agonists, ACh and ADP, were examined cumulatively in endothelium-intact rings precontracted with 1 µmol/L norepinephrine. The responses to ACh were also elicited in the presence of 0.1 mmol/L L-NAME; L-NAME and 10 µmol/L indomethacin; L-NAME, indomethacin, and 1 µmol/L apamin; and L-NAME, indomethacin, apamin, and 10 µmol/L glyburide (inhibitors of NO synthase, cyclooxygenase, Ca²⁺-activated K⁺ channels, and ATP-dependent K⁺ channels, respectively).

Endothelium-Dependent Relaxation After Depolarization-Mediated Precontraction
An endothelium-intact arterial ring was used to study relaxations to ACh and ADP after precontraction with 60 mmol/L KCl (thus preventing endothelium-dependent hyperpolarization).²⁰ The responses were also generated in the presence of 0.1 mmol/L L-NAME.

Endothelium-Independent Arterial Relaxation
Responses to sodium nitroprusside, SIN-1, and isoproterenol were determined in endothelium-denuded rings after precontraction with 1 µmol/L norepinephrine.

Effect of Endothelium on Arterial Contractions
Concentration-response curves for norepinephrine and 5-HT were cumulatively determined from an endothelium-intact ring in the absence and presence of 0.1 mmol/L L-NAME. Contractions to 5-HT were also studied in the presence of 0.1 mmol/L L-NAME and 10 µmol/L indomethacin.

Depolarization-Mediated Arterial Contractions After Elimination of the Influence of Adrenergic Nerve Endings
Endothelium was denuded, then a chemical sympathectomy was performed: the rings were exposed in vitro to 1.2 mmol/L 6-hydroxydopamine in buffer-free solution, which was vigorously gassed with nitrogen for 15 minutes, after which a 2-hour recovery period in normal PSS was allowed.²¹ Then, contractions to KCl were determined. In solutions containing high concentrations of K⁺ (20 to 125 mmol/L), NaCl was replaced with KCl on an equimolar basis.

Arterial Smooth Muscle Sensitivity to Calcium Entry Blockade by Nifedipine
Calcium was omitted from the buffer solution, and endothelium-denuded rings were contracted with 10 µmol/L norepinephrine to empty the cellular calcium stores.²² The rings were challenged again with 10 µmol/L norepinephrine in calcium-free buffer, and calcium was returned to the organ bath in increasing concentrations (0.05 to 2.5 mmol/L). The subsequent contraction was registered. The procedure was repeated in the presence of the dihydropyridine nifedipine (0.5 nmol/L).

Unless otherwise stated, all rings were allowed a 30-minute recovery period in resting tension between the responses. Relaxations to ACh, ADP, nitroprusside, SIN-1, and isoproterenol were presented as percentage of preexisting precontraction. Contractions to 5-HT, norepinephrine, KCl, and calcium accumulation were expressed in grams, and responses to 5-HT and KCl were also depicted as percentage of maximum. The pD₂ for 5-HT, norepinephrine, and KCl was the negative logarithm of the concentration producing 50% of maximal response in each ring. The effect of nifedipine on calcium contractions was calculated by comparing the response with nifedipine to the maximal response without it.

Drugs
The following drugs were used: ADP (Boehringer Mannheim GmbH), DOC (Ciba-Geigy Ltd), sodium nitroprusside (E. Merck AG), norepinephrine hydrogentartrate (Fluka Chemie AG), SIN-1 (GEA Ltd), nifedipine (Orion Pharmaceutical Co), and acetylcholine chloride, 5-HT, L-NAME, indomethacin, 6-hydroxydopamine, isoprenaline, apamin, and glyburide (Sigma Chemical Co).

Analysis of Results
Statistical analysis was performed with one-way ANOVA supported by the Bonferroni test for pairwise between-group comparisons. When the data consisted of repeated observations at successive time points, ANOVA for repeated measurements was applied to investigate between-group differences. All results were expressed as mean±SEM, with P<.05 considered significant. The data were analyzed with BMDP statistical software.

	Results

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Blood Pressure, Heart Weight, and Body Weight
Calcium supplementation markedly attenuated the development of two-kidney DOC-NaCl hypertension during the 10-week study, whereas blood pressure in normotensive rats was not significantly affected (Fig 1). Heart weight and ratio of heart to body weight were clearly increased by the DOC-NaCl treatment, but heart weight in the Ca-DOC group was lower than in the DOC group, and a trend toward reduced heart weight–to–body weight ratio was also observed after the high-calcium diet (P=.062). Animals in the Ca-WKY, DOC, and Ca-DOC groups gained slightly less weight than the untreated WKY rats (Table 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Systolic blood pressures during the 10-week study. The groups are untreated WKY rats and DOC-NaCl–treated WKY (DOC) on normal diet and untreated WKY (Ca-WKY) and DOC-NaCl–treated WKY (Ca-DOC) on high-calcium diet. Symbols indicate mean±SEM, n=10 to 12 in each group. *P<.05, ANOVA for repeated measurements.

View this table: [in this window] [in a new window]   Table 1. Experimental Group Data at Close of the Study

Mesenteric Arterial Responses In Vitro
The relaxations induced by ACh and ADP in endothelium-intact norepinephrine-precontracted arterial rings were impaired in the DOC group, but these responses were clearly improved by calcium supplementation, the relaxations to ACh not differing from those of normotensive controls (Figs 2A and 3A). The NO synthase inhibitor L-NAME effectively diminished the relaxations to ACh in all groups, the influence being most pronounced in the DOC group (Fig 2B). Cyclooxygenase inhibition with indomethacin (in the presence of L-NAME) did not significantly affect the relaxations to ACh (Fig 2C). In contrast, apamin, an inhibitor of Ca²⁺-activated K⁺ channels, reduced the L-NAME and indomethacin-resistant relaxation to higher concentrations of ACh by 30% to 50% in the WKY, Ca-WKY, and Ca-DOC groups and totally inhibited the marginal response to ACh in the DOC group (Fig 2D). Further addition of glyburide, a blocker of ATP-dependent K⁺ channels, abolished the L-NAME– and indomethacin-resistant relaxation to ACh in the Ca-DOC group, whereas a small residual response was still observed in the WKY and Ca-WKY groups (Fig 2E).

View larger version (23K): [in this window] [in a new window]   Figure 2. Cumulative relaxation responses to ACh after precontraction with 1 µmol/L norepinephrine in endothelium-intact mesenteric arterial rings (A through E) and the effect of 0.l mmol/L L-NAME (B) and L-NAME, indomethacin, apamin, and 10 µmol/L glyburide (E) on the response. F, Relaxation to ACh was generated after precontraction with 60 mmol/L KCl. Groups as in Fig 1. Bar graph inset in B shows the reduction in relaxation to 1 µmol/L ACh caused by L-NAME in norepinephrine-precontracted rings. Symbols indicate mean±SEM, n=7 to 10 in each group. *P<.05, ANOVA for repeated measurements; +P<.05 compared with the WKY group and #P<.05 compared with the DOC group, Bonferroni test.

View larger version (13K): [in this window] [in a new window]   Figure 3. Cumulative relaxation responses to ADP after precontraction with 1 µmol/L norepinephrine (NE) (A) and after precontraction with 60 mmol/L KCl (B) in endothelium-intact mesenteric arterial rings. Groups as in Fig 1. Symbols indicate mean±SEM, n=7 to 10 in each group. *P<.05, ANOVA for repeated measurements.

Interestingly, when the relaxations to ACh were elicited after precontraction with KCl, ie, under conditions of prevented endothelium-dependent hyperpolarization, the responses were slightly augmented in both of the DOC-NaCl–treated groups compared with the WKY group (Fig 2F). Furthermore, the relaxation to ADP in KCl-precontracted rings was comparable in all four study groups (Fig 3B). The responses to ACh and ADP in KCl-precontracted rings were completely abolished in the presence of L-NAME (data not shown).

The relaxations to the endothelium-independent agents nitroprusside, SIN-1, and isoproterenol in norepinephrine-precontracted endothelium-denuded arterial rings were also impaired in the DOC group compared with the WKY group (Fig 4). Calcium supplementation markedly enhanced these responses, the relaxations in the Ca-DOC group not differing from those of normotensive controls (Fig 4).

View larger version (15K): [in this window] [in a new window]   Figure 4. Cumulative relaxation responses to sodium nitroprusside (A), SIN-1 (B), and isoproterenol (C) after full precontraction with 1 µmol/L norepinephrine in endothelium-denuded mesenteric arterial rings. Groups as in Fig 1. Symbols indicate mean±SEM, n=7 to 10 in each group. *P<.05, ANOVA for repeated measurements.

The DOC-NaCl treatment enhanced arterial contractile sensitivity to 5-HT (ie, increased pD₂ values) whether the vascular endothelium was present or not (Table 2 and Fig 5A). In endothelium-intact rings of DOC-NaCl–treated rats, higher sensitivity to 5-HT was also observed after pretreatment with L-NAME and indomethacin, and the increases in pD₂ values caused by these two inhibitors were comparable in all groups (Fig 5B and 5C). The DOC-NaCl treatment also enhanced contractile sensitivity to norepinephrine in endothelium-intact rings, and the increase in sensitivity induced by L-NAME to this agonist was comparable in all groups. Maximal contractile forces generated by 5-HT and norepinephrine corresponded between the study groups (Table 2). The high-calcium diet was without effect on the 5-HT– and norepinephrine-elicited contractions and the endothelial modulation thereof (Table 2 and Fig 5).

View this table: [in this window] [in a new window]   Table 2. Parameters of Contractile Responses of Isolated Mesenteric Arterial Rings

View larger version (19K): [in this window] [in a new window]   Figure 5. Concentration-response curves of endothelium-intact mesenteric arterial rings to 5-HT (A), and the effects of 0.1 mmol/L L-NAME (B) and L-NAME and 10 µmol/L indomethacin (C) on the contraction. Groups as in Fig 1. Bar graph insets show the increase in pD2 value (pD2) induced by L-NAME (B) and indomethacin (C). pD2 is the negative logarithm of concentration of agonist inducing 50% response compared with maximum. Symbols indicate mean±SEM, n=8 to 10 in each group. +P<.05, DOC and Ca-DOC groups compared with WKY group, Bonferroni test.

The contractions to 20 mmol/L KCl in chemically sympathectomized endothelium-denuded vascular rings were enhanced in both DOC-NaCl–treated groups (Fig 6A). However, the pD₂ values and maximal contractile force generation in response to KCl were comparable in the study groups (Table 2). Maximal contractions of endothelium-denuded rings in response to cumulative Ca²⁺ addition (with norepinephrine as the agonist) were also similar in all groups (2.31±0.17, 2.43±0.28, 2.37±0.37, and 2.29±0.25 g in the WKY, Ca-WKY, DOC, and Ca-DOC groups, respectively), while the inhibitory effect of the calcium entry blocker nifedipine was more pronounced in the DOC and Ca-DOC groups than in normotensive controls (Fig 6B).

View larger version (17K): [in this window] [in a new window]   Figure 6. Concentration-response curves of sympathectomized and endothelium-denuded mesenteric arterial rings to KCl (A). Effect of nifedipine (0.5 nmol/L) on contractile responses of endothelium-denuded mesenteric arterial rings to cumulative addition of calcium to the organ bath after precontraction with 10 µmol/L norepinephrine in calcium-free medium (B). Groups as in Fig 1. Symbols indicate mean±SEM, n=8 to 10 in each group. *P<.05, ANOVA for repeated measurements. +P<.05, DOC and Ca-DOC groups compared with WKY group, Bonferroni test.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study showed that a high-calcium diet markedly attenuated the development of experimental two-kidney DOC-NaCl hypertension in rats. Moreover, calcium supplementation not only slowed the rate of ascending blood pressure¹⁰ but also had a persistent long-term antihypertensive effect. Also, the DOC-NaCl–induced cardiac hypertrophy was alleviated, thus supporting the concept of reduced cardiac afterload and peripheral resistance after calcium supplementation.²³

ACh relaxes arteries via the release of several factors from endothelial cells, the most prominent autacoids being NO, prostacyclin, and EDHF. Subsequently, NO stimulates soluble guanylate cyclase, elevating intracellular cGMP in smooth muscle; prostacyclin acts via adenylate cyclase and cAMP, and EDHF dilates arteries via the opening of K⁺ channels.^{15 24} Impaired endothelium-dependent relaxation to ACh has often been observed in studies of experimental hypertension,^{25 26 27} and in the present investigation, the relaxations to ACh in norepinephrine-precontracted arterial rings were impaired in the DOC group, whereas these responses were normalized by calcium supplementation. Moreover, inhibition of NO synthesis diminished dilatations to ACh most effectively in the DOC group, in which L-NAME practically abolished the responses, indicating that the relaxations to ACh were mediated predominantly by NO in this group. In the Ca-DOC group, however, a distinct L-NAME–resistant relaxation was observed, suggesting that endothelial product(s) other than NO took part in the enhanced relaxation to ACh in the calcium-supplemented DOC-NaCl–treated rats.

EDCF has been proposed to be involved in impaired endothelium-dependent relaxations in experimental hypertension. The production of EDCF requires the activity of cyclooxygenase, since indomethacin has been found to normalize the impaired endothelium-dependent relaxations in resistance arteries of spontaneously hypertensive rats and DOC-NaCl hypertensive rats.^{28 29} In the present study, however, indomethacin was without a significant effect on the responses to ACh. Thus, the role of cyclooxygenase-derived substances appeared to be negligible in these endothelium-mediated relaxations.

ACh has been shown to cause hyperpolarization of arterial smooth muscle in vitro,^{30 31 32} which remains unaffected by N^G-nitro-L-arginine, indomethacin, or hemoglobin, whereas the hyperpolarization generated by NO can be blocked with hemoglobin.³⁰ Furthermore, in guinea pig coronary arteries, ACh hyperpolarizes the cell membrane independently of changes in tissue cGMP.³³ These findings suggest that the hyperpolarization induced by ACh is not caused by NO, and a distinct substance, EDHF, is released from the endothelial cells. The chemical characteristics of EDHF remain unknown, but it has been reported to be an endogenous K⁺ channel opener,^{31 34} the action of which can be inhibited by membrane depolarization with KCl.^{20 32} Thus, the relaxation to ACh during precontraction with KCl reflects mainly the effects of NO, whereas during precontraction with norepinephrine, EDHF also remains operative. In the present study, the relaxations to ACh and ADP during KCl precontraction were not impaired in the DOC-NaCl–treated rats. Moreover, NO synthesis inhibition by L-NAME totally inhibited these dilatations in all groups, suggesting that the release of NO was indeed responsible for the ACh- and ADP-induced relaxations of KCl-precontracted preparations. Since the relaxations to ACh in the DOC group during KCl precontraction corresponded to those in the other groups but were clearly impaired in norepinephrine-precontracted rings and also more effectively inhibited by L-NAME, the attenuated relaxations in the DOC-NaCl–treated rats could be attributed to reduced endothelium-dependent hyperpolarization, while the role of NO in the responses appeared to be well preserved. This finding parallels previous results whereby the production of endothelium-derived NO was even enhanced in mineralocorticoid hypertension.³⁵ Interestingly, deficient endothelium-dependent hyperpolarization has also been observed in spontaneously hypertensive rats.^{24 26 36} The fact that the endothelium-mediated relaxations of KCl-precontracted rings were not affected in the Ca-DOC group, whereas those induced in norepinephrine-precontracted rings were enhanced compared with the DOC group, suggests that endothelium-dependent hyperpolarization was augmented by oral calcium supplementation.

The nature of the K⁺ channels opened by EDHF is not fully characterized. Glyburide inhibits ACh-induced hyperpolarization in rabbit cerebral artery³⁷ and exhibits partial antagonism of ACh-induced relaxation in rat aorta,³⁸ suggesting an involvement of ATP-sensitive K⁺ channels. However, in rat mesenteric artery, apamin has been reported to reduce the L-NAME–insensitive ACh-elicited relaxation by 55%, and apamin together with another Ca²⁺-activated K⁺ channel blocker, charybdotoxin, to completely abolish these relaxations,³⁹ whereas glyburide was ineffective in blocking the hyperpolarization and relaxation to ACh.^{30 40} These findings suggest that EDHF relaxes arterial smooth muscle via activation of Ca²⁺-activated K⁺ channels. In the present study, apamin reduced the L-NAME– and indomethacin-resistant relaxation to ACh by 30% to 50% in the WKY, Ca-WKY, and Ca-DOC groups, whereas the marginal remaining relaxation to ACh was totally abolished by apamin in the DOC group. Complete inhibition of the relaxation was observed in the Ca-DOC group when glyburide was added to the medium, whereas in the WKY and Ca-WKY groups, minute relaxations to ACh still remained. These findings further support the notion of augmented endothelium-dependent hyperpolarization by oral calcium supplementation in DOC-NaCl hypertension, mediated via Ca²⁺-activated and ATP-dependent K⁺ channels.

The endothelium-independent arterial relaxations induced by nitroprusside, SIN-1, and isoproterenol were also attenuated in the DOC group. These responses, too, were enhanced by oral calcium supplementation, and the relaxations in the Ca-DOC group did not differ from those of the WKY group. The normalization of arterial relaxation to exogenous NO and nonselective ß-adrenergic receptor activation, which most likely reflected an enhancement of general relaxation properties of vascular smooth muscle, may also have contributed to the enhanced endothelium-dependent relaxations in the Ca-DOC group. Moreover, exogenous NO has been shown to hyperpolarize guinea pig uterine artery⁴¹ and rat mesenteric artery,³⁰ the hyperpolarization of the latter being inhibited by glyburide. The blockers of Ca²⁺-activated K⁺ channels charybdotoxin and tetraethylammonium have also been shown to decrease NO donor–mediated relaxation in guinea pig pulmonary arterial and tracheal smooth muscle.⁴² In addition, isoproterenol has been reported to open ATP-dependent K⁺ channels in the smooth muscle of canine saphenous vein⁴³ and to cause endothelium-independent hyperpolarization of smooth muscle in porcine coronary artery.⁴⁴ Thus, augmented function of K⁺ channels in smooth muscle could also partially explain the enhanced relaxations to the endothelium-independent agonists as well as the improved endothelium-mediated hyperpolarization in the Ca-DOC group in this study. Nevertheless, the slightly augmented relaxation to ACh after precontraction with KCl in the DOC group, despite the impaired arterial relaxation sensitivity to exogenous NO, supports the conclusion that the production of endogenous NO was not reduced in DOC-NaCl hypertension.

Hypertension induced by DOC-NaCl administration has been shown to enhance vascular contractility,^{45 46} and abnormalities in endothelial function might contribute to this phenomenon. In the present study, the sensitivity to 5-HT in endothelium-intact arterial rings was increased in both DOC-NaCl–treated groups. 5-HT is known to activate serotonergic receptors in the endothelium too, causing the release of NO as well as of EDCF.⁴⁷ Since the sensitivity to 5-HT was comparably increased by pretreatment with L-NAME and indomethacin in all groups, enhanced vascular sensitivity to 5-HT in DOC-NaCl hypertension was probably not caused by an abnormal release of endothelial NO or products of the cyclooxygenase pathway. Increased sensitivity to 5-HT in the DOC-NaCl–treated groups was also present after endothelial denudation, indicating that the underlying abnormality was located in the smooth muscle. Furthermore, the sensitivity to norepinephrine of endothelium-intact rings from the DOC-NaCl–treated groups was enhanced in the absence and presence of L-NAME, but these differences in sensitivity were no longer observed in endothelium-denuded preparations. Thus, contractile sensitivity to norepinephrine, unlike that to 5-HT, was increased in DOC-NaCl hypertension because of an endothelial factor, the nature of which was not apparent from the present results, but a possible candidate would be EDCF.⁴⁸ Finally, in addition to enhanced receptor-mediated contractions, the DOC-NaCl treatment also augmented voltage-dependent Ca²⁺ entry, as suggested by enhanced contractile responses to depolarization with 20 mmol/L KCl and increased inhibitory effect of nifedipine on Ca²⁺ contractions in the DOC and Ca-DOC groups. Since the high-calcium diet failed to prevent the DOC-NaCl–induced enhancement of arterial contractions and Ca²⁺ entry, the present results indicate that calcium supplementation especially augmented vascular relaxations and that the enhanced relaxations could not be explained by changes in contractility.

This study demonstrated that calcium supplementation exerted an effective and long-term antihypertensive action in experimental two-kidney DOC-NaCl hypertension. The blood pressure–lowering effect was associated with improved arterial relaxation, whereas the abnormalities of contractile properties remained unaffected. During both receptor-mediated contractions and endothelium-dependent relaxations, the role of endothelial NO was well preserved in DOC-NaCl hypertension, whereas the augmented endothelium-dependent relaxations following calcium supplementation most likely resulted from enhanced endothelium-dependent hyperpolarization of vascular smooth muscle. The high-calcium diet also increased arterial sensitivity to exogenous NO and ß-adrenergic receptor activation, suggesting improvement of general relaxation properties in smooth muscle. Taken together, enhanced vascular relaxation may play a significant role in the mechanisms whereby calcium supplementation lowers blood pressure in sodium-volume–dependent hypertension.

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine DOC = deoxycorticosterone trimethylacetate EDCF = endothelium-derived contracting factor(s) EDHF = endothelium-derived hyperpolarizing factor(s) 5-HT = 5-hydroxytryptamine L-NAME = NG-nitro-L-arginine methyl ester pD2 = negative logarithm of concentration producing 50% of maximal response PSS = physiological salt solution SIN-1 = 3-morpholinosydnonimine WKY = Wistar-Kyoto

	Acknowledgments

 
This study was supported by the Foundation for Nutrition Research, the Aarne Koskelo Foundation, the Emil Aaltonen Foundation, and the Medical Research Fund of Tampere University Hospital, Finland.

Received June 6, 1995; revision received September 27, 1995; accepted October 4, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. McCarron DA, Morris CD, Henry HJ, Stanton JL. Blood pressure and nutrient intake in the United States. Science. 1984;224:1392-1398. [Abstract/Free Full Text]
   
2. Gruchow HW, Sobocinski KA, Barboriak JJ. Calcium intake and the relationship of dietary sodium and potassium to blood pressure. Am J Clin Nutr. 1988;48:1463-1470. [Abstract/Free Full Text]
   
3. Cutler JA, Brittain E. Calcium and blood pressure: an epidemiological perspective. Am J Hypertens. 1990;3(8 pt 2):137S-146S.
   
4. McCarron DA, Morris CD. Blood pressure response to oral calcium in persons with mild to moderate hypertension: a randomized double-blind, placebo-controlled, cross-over trial. Ann Intern Med. 1985;103:825-831.
   
5. Saito K, Sano H, Furuta Y, Fukuzaki H. Effect of oral calcium on blood pressure response in salt-loaded borderline hypertensive patients. Hypertension. 1989;13:219-226. [Abstract/Free Full Text]
   
6. Lasaridis AN, Kaisis CN, Zananiri KI, Syrganis CD, Tourkantonis AA. Increased natriuretic ability and hypotensive effect during short-term high calcium intake in essential hypertension. Nephron. 1989;51:517-523. [Medline] [Order article via Infotrieve]
   
7. Belizán JM, Villar J, Gonzales L, Campodonico L, Bergel E. Calcium supplementation to prevent hypertensive disorders of pregnancy. N Engl J Med. 1991;325:1399-1405. [Abstract]
   
8. Hatton DC, McCarron DA. Dietary calcium and blood pressure in experimental models of hypertension. Hypertension. 1994;23:513-530. [Abstract/Free Full Text]
   
9. Resnick LM, Sosa RE, Corbett ML, Gertner JM, Sealey JE, Laragh JH. Effect of dietary calcium on sodium volume vs. renin-dependent forms of experimental hypertension. Trans Assoc Am Physicians. 1986;99:172-179. [Medline] [Order article via Infotrieve]
   
10. Arvola P, Ruskoaho H, Pörsti I. Effects of high calcium diet on arterial smooth muscle function and electrolyte balance in mineralocorticoid-salt hypertensive rats. Br J Pharmacol. 1993;108:948-958. [Medline] [Order article via Infotrieve]
    
11. Mäkynen H, Arvola P, Vapaatalo H, Pörsti I. High calcium diet effectively opposes the development of deoxycorticosterone-salt hypertension in rats. Am J Hypertens. 1994;7:520-528. [Medline] [Order article via Infotrieve]
    
12. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376. [Medline] [Order article via Infotrieve]
    
13. Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J. 1989;3:2007-2018. [Abstract]
    
14. Katusic ZS, Shepherd JT. Endothelium-derived vasoactive factors, II: endothelium-dependent contraction. Hypertension. 1991;18(suppl III):III-86-III-92.
    
15. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109-142. [Medline] [Order article via Infotrieve]
    
16. Lüscher TF, Dohi Y, Tschudi M. Endothelium-dependent regulation of resistance arteries: alterations with aging and hypertension. J Cardiovasc Pharmacol. 1992;19(suppl 5):S34-S42.
    
17. Clozel M, Kuhn H, Hefti F. Effects of angiotensin converting enzyme inhibitors and of hydralazine on endothelial function in hypertensive rats. Hypertension. 1990;16:532-540. [Abstract/Free Full Text]
    
18. Kähönen M, Mäkynen H, Arvola P, Pörsti I. Enhancement of arterial relaxation by long-term atenolol treatment in spontaneously hypertensive rats. Br J Pharmacol. 1994;112:925-933. [Medline] [Order article via Infotrieve]
    
19. Kähönen M, Mäkynen H, Arvola P, Wuorela H, Pörsti I. Arterial function after trichlormethiazide therapy in spontaneously hypertensive rats. J Pharmacol Exp Ther. 1995;272:1223-1230. [Abstract/Free Full Text]
    
20. Adeagbo ASO, Triggle CR. Varying extracellular [K⁺]: a functional approach to separating EDHF- and EDNO-related mechanism in perfused rat mesenteric arterial bed. J Cardiovasc Pharmacol. 1993;21:423-429. [Medline] [Order article via Infotrieve]
    
21. Aprigliano O, Hermsmeyer K. In vitro denervation of the portal vein and caudal artery of the rat. J Pharmacol Exp Ther. 1976;94:325-334.
    
22. Kähönen M, Arvola P, Wu X, Pörsti I. Arterial contractions induced by cumulative addition of calcium in hypertensive and normotensive rats: influence of endothelium. Naunyn Schmiedebergs Arch Pharmacol. 1994;349:627-636. [Medline] [Order article via Infotrieve]
    
23. DiPette DJ, Greilich PE, Kerr NE, Graham GA, Holland OB. Systemic and regional hemodynamic effects of dietary calcium supplementation in mineralocorticoid hypertension. Hypertension. 1989;13:77-82. [Abstract]
    
24. Fujii K, Tominaga M, Ohmori S, Kobayashi K, Koga T, Takata Y, Fujishima M. Decreased endothelium-dependent hyperpolarization to acetylcholine in smooth muscle of the mesenteric artery of spontaneously hypertensive rats. Circ Res. 1992;70:660-669. [Abstract/Free Full Text]
    
25. Hoshino J, Sakamaki T, Nakamura T, Kobayashi M, Kato M, Sakamoto H, Kurashina T, Yagi A, Sato K, Ono Z. Exaggerated vascular response due to endothelial dysfunction and role of the renin-angiotensin system at early stage of renal hypertension in rats. Circ Res. 1994;74:130-138. [Abstract/Free Full Text]
    
26. Fujii K, Ohmori S, Tominaga M, Abe I, Takata Y, Ohya Y, Kabayashi K, Fujishima M. Age-related changes in endothelium-dependent hyperpolarization in the rat mesenteric artery. Am J Physiol. 1993;265:H509-H516. [Abstract/Free Full Text]
    
27. Hayakawa H, Hirata Y, Suzuki E, Sugimoto T, Matsuoka H, Kikuchi K, Nagano T, Hirobe M, Sugimoto T. Mechanisms for altered endothelium-dependent vasorelaxation in isolated kidneys from experimental hypertensive rats. Am J Physiol. 1993;264:H1535-H1541. [Abstract/Free Full Text]
    
28. Jameson M, Dai F, Lüscher T, Skopec J, Diederich A, Diederich D. Endothelium-derived contracting factors in resistance arteries of young spontaneously hypertensive rats before development of overt hypertension. Hypertension. 1993;21:280-288. [Abstract/Free Full Text]
    
29. Fortes ZB, Nigro D, Scivoletto R, de Carvalho MHC. Indirect evidence for an endothelium-derived contracting factor released in arterioles of deoxycorticosterone acetate salt hypertensive rats. J Hypertens. 1990;8:1043-1048. [Medline] [Order article via Infotrieve]
    
30. Garland CJ, McPherson GA. Evidence that nitric oxide does not mediate the hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery. Br J Pharmacol. 1992;105:429-435. [Medline] [Order article via Infotrieve]
    
31. Chen G, Suzuki H, Weston AH. Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from rat blood vessels. Br J Pharmacol. 1988;95:1165-1174. [Medline] [Order article via Infotrieve]
    
32. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol. 1988;93:515-524.[Medline] [Order article via Infotrieve]
    
33. Eckman DM, Weinert JS, Buxton ILO, Keef KD. Cyclic GMP-independent relaxation and hyperpolarization with acetylcholine in guinea-pig coronary artery. Br J Pharmacol. 1994;111:1053-1060. [Medline] [Order article via Infotrieve]
    
34. Bray K, Quast U. Differences in the K⁺-channels opened by cromakalim, acetylcholine and substance P in rat aorta and porcine coronary artery. Br J Pharmacol. 1991;102:585-594. [Medline] [Order article via Infotrieve]
    
35. Bockman CS, Jeffries WB, Pettinger WA, Abel PW. Enhanced release of endothelium-derived relaxing factor in mineralocorticoid hypertension. Hypertension. 1992;20:304-313. [Abstract/Free Full Text]
    
36. Li J, Bukoski RD. A non-cyclo-oxygenase, non-nitric oxide relaxing factor is present in resistance arteries of normotensive but not spontaneously hypertensive rats. Am J Med Sci. 1994;307:7-14. [Medline] [Order article via Infotrieve]
    
37. Standen NB, Quayle JM, Davis NW, Brayden JE, Huang Y, Nelson TM. Hyperpolarizing vasodilators activate ATP-sensitive K⁺ channels in arterial smooth muscle. Science. 1989;245:177-180. [Abstract/Free Full Text]
    
38. Edwards G, Weston AH. Potassium channel openers and vascular smooth muscle relaxation. Pharmacol Ther. 1990;48:237-258. [Medline] [Order article via Infotrieve]
    
39. Waldron GJ, Garland CJ. Effect of potassium channel blockers on L-NAME insensitive relaxations in rat small mesenteric artery. Can J Physiol Pharmacol. 1994;72(suppl 1):115.
    
40. McPherson GA, Angus J. Evidence that acetylcholine mediated hyperpolarization of the rat small mesenteric artery does not involve the K⁺ channel opened by cromakalim. Br J Pharmacol. 1991;103:1184-1190. [Medline] [Order article via Infotrieve]
    
41. Tare M, Parkington IA, Coleman HA, Neild TO, Dusting GJ. Hyperpolarisation and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium. Nature. 1990;346:69-71. [Medline] [Order article via Infotrieve]
    
42. Bialecki RA, Stinson-Fisher C. K_Ca channel antagonists reduce NO donor-mediated relaxation of vascular and tracheal smooth muscle. Am J Physiol. 1995;268:L152-L159. [Abstract/Free Full Text]
    
43. Nakashima M, Vanhoutte PM. Isoproterenol causes hyperpolarization through opening of ATP-sensitive potassium channels in vascular smooth muscle of the canine saphenous vein. J Pharmacol Exp Ther. 1995;272:379-384. [Abstract/Free Full Text]
    
44. Beny JL, Pacicca C. Bidirectional electrical communication between smooth muscle and endothelial cells in the pig coronary artery. Am J Physiol. 1994;266:H1465-H1472. [Abstract/Free Full Text]
    
45. Perry PA, Webb CR. Sensitivity and adrenoreceptor affinity in the mesenteric artery of the deoxycorticosterone acetate hypertensive rat. Can J Physiol Pharmacol. 1988;66:1095-1099. [Medline] [Order article via Infotrieve]
    
46. Bruner CA. Vascular responsiveness in rats resistant to aldosterone-salt hypertension. Hypertension. 1992;20:59-66. [Abstract/Free Full Text]
    
47. Vanhoutte PM, Lüscher TF. Serotonin and the blood vessel wall. J Hypertens. 1986;4(suppl 1):S29-S30.
    
48. Li J, Bukoski RD. Endothelium-dependent relaxation of hypertensive resistance arteries is not impaired under all conditions. Circ Res. 1993;72:290-296.[Abstract/Free Full Text]
    

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