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Cardiovascular Research 1998 40(2):364-374; doi:10.1016/S0008-6363(98)00180-1
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Comparison of the effects of supplementation with whey mineral and potassium on arterial tone in experimental hypertension

Xiumin Wua, Jari-Petteri Tolvanena,d, Nina Hutri-Kähönena,e, Mika Kähönena,f, Heikki Mäkynena,h, Riitta Korpelab,c, Heikki Ruskoahoi, Kirsi Karjalac and Ilkka Pörstia,g,*

aUniversity of Tampere, Department of Pharmacological Sciences, P.O. Box 607, FIN-33101 Tampere, Finland
bResearch and Development Centre, Valio Ltd, Helsinki, Finland
cDepartment of Pharmacology and Toxicology, University of Helsinki, Helsinki, Finland
dDepartment of Clinical Chemistry, Tampere University Hospital, Tampere, Finland
eDepartment of Pediatrics, Tampere University Hospital, Tampere, Finland
fDepartment of Clinical Physiology, Tampere University Hospital, Tampere, Finland
gDepartment of Internal Medicine, Tampere University Hospital, Tampere, Finland
hDepartment of Medicine, Helsinki University Hospital, Helsinki, Finland
iDepartment of Pharmacology and Toxicology, Biocenter Oulu, University of Oulu, Oulu, Finland

* Corresponding author. Tel.: +358-3-215-6111; Fax: +358-3-215-6170; E-mail: blilpo@uta.fi

Received 14 January 1998; accepted 14 April 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of this work was to compare the effects of supplementation of rat chow diet with potassium (K+) and whey mineral concentrate (Whey), a diet rich in milk minerals, on blood pressure and arterial responses in vitro in spontaneously hypertensive rats (SHR). Methods: Thirty young SHR and twenty Wistar–Kyoto rats (WKY) were allocated into five groups: SHR, Whey-SHR, K+-SHR, WKY and Whey-WKY. Whey-supplementation was performed by adding 25% whey mineral concentrate to the chow, which in particular increased the intake of potassium (from 1.0% to 3.6%) and also that of calcium (from 1.0% to 1.3%) and magnesium (from 0.2% to 0.3%) in the rats. The K+-SHR were given extra potassium chloride (KCl) so that the final potassium content in the chow was 3.6%. Blood pressures were measured indirectly by the tail-cuff method. Responses of mesenteric arterial rings were examined in standard organ chambers after 12 study weeks. Results: During the 12-week study systolic blood pressures in control SHR increased steadily from 160 to about 230 mmHg, while supplementation with either Whey or potassium had a clear antihypertensive effect of about 50 mmHg in the hypertensive rats. Blood pressures in the WKY and Whey-WKY groups remained comparable during the whole study. In noradrenaline-precontracted arterial rings, endothelium-dependent relaxations to acetylcholine (ACh), as well as endothelium-independent relaxations to nitroprusside and isoprenaline were attenuated in untreated SHR, while all these dilatory responses were similarly improved by Whey and potassium supplementation. The cyclooxygenase inhibitor diclofenac, which reduces the synthesis of dilatory and constricting prostanoids, clearly enhanced the relaxation to ACh in untreated SHR, but was without effect in the other groups. In the presence of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester the relaxation to ACh was markedly reduced in all SHR groups, whereas in the two WKY groups, distinct relaxations to ACh were still present. The remaining responses were partially prevented by tetraethylammonium, an inhibitor of calcium-activated potassium channels, and the difference between untreated and potassium-supplemented SHR was abolished. When endothelium-mediated hyperpolarization of smooth muscle was prevented by precontracting the preparations with 50 mM KCl, only marginal differences were observed in relaxations to ACh between untreated SHR and the other groups. Interestingly, the impaired endothelium-independent relaxations to cromakalim, a hyperpolarizing vasodilator acting via ATP-sensitive potassium channels, were normalized by Whey mineral and potassium diets. Conclusion: Supplementation with Whey mineral and a comparable dose of potassium similarly opposed the development of experimental genetic hypertension, an effect which was associated with improved arterial dilatory properties. Both supplements augmented the hyperpolarization-related component of arterial relaxation, increased the sensitivity of smooth muscle to nitric oxide, and decreased the production of vasoconstrictor prostanoids. Therefore, the beneficial effects of the Whey diet could be attributed to increased intake of potassium in SHR.

KEYWORDS ANP; Arterial smooth muscle; Blood pressure; Dietary potassium; Dietary sodium; Endothelium; Hyperpolarization; Spontaneously hypertensive rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Potassium supplementation has been reported to lower blood pressure in essential [1–3]as well as in experimental hypertension [4, 5], although contradictory results have also been published [6–8]. The mechanisms by which potassium supplements reduce blood pressure are not fully understood, but both vascular and nonvascular explanations have been suggested. Potassium supplementation has been proposed to augment natriuresis in the absence of alterations in plasma renin [1, 4, 9], reduce sympathetic nervous activity [5, 10], improve endothelium-dependent vascular relaxation [8, 11, 12], stimulate Na+,K+-ATPase in adrenergic nerve terminals and vascular smooth muscle [13], increase prostacyclin synthesis [14], and enhance arterial compliance [11]. Also dietary calcium intake has been found to inversely correlate with blood pressure in humans and especially in different forms of experimental hypertension [15–17], and to enhance arterial relaxation in experimental hypertension [16–19]. Magnesium deficiency, in turn, has been shown to elevate blood pressure and increase peripheral vascular resistance in rats [20], whereas increased magnesium intake has been reported to reduce blood pressure in patients with essential hypertension [21].

Whey mineral concentrate supplementation, a diet rich in milk minerals, has been found to lower blood pressure and enhance urinary excretion of sodium in spontaneously hypertensive rats (SHR) [22], and to have a protective effect on endothelium-mediated control of arterial tone in mineralocorticoid–NaCl hypertension [23]. The major electrolyte in whey mineral is potassium, but it also increases the intakes of calcium and magnesium, all of which are regarded as beneficial nutritional factors in hypertension (see above). However, it has not been determined which of the above factors are responsible for the observed beneficial changes in arterial function following the whey mineral diet [23]. Therefore, the aim of the present work was to compare the vascular effects of whey mineral diet with those of potassium supplementation in SHR ingesting a moderate sodium diet. The effects of these supplements on blood pressure, and arterial contractile and dilatory responses in vitro were examined. Here we report for the first time that the beneficial effects of whey mineral diet on arterial dilation in hypertension can be attributed to the increased intake of potassium.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals, experimental design and atrial natriuretic peptide (ANP) determinations
Male SHR (Okamoto–Aoki strain, n=30) and age-matched Wistar–Kyoto rats (WKY) (n=20) were obtained from Møllegaard's Breeding Centre (Ejby, Denmark), which uses the standard brother–sister mating system [24]for the production of their inbred rats. The animals were housed four to a cage in a standard experimental animal laboratory (illuminated 06 h 00 min–18 h 00 min, temperature +22°C), and provided standard chow (Altromin Nr. 1314, Chr. Petersen, Ringsted, Denmark) and drinking fluid (tap water) ad libitum. The systolic blood pressures of conscious animals held in plastic restrainers were measured at +28°C using the tail-cuff method (Model 129 Blood Pressure Meter; IITC, Woodland Hills, CA, USA) with an acclimation period of about 30 min preceding the measurements. At 7 weeks of age, the SHR were divided into three groups (SHR, Whey-SHR, K-SHR) and the WKY into two groups (WKY, Whey-WKY) of equal mean systolic blood pressures (n=10).

The diets in the study were as follows. In the Whey-SHR and Whey-WKY groups, the chow contained 25% whey mineral concentrate (w/w; Valio SUVALTM whey salt, Valio, Helsinki, Finland). Whey mineral concentrate contained most of the milk minerals and low molecular weight whey protein, and it was prepared from lactose-rich solution which is generated from cow milk as a side product in the process of making cheese in dairy industry. The amount of lactose and protein in the solution was reduced by crystallization, ultrafiltration and chromatographic separation, whereafter the whey products were dried using standard dairy equipment. The composition of the whey mineral concentrate was (%): potassium 11.1, calcium 2.17, magnesium 0.37, sodium 3.94, phosphorus 1.94, water 1.8, protein (low molecular weight whey protein) 31.8, lactose 19.3, chloride 13.4, nitrate 0.15, sulphate 0.6, and organic acids 11.0 (including lactic acid 0.8, orotic acid 0.06, citric acid 4.0). In addition, it contained minute amounts (mg/kg) of riboflavin 5.6, copper 1.0, and iron 7.7. The major constituents of the diets in the study groups are summarized in Table 1. The dietary contents of potassium in the control SHR and K-SHR groups were 1% potassium and 3.6% potassium, respectively. In the SHR, K-SHR and control WKY groups the sodium content of the diet was adjusted to match that in the whey mineral concentrate-supplemented groups by the addition of NaCl (22.5 g/kg). Thus, all diets contained 1.1% of Na+. Control WKY received the same diet as control SHR. The extra potassium and sodium were added to standard food pellets (Altromin no. 1314, Chr. Petersen) as KCl and NaCl, respectively. Without the moderate addition of NaCl this laboratory chow can be regarded as a low Na+ diet since it only contains 0.2% Na+, and thus it does not well correspond to the diet consumed in most industrialized societies [25].


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  		Table 1 Compositions of the NaCl-enriched normal chow and the chow containing 25% whey mineral concentrate showing the major differences betwen these two diets

 
The supplementations and indirect blood pressure measurements were continued for 12 more weeks until the animals were 19 weeks old. Thereafter, the rats were anaesthetized by intraperitoneal administration of urethane (1.3 g/kg) and exsanguinated. Blood samples were drawn into chilled tubes on ice containing 2.7 mM EDTA for plasma ANP assays, whereafter the samples were centrifuged, and plasma stored at –70°C until analysis. ANP was extracted from plasma as previously described [26]. The plasma samples were incubated in duplicates of 100 µl with 100 µl of the specific rabbit ANP antiserum in the final dilution of 1:2.5·104. ANP was determined by radioimmunoassay as previously described [26]. The hearts were removed and weighed, and the superior mesenteric arteries carefully excised and cleaned of adherent connective tissue. The experimental design of the study was approved by the Animal Experimentation Committee of the University of Tampere, Finland. Moreover, the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.2 Mesenteric arterial responses in vitro
Four successive standard sections (3 mm in length) of the mesenteric artery from each animal were cut, beginning 5 mm distally from the mesenteric artery–aorta junction. The endothelium of the most distal ring was removed by gently rubbing the preparation with a jagged injection needle [27]. The rings were placed between stainless steel hooks (diameter 0.3 mm) and suspended in an organ bath chamber (volume 20 ml) in physiological salt solution (PSS) (pH 7.4) of the following composition (mM): NaCl 119.0, NaHCO3 25.0, glucose 11.1, CaCl2 1.6, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, and aerated with 95% O2 and 5% CO2. The rings were initially equilibrated for 30 min at 37°C with a resting tension of 1.5 g. The force of contraction was measured with an isometric force–displacement transducer and registered on a polygraph (FT03 transducer and model 7E Polygraph; Grass Instrument Co., Quincy, MA, USA). The presence of intact endothelium in vascular preparations was confirmed by clear relaxation responses to 1 µM acetylcholine (ACh) in rings that were precontracted with 1 µM noradrenaline (NA), and the absence of endothelium by the lack of this response. If any relaxation was observed in endothelium-denuded rings, the endothelium was further rubbed.

2.2.1 Receptor and depolarization-mediated contractions
After the equilibration period, concentration–response curves for NA and KCl were cumulatively determined. The next concentration of the agonist was added only when the previous level of the response was stable. After the maximal response had been reached, rings were rinsed with PSS and allowed a 20 min recovery period at resting tension. The contractions were then elicited in the presence of 3 µM diclofenac (cyclooxygenase inhibitor), and after this 0.1 mM NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor] was also added to the bath and responses to NA and KCl were retested. A 30 min period was always allowed after a new drug was introduced. In solutions containing high concentrations of potassium (20–125 mM), NaCl was replaced with KCl on an equimolar basis.

2.2.2 Endothelium-dependent relaxation after receptor-mediated precontraction
After the equilibration period, vascular responses to ACh and ADP were examined. The rings were precontracted with 1 µM NA, and after the contraction had fully developed increasing concentrations of the relaxing agent were cumulatively added to the organ bath. Responses to ACh were then elicited in the presence of 3 µM diclofenac, after this 0.1 mM L-NAME was also added to the bath and responses to ACh were re-tested. Finally, 1 mM tetraethylammonium [TEA; inhibitor of large-conductance Ca2+-activated potassium channels (KCa)] was added to the bath and the responses were again tested.

2.2.3 Endothelium-dependent relaxation after depolarization-mediated precontraction
After the equilibration period, vascular responses to ACh and ADP were examined. The rings were precontracted with 50 mM KCl, and increasing concentrations of the relaxing agent were cumulatively added to the organ bath. Responses to ACh were then elicited in the presence of 3 µM diclofenac, after this 0.1 mM L-NAME was also added to the bath and responses to ACh were re-tested.

2.2.4 Endothelium-independent relaxations, and calcium sensitivity during depolarization
After removal of endothelium and the equilibration period, cumulative relaxations to sodium nitroprusside were examined in rings precontracted with 1 µM NA and 50 mM KCl. The responses to isoprenaline and cromakalim were then cumulatively determined after precontraction with 1 µM NA. In additional experiments vasodilatation to isoprenaline was examined after precontractions induced by 50 mM KCl. We found that the endothelium-independent relaxations to isoprenaline were abolished in arterial rings from both SHR and WKY when the precontractions were elicited by depolarization with KCl.

The maximal contractions to NA and KCl were presented in grams and related to tissue dry weight (g/mg). The EC50 values for NA and KCl were calculated with a computer program and presented as the negative logarithm (pD2), which values were also used in the statistical analysis. The relaxations to ACh, nitroprusside, isoprenaline and cromakalim were presented as a percentage of the pre-existing contraction force.

2.3 Compounds
The following drugs were used: acetylcholine chloride, sodium salt of adenosine 5'-diphosphate, (±)-cromakalim, (±)-isoprenaline hydrochloride, NG-nitro-L-arginine methyl ester hydrochloride, tetraethylammonium chloride (Sigma, St. Louis, MO, USA), diclofenac (Voltaren injection solution; Ciba–Geigy, Basel, Switzerland), EDTA, L-noradrenaline L-hydrogentartrate (Fluka, Buchs, Switzerland) and sodium nitroprusside (Merck, Darmstadt, Germany). The stock solutions of the compounds used in the in vitro studies were dissolved in distilled water, with the exception of nifedipine (in 50% ethanol). All solutions were freshly prepared before use and protected from light.

2.4 Analysis of results
Statistical analysis was carried out by one-way analysis of variance (ANOVA) supported by Bonferroni confidence intervals in the case of pairwise between-group comparisons (Tables 2 and 3Go). When the data consisted of repeated observations at successive time points ANOVA for repeated measurements was applied (comparisons within and between panels in Figs. 1–4GoGoGo). Differences were considered significant when P<0.05. All results were expressed as mean±SEM. The data were analyzed with BMDP statistical software.


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  		Table 2 Physiological variables in experimental groups at close of the study

 

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  		Table 3 Parameters of contractile responses of isolated endothelium-intact mesenteric arterial rings

 

Figure 1
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  		Fig. 1 Line graphs show blood pressures in untreated spontaneously hypertensive rats (SHR, bullet), potassium-supplemented SHR ({diamondsuit}), Whey-supplemented SHR ({blacktriangleup}), untreated Wistar–Kyoto (WKY; {circ}), and Whey-supplemented WKY rats ({Delta}). Values represent mean±SEM, n=10–12 in each group; * P<0.05, ANOVA for repeated measurements.

 

Figure 2
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  		Fig. 2 Line graphs show relaxations to acetylcholine in isolated endothelium-intact mesenteric arterial rings from untreated spontaneously hypertensive rats (SHR, bullet), potassium-supplemented SHR ({diamondsuit}), Whey-supplemented SHR ({blacktriangleup}), untreated Wistar–Kyoto (WKY; {circ}), and Whey-supplemented WKY rats ({Delta}). The relaxations were induced after precontraction with 1 µM noradrenaline in the absence (a) and presence (b) of 3 µM diclofenac, in the presence of diclofenac and 0.1 mM NG-nitro-L-arginine methyl ester (L-NAME; c), and in the presence of diclofenac, L-NAME and 1 mM tetraethylammonium (d). Values represent mean±SEM, n=8–10 in each group; * P<0.05, ANOVA for repeated measurements.

 

Figure 3
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  		Fig. 3 Line graphs show relaxations to acetylcholine in isolated endothelium-intact mesenteric arterial rings from untreated spontaneously hypertensive rats (SHR, bullet), potassium-supplemented SHR ({diamondsuit}), Whey-supplemented SHR ({blacktriangleup}), untreated Wistar–Kyoto (WKY; {circ}), and Whey-supplemented WKY rats ({Delta}). The relaxations were induced after precontraction with 50 mM KCl in the absence (a) and presence (b) of 3 µM diclofenac, and in the presence of diclofenac and 0.1 mM NG-nitro-L-arginine methyl ester (c). Values represent mean±SEM, n=8–10 in each group; * P<0.05, ANOVA for repeated measurements.

 

Figure 4
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  		Fig. 4 Line graphs show relaxations to nitroprusside after precontraction with 1 µM noradrenaline (a) and 50 mM KCl (b), and relaxations to cromakalim (c) and isoprenaline (d) after precontraction with 1 µM noradrenaline. All responses were elicited in isolated endothelium-denuded mesenteric arterial rings from untreated spontaneously hypertensive rats (SHR, bullet), potassium-supplemented SHR ({diamondsuit}), Whey-supplemented SHR ({blacktriangleup}), untreated Wistar–Kyoto (WKY, {circ}), and Whey-supplemented WKY rats ({Delta}). Values represent mean±SEM, n=8–10 in each group; * P<0.05, ANOVA for repeated measurements.

 

   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Whey mineral diet
The compositions of the diets are shown in Table 1. The content of potassium was elevated by 3.6-fold, while that of calcium, magnesium, and phosphorus were approximately 25–30% higher in the whey mineral concentrate diet when compared with the control chow. The whey mineral-enriched chow contained an almost twofold higher amount of chloride, and somewhat more protein and less fat than the control food. Thus, the most prominent nutritional alteration in the Whey-WKY and Whey-SHR groups was the marked increase in the proportion of potassium in the diet.

3.2 Blood pressure, plasma ANP, and heart and body weights
The systolic blood pressures in SHR were already higher at the beginning of the study than in WKY. During the 12-week-long follow up, supplementation with either whey and potassium markedly and comparably attenuated the development of hypertension in SHR. Nevertheless, blood pressures still remained higher in all SHR groups than in the WKY groups (Fig. 1).

The concentration of ANP in plasma was comparable in the untreated SHR and WKY groups. In contrast, high potassium and whey diets clearly elevated plasma ANP in SHR (Table 2). The heart weights in WKY and in SHR receiving the supplements were corresponding and lower than in control SHR (Table 2). However, all supplemented hypertensive rats gained somewhat less weight than control SHR and WKY, and the heart/body weight ratios did not differ between the SHR groups. In contrast, the heart/body weight ratio was clearly lower in the normotensive WKY when compared with all SHR groups (Table 2). No signs of compromised well-being of the animals were observed by our experienced experimental animal laboratory staff. Chow intakes were comparable in the study groups (data not shown).

3.3 Mesenteric arterial responses in vitro
The endothelium-mediated relaxations of NA-precontracted mesenteric arterial rings to ACh were markedly impaired in control SHR when compared with the WKY group (Fig. 2). The response to ACh was clearly and comparably improved in SHR by either whey or potassium supplementation, but the relaxation still remained less marked than in WKY. Cyclooxygenase inhibition with diclofenac enhanced the relaxation elicited by ACh in control SHR, but did not affect the response in the other groups (comparison between panel a and b). However, the relaxation to ACh remained diminished in control SHR when compared with the rest of the groups. The addition of the NO synthase inhibitor L-NAME (in the presence of diclofenac) to the organ bath attenuated the response to ACh in all groups (comparison between panel b and c), and only a minute relaxation was observed in the control SHR group. TEA, an inhibitor of large-conductance KCa [28], reduced the diclofenac and L-NAME-resistant relaxation induced by ACh in all groups (comparison between panel c and d), and almost completely abolished them in control SHR, Whey-SHR and K-SHR, while the WKY groups still showed distinct relaxations (Fig. 2).

When endothelium-mediated hyperpolarization of arterial smooth muscle was eliminated by precontracting arterial rings with 50 mM KCl, the relaxations elicited by ACh in the absence and presence of diclofenac were again impaired in control SHR when compared with the other groups (Fig. 3). Moreover, in the presence of diclofenac the responses to ACh were somewhat augmented in control SHR, Whey-SHR and K-SHR when compared with the responses elicited in the absence of diclofenac. The addition of L-NAME markedly attenuated the relaxations to ACh in all groups (comparison between panel b and c), and almost completely abolished them in control SHR, while the Whey and K-SHR showed minute diclofenac, L-NAME and depolarization-resistant relaxations to ACh (Fig. 3). In addition, the results on arterial relaxation which were obtained with ADP, another endothelium-dependent agonist, were corresponding to those observed with ACh in both NA and KCl-precontracted vascular rings (data not shown).

The relaxation of NA or KCl-precontracted endothelium-denuded rings to nitroprusside, an agent that mediates arterial relaxation via the formation of exogenous NO, was markedly impaired in control SHR when compared with the WKY. These responses were restored in SHR by the supplementations. Furthermore, the supplementations also clearly improved the vasorelaxation elicited by the β-adrenoceptor agonist isoprenaline and the ATP-sensitive potassium channel opener cromakalim (KATP; Fig. 4).

Control SHR were more sensitive to NA- and KCl-induced contractions than the supplemented SHR (Table 3). The addition of diclofenac reduced sensitivity to NA and KCl in all SHR, and abolished the difference between control and supplemented SHR. The addition of L-NAME did not significantly affect contractile sensitivity to NA or KCl in this study (Table 3). The maximal force generation in response to NA and KCl in the absence and presence of diclofenac, and in the presence of diclofenac and L-NAME, was comparable in the SHR groups (Table 3). The contractile force generation in response to 1 µM NA did not differ between endothelium-intact or endothelium-denuded arterial rings in any group (data not shown).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The development of hypertension in SHR is not sodium dependent, but moderate changes in blood pressure can be achieved by alterations in sodium intake [29]. Accordingly, the present SHR on a moderate sodium diet developed a more severe hypertension than in previous investigations [30, 31]. Whey mineral diet and potassium supplementation have previously been reported to moderately lower blood pressure in SHR [14, 22], the findings of which were confirmed in the present study. Thus, the antihypertensive effect of neither whey nor potassium was compromised by increased sodium intake, rather the effects appeared more pronounced when compared with earlier reports [14, 22].

The potassium content in whey diet was elevated 3.6-fold (thus being the major nutritional alteration), while that of calcium and magnesium were approximately 30% higher when compared with the control chow. Moreover, the whey mineral diet also increased the intakes of chloride, phosphorus, organic acids, and protein, and reduced the intake of fat. Increased intakes of potassium, calcium and magnesium have been regarded as beneficial nutritional alterations in hypertension [1, 20, 32], whereas enhanced chloride intake has been found to elevate blood pressure in both hypertensive humans and experimental animals [33]. In the present study the effects on blood pressure of whey mineral diet with those of potassium supplementation in SHR were very similar. This suggests that the beneficial effects of whey mineral diet on high blood pressure can be attributed to the increased intake of potassium.

The present results did not show differences in plasma ANP between SHR and WKY, even though hypertension-induced ventricular hypertrophy is associated with increased synthesis and release of ANP [34]. This discrepancy can be explained by the moderately elevated sodium content of the present diet, which results in stimulated ANP release from the heart [34]. Interestingly, high potassium diet has been reported to enhance natriuresis [13], and in this study whey and potassium supplementations elevated plasma ANP in SHR, which may partially explain the observed changes in blood pressure in these animals. Previously, potassium supplementation have been shown to attenuate sodium retention and weight gain in essential hypertensive patients on a high sodium diet [35]. Correspondingly, in this study both SHR receiving the whey mineral and potassium diets gained less weight than control SHR.

The heart/body weight ratios were not decreased by the present supplements. It is known that cardiac hypertrophy in SHR is not only governed by the level of blood pressure, but also by enhanced cellular responses to growth factors such as endothelin [36]. More importantly, sodium intake is a strong and blood pressure-independent determinant of cardiac hypertrophy in experimental animals and even in human subjects [37, 38]. Thus, the increased sodium content of the present diet provides an explanation why the heart/body weight ratios remained comparable in all SHR groups despite the clear differences in blood pressure.

Previously, endothelium-dependent dilation has been shown to be impaired in hypertension [30, 39]. Interestingly, high potassium diet has been found to augment endothelium-dependent relaxations in hypertensive rats via a mechanism not related to alterations in blood pressure [8]. In the present investigation, supplementation with either whey or potassium was accompanied by enhancement of endothelium-mediated relaxation in SHR, which thus agrees with previous findings [8, 23]. Since the effects of whey mineral diet and potassium supplementation on endothelium-mediated dilatation were similar in SHR in the present study, the beneficial effects of whey mineral diet on endothelial function may be attributed to the increased intake of potassium.

Endothelium-derived contractile factors (EDCF), the production of which depends on cyclooxygenase, have been suggested to be involved in impaired endothelium-mediated vasomotion in SHR [40]. In the present study diclofenac improved the dilator response to ACh in control SHR, thus supporting the concept whereby EDCF were released from the endothelium of these animals. On the other hand, diclofenac was without significant effect on the response to ACh in all mineral-supplemented rats, suggesting that whey and potassium supplements diminished the production of EDCF in SHR. However, reduced EDCF release did not entirely account for the enhanced relaxations to ACh in the whey- and potassium-supplemented rats.

Inhibition of NO synthase by L-NAME effectively diminished the relaxations to ACh in all study groups. Since the endothelium-mediated response in control SHR was nearly abolished by L-NAME, it was predominantly mediated via NO, whereas all other groups showed distinct diclofenac- and L-NAME-resistant relaxations, suggesting that endothelial products other than NO were mediating the enhanced response to ACh. Recent investigations have indicated that endothelium-mediated relaxations which remain resistant to both NO synthase and cyclooxygenase inhibitions are mediated by another vasoactive autacoid, the endothelium-derived hyperpolarizing factor (EDHF) [28]. The chemical characteristics of EDHF remain unknown, but functionally this factor is a potassium channel opener [28], the action of which can be inhibited by potassium channel blockers or by depolarizing the cell membrane with high concentrations of KCl [41]. Interestingly, endothelium-dependent hyperpolarization has been reported to be impaired in SHR [42].

The KCa have been found to be active during EDHF-induced relaxation [41], and apamin (an inhibitor of small-conductance KCa) has been shown to significantly reduce the L-NAME-insensitive dilatation in the rat mesenteric artery, and apamin together with charybdotoxin (an inhibitor of large-conductance KCa), to completely abolish these relaxations [43]. In the present study, inhibition of large-conductance KCa by TEA reduced the diclofenac and L-NAME-resistant relaxation in all groups, and abolished the difference between control SHR and the group supplemented with potassium. This suggests that endothelium-mediated hyperpolarization via KCa was enhanced after high potassium diet in SHR. When endothelium-mediated hyperpolarization of arterial smooth muscle was eliminated by precontractions with KCl (as described by Adeagbo and Triggle [41]), we found only small differences in ACh-induced relaxations between control SHR and the other groups, and the responses were similar in all mineral-supplemented SHR and the WKY. Collectively these findings support the view that supplementations with whey and potassium normalized the endothelium-mediated relaxations in SHR via enhanced hyperpolarization mechanisms.

Arterial relaxations to nitroprusside were augmented in both supplemented SHR groups, indicating enhanced sensitivity to NO following increased whey and potassium intakes. Recently, NO has also been reported to activate potassium channels in vascular smooth muscle [44]. However, the fact that the relaxations to nitroprusside were similarly improved whether the precontractions were induced by NA or KCl suggests that NO-mediated hyperpolarization was not playing a significant role in this response. Previously, the relaxation to nitroprusside has been reported to remain unaffected after potassium supplementation in stroke-prone SHR [8].

Vasodilation to isoprenaline is predominantly endothelium-independent via the stimulation of β-adrenoceptors and the subsequent increase in cyclic AMP in smooth muscle [45]. However, isoprenaline also hyperpolarizes blood vessels via KATP and KCa in smooth muscle [46, 47]. Thus, enhanced hyperpolarization could partially explain the improved relaxation to isoprenaline in the mineral-supplemented SHR in this study. Indeed, the present results whereby the responses to cromakalim, an opener of KATP, were improved in all supplemented groups further support the view of augmented arterial hyperpolarization following increased whey and potassium intakes. The fact that the above agonists mediate vasodilation via three different mechanisms suggests that the improvement of general vascular relaxation properties (e.g. regulation of intracellular calcium) may also have played a role in the enhanced endothelium-dependent relaxation in the supplemented groups.

Arterial contractile sensitivity to NA and KCl was lower in both supplemented SHR when compared with control SHR. This difference in contractile sensitivity between control and mineral-supplemented SHR was abolished by cyclooxygenase inhibition, suggesting that diminished release of vasoconstrictor prostanoids was responsible for the lower sensitivity to NA and KCl after whey and potassium supplementations. However, the increased contractile sensitivity to NA observed in control SHR may also have resulted from decreased β-adrenoceptor responsiveness, since the β-adrenoceptor-mediated vasodilatation was found to be attenuated in control SHR.

In conclusion, the present results showed that dietary whey and potassium supplements comparably lowered blood pressure in SHR on a moderate sodium diet. This effect was accompanied by comparably enhanced endothelium-dependent and -independent arterial relaxation and attenuated receptor- and depolarization-mediated contractile sensitivity in both supplemented groups. The vascular mechanisms underlying the antihypertensive effects of these supplements may involve improved arterial hyperpolarization, increased sensitivity of smooth muscle to NO, and decreased production of vasoconstrictor prostanoids. Finally, since the effects of these supplementations on arterial function were very similar, the beneficial effects of whey mineral diet on arterial dilation in hypertension may largely be attributed to the increased intake of potassium.

Time for primary review 23 days.


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


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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