Arterial Responses in vitro and Plasma Digoxin Immunoreactivity after Losartan and Enalapril Treatments in Experimental Hypertension

C Pharmacology & Toxicology 2000, 86, 36–43. Printed in Denmark . All rights reserved

Copyright C ISSN 0901-9928

Arterial Responses in vitro and Plasma Digoxin Immunoreactivity after Losartan and Enalapril Treatments in Experimental Hypertension Jarkko Kalliovalkama1, Mika Ka¨ho¨nen1,3, Jari-Petteri Tolvanen1,4, Xiumin Wu1, Juha Voipio6, Anu Pekki2, Peter A. Doris7, Pauli Ylitalo1 and Ilkka Po¨rsti1,5 1

Departments of Pharmacological Sciences and 2Anatomy, University of Tampere, Finland; Departments of 3Clinical Physiology, 4Clinical Chemistry and 5Internal Medicine, Tampere University Hospital, Finland; 6Department of Biosciences, Division of Animal Physiology, University of Helsinki, Finland; 7Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX, USA (Received June 1, 1999; Accepted August 23, 1999)

Abstract: Treatment with the angiotensin-converting enzyme inhibitor, quinapril, has been shown to normalize increased dihydropyridine sensitivity and impaired potassium relaxation, characteristic features of arterial smooth muscle in spontaneously hypertensive rats, and also reduce the concentration of plasma digoxin-like immunoreactivity in these animals. However, whether angiotensin II receptor blocker therapy can beneficially influence these variables is not known. Therefore, we compared the effects of 10-week losartan and enalapril treatments (15 and 4 mg/kg/day, respectively) on functional responses of mesenteric arterial rings in spontaneously hypertensive rats and Wistar-Kyoto rats. Both losartan and enalapril normalized blood pressure, cardiac mass, and media to lumen ratio without significantly changing the media crosssectional area in the mesenteric artery of spontaneously hypertensive rats (i.e. induced outward remodelling). The inhibitory effect of the calcium entry blocker nifedipine on calcium-evoked contractions was similar and less marked in arterial preparations from Wistar-Kyoto rats and losartan- and enalapril-treated spontaneously hypertensive rats than in those from untreated spontaneously hypertensive rats. Furthermore, the relaxations of arterial rings induced by the return of potassium to the organ bath (upon precontractions elicited by potassium-free solution) were used to evaluate the function of vascular Naπ,Kπ-ATPase. The rate of potassium relaxation was faster in losartan- and enalapril-treated spontaneously hypertensive rats and all Wistar-Kyoto groups than in untreated spontaneously hypertensive rats, and the response was effectively inhibited by the sodium pump inhibitor ouabain. Both treatments especially augmented the ouabain-sensitive part of the potassium-relaxation in spontaneously hypertensive rats, indicating the involvement of the sodium pump in this response. However, no significant changes in plasma digoxin-like immunoreactivity were observed. In conclusion, the outward remodelling following long-term AT1-receptor blockade and angiotensin-converting enzyme inhibition in spontaneously hypertensive rats was associated with normalization of the increased dihydropyridine sensitivity of arteries. Both losartan and enalapril treatments also augmented arterial potassium relaxation in spontaneously hypertensive rats, suggesting enhanced function of Naπ,Kπ-ATPase, but this effect could not be attributed to changes in circulating sodium pump inhibitor concentration.

The antihypertensive action of angiotensin-converting enzyme inhibitors is based on the inhibition of angiotensin II formation, while angiotensin II receptor antagonists compete with angiotensin II at the AT1 receptor. Treatment with these classes of drugs has normalized the structure of mesenteric resistance arteries in spontaneously hypertensive rats by inducing outward eutrophic remodelling (Rizzoni et al. 1998). AT1 receptor blockade and angiotensin-converting enzyme inhibition have also attenuated vasoconstrictor reactivity in experimental hypertension (Mulvany et al. 1991; Arvola et al. 1993; Major et al. 1993; Soltis 1993). The changes in arterial tone following angiotensin-converting enzyme inhibition have been attributed to a normalized function of voltage-dependent calcium channels and vascuAuthor for correspondence: Jarkko Kalliovalkama, University of Tampere, Medical School, Department of Pharmacological Sciences, P.O. Box 607, FIN-33101 Tampere, Finland (fax π358 3 215 6170, e-mail: jarkko.kalliovalkama/uta.fi).

lar structure, sympathoinhibitory action, and enhanced endothelial nitric oxide and prostacyclin release (Major et al. 1993; Cachofeiro et al. 1995; Ka¨ho¨nen et al. 1996). In contrast to angiotensin-converting enzyme inhibition, the influences of AT1-receptor antagonism on vasoconstrictor responses and voltage-dependent calcium channels in experimental hypertension have not been studied in detail. The altered function of Naπ,Kπ-ATPase may play a role in the pathogenesis of hypertension, and elevated levels of circulating sodium pump inhibitors have been found in hypertensive patients (Hamlyn & Manunta 1992) and experimental animals (Wauquier et al. 1988; Doris 1994). This digitalis-like factor is a natriuretic agent, but it depolarizes arterial smooth muscle via Naπ,Kπ-ATPase inhibition, and thus elevates peripheral resistance (Zhu et al. 1994). Quinapril treatment has been found to decrease plasma digoxinlike immunoreactivity in spontaneously hypertensive rats (Ka¨ho¨nen et al. 1996). Furthermore, potassium-induced vasodilatation, a response which evaluates the function of

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LOSARTAN, ENALAPRIL AND ARTERIAL RESPONSES

Naπ,Kπ-ATPase, is impaired in mesenteric arterial rings from spontaneously hypertensive rats (Arvola et al. 1992), and is augmented by long-term angiotensin converting enzyme inhibition (Arvola et al. 1993; Ka¨ho¨nen et al. 1996). We tested the hypothesis whether the long-term effects of AT1-receptor blockade with losartan on vascular structure are associated with normalization of the increased dihydropyridine sensitivity and the impaired potassium relaxation in arterial smooth muscle of spontaneously hypertensive rats. We also examined whether the possible changes in sodium pump function are associated with alterations in the concentration of plasma digoxin-like immunoreactivity. For comparison, treatment with the angiotensin-converting enzyme inhibitor, enalapril, was included in the study. WistarKyoto rats served as the normotensive controls. Materials and Methods Animals and experimental design. Male spontaneously hypertensive rats (Okamoto-Aoki strain) and age-matched Wistar-Kyoto rats were obtained from Møllegaard’s Breeding Centre, Ejby, Denmark. The animals were housed four to a cage in an experimental animal laboratory (illuminated 6 a.m.–6 p.m., temperature 22æ) with free access to drinking fluid and food pellets (Ewos, So¨derta¨lje, Sweden). The systolic blood pressures of conscious animals were measured at 28æ by the tail-cuff method (Model 129 Blood Pressure Meter; IITC Inc., Woodland Hills, CA, USA). At 7 weeks of age both spontaneously hypertensive rats and Wistar-Kyoto rats were divided into three groups of equal mean systolic blood pressures. Thereafter, spontaneously hypertensive rats and Wistar-Kyoto rats were treated with losartan or enalapril (nΩ10 in all 4 treatment groups). The drugs were given in drinking water in light-proof bottles (average doses 15 and 4 mg/kg/day, respectively), fresh drug solutions being prepared daily. Untreated spontaneously hypertensive rats (nΩ15) and Wistar-Kyoto rats (nΩ15) were kept on normal drinking fluid. Losartan and enalapril therapies continued for 10 more weeks. The indirect blood pressure measurements were performed every two weeks. Losartan and enalapril administrations were withdrawn 1 day before the rats were anaesthetized by intraperitoneal administration of urethane (1.3 g/kg) and exsanguinated. 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.

Mesenteric arterial responses in vitro. Three successive standard sections (3 mm in length) of the superior mesenteric artery were cut from each animal. In the most distal ring the endothelium was left intact, and from the first two pieces it was removed (Arvola et al. 1992). The rings were placed between small hooks and suspended in an organ bath chamber in a physiological salt solution (pH 7.4) containing (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 equilibrated for 11/2 hr at 37æ with a resting preload of 1.5 g. The force of contraction was measured with an isometric force-displacement transducer (FT 03 transducer and Model 7 E Polygraph; Grass Instrument Co., Quincy, MA, USA). The presence of intact endothelium in vascular preparations was confirmed by a clear relaxation to 1 mM acetylcholine in 1 mM noradrenaline-precontracted rings, and the absence of endothelium by the lack of this response. Vascular preparation 1. Concentration-response curves of endothelium-denuded rings for KCl were determined in the absence and presence of 1 mM phentolamine and 10 mM atenolol (Ka¨ho¨nen et al. 1993). In solutions containing high concentrations of potassium (20–125 mM), NaCl was replaced with KCl on an equimolar basis. The rings were rinsed with Ca2π-free physiological salt solution, and repeatedly contracted with noradrenaline in Ca2π-free medium to deplete the cellular calcium stores. Subsequently, the preparations were challenged with KCl (125 mM) at 0 mM calcium in the presence of 1 mM phentolamine and 10 mM atenolol, whereafter calcium was cumulatively added (0.01–2.5 mM) and the contraction registered. After the maximal response, the rings were rinsed with Ca2π-free physiological salt solution and allowed a 20 min. recovery. Thereafter the same scheme was elicited in the presence of 0.5 nM nifedipine. Due to the light sensitivity of nifedipine, the cuvettes were protected from light, and lighting was reduced to a minimum. Vascular preparation 2. The endothelium-denuded ring was contracted with 125 mM KCl (reference response), and after 30 min. exposed to Kπ-free buffer solution (pH 7.4), which was prepared by substituting KH2PO4 and KCl with NaH2PO4 and NaCl, respectively. The omission of potassium induced gradual contractions in all vascular rings. Once the contraction had reached a plateau, 1 mM potassium was readded and the subsequent relaxation reflecting the action of Naπ,Kπ-ATPase was registered (Arvola et al. 1992). Then 30 min. later the contractions induced by Kπ-free buffer solution and relaxation induced by potassium repletion were repeated in the presence of 1 mM ouabain. Vascular preparation 3. Concentration-response curves to 5-hydroxytryptamine were determined in endothelium-intact rings. The responses to 5-hydroxytryptamine were also elicited in the presence

Table 1. Experimental group data at the end of the study (week 17) and systolic blood pressure both at the beginning and at the end of the study (weeks 7 and 17). Variable Body weight (g) Heart weight (mg) Heart-body weight ratio (mg/g) Media to lumen ratio Media cross-sectional area (mm2) Preparation weight (mg) Heart rate (beats/min.) Systolic blood pressure (mmHg) Week 7 Week 17

SHR

LSHR

ESHR

303∫2 859∫42 2.84∫0.14 0.17∫0.02 22067∫3290 0.35∫0.04 338∫6

WKY

305∫11 708∫28*† 2.34∫0.12*† 0.11∫0.01† 20198∫3378 0.29∫0.02† 331∫3

LWKY

311∫6 720∫42*† 2.32∫0.15*† 0.11∫0.01† 26422∫3358 0.39∫0.06† 336∫7

EWKY

305∫3 1163∫410* 3.83∫0.15* 0.34∫0.07* 30630∫4531 0.59∫0.03* 349∫7

295∫5 780∫34† 2.64∫0.11† 0.17∫0.04† 25676∫5345 0.38∫0.05† 326∫4†

294∫5 780∫44† 2.66∫0.15† 0.11∫0.01† 33839∫4378 0.36∫0.04† 326∫7†

109∫10 143∫8

110∫10† 135∫6†

110∫10† 134∫11†

133∫9* 234∫9*

134∫8* 135∫7†

134∫8* 136∫7†

Values are mean∫S.E.M., nΩ10–11, for morphological studies nΩ5. WKY, LWKY and EWKY, untreated, losartan-treated and enalapriltreated Wistar-Kyoto rats, respectively; SHR, LSHR and ESHR, untreated, losartan-treated and enalapril-treated spontaneously hypertensive rats, respectively. * P∞0.05 compared to the WKY group, † P∞0.05 versus SHR (Bonferroni test).

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JARKKO KALLIOVALKAMA ET AL.

of 3 mM diclofenac, and in the presence of 3 mM diclofenac and 0.1 mM NG-nitro-L-arginine methyl ester (L-NAME). By the use of diclofenac and L-NAME the modulatory influence of prostanoids and nitric oxide, respectively, on the contractile response was examined. Contractile responses to 5-hydroxytryptamine, calcium, KCl and Kπ-free medium were expressed in mN/mm (i.e. wall tension). The calcium-induced contractions were also expressed as percentage of maximal response. The calculation of EC50 for 5-hydroxytryptamine and KCl in each ring was based on each individual maximal response. The EC50 values were presented as the negative logarithm (pD2), which values were used in the statistical analysis. The Kπ relaxation was presented as percentage of pre-existing contractile force induced by the Kπ-free medium. The ouabain-sensitive part of the Kπ relaxation in each vascular ring was calculated by subtracting the Kπ relaxation in the presence of ouabain (i.e. the ouabain-insensitive part) from the Kπ relaxation without ouabain.

ments was applied to investigate between-group differences. Differences were considered significant when P∞0.05.

Results Blood pressure, heart and body weights, arterial structure. The systolic blood pressure was already higher at the beginning of the study in spontaneously hypertensive rats than in Wistar-Kyoto rats, and during the 10-week follow-up a marked increase in blood pressure was observed in un-

Morphological studies. From 5 animals in each study group, vascular rings were prepared for light microscopy from a standard section of the proximal part of each superior mesenteric artery. The rings were fixed in 2% glutaraldehyde at 4æ and postfixed in 2% osmiumtetroxide. After washing, they were stained with 1% uranyl acetate and dehydrated with acetone series. Thereafter the samples were embedded in Epon (LX-112 Resin, Ladd, Burlington, VT, USA). Thin (2 mM) transverse sections were stained with 1% toluidine blue, and examined and photographed under light microscopy (Nikon Microphot-FXA, Japan). In each vascular ring the diameter of lumen and thickness of medial smooth muscle were measured from the photographs. Digoxin-like immunoreactivity. Plasma digoxin-like immunoreactivity was determined by ELISA of C18 (Bond Elut, Varian, Harbor City, CA, USA) extracts of plasma. The ELISA employed a digoxin antibody with cross-reactivity characteristics which have been reported previously (Doris 1992). The antibody was raised in rabbits against digoxin coupled via reductive amination after periodate oxidation to bovine serum albumin. ELISA plates were coated with digoxin coupled to ovalbumin. The assay was incubated at room temperature for 2 hr. Unbound antibody was removed by washing. Peroxidase-coupled goat-anti-rabbit gammaglobulin was added to each well. After a further 30 min. incubation the plates were rinsed and TMB reagent (Kirkegaard and Perry, Gaithersburg, MD, USA) added as a colour indicator of peroxidase activity. Assays were read at end-point after the addition of phosphoric acid to stop the peroxidase reaction. Standard curves were fitted to a 4-parameter logistic model and unknown values interpolated using Delta-Soft software. Drugs. The following drugs were used: enalapril maleate, losartan potassium (Merck Pharmaceutical Co., Wilmington, DE, USA), NG-nitro-L-arginine methyl ester hydrochloride, 5-hydroxytryptamine, ouabain, phentolamine hydrochloride (Sigma Chemical Co., St. Louis, MO, USA), L-noradrenaline L-hydrogentartrate and sodium nitroprusside (Fluka Chemie AG, Buchs SG, Switzerland), diclofenac (VoltarenA intravenous injection solution, Ciba-Geigy, Basel, Switzerland), atenolol (Leiras Oy, Turku, Finland), and nifedipine (Orion Pharma Ltd., Espoo, Finland). Losartan and enalapril were dissolved directly in tap water. The stock solutions of the compounds used in the in vitro studies were prepared in distilled water, with the exception of ouabain (directly in physiological salt solution), and nifedipine (in 50% ethanol). All solutions were freshly prepared before use and protected from light. Analysis of results. The results are expressed as means with S.E.M. Statistical analysis was carried out by one-way analysis of variance supported by Bonferroni test in the case of pairwise between-group comparisons. When the data consisted of repeated observations at successive time points, analysis of variance for repeated measure-

Fig. 1. Contractile responses of endothelium-denuded mesenteric arterial rings to cumulative addition of calcium to the organ bath after precontraction with 125 mM KCl in calcium-free medium in the presence of 1 mM phentolamine and 10 mM atenolol in WistarKyoto rats (WKY), spontaneously hypertensive rats (SHR), losartan-treated SHR (LSHR), and enalapril-treated SHR (ESHR). The responses were induced in the absence (A) and presence (B) of 0.5 nM nifedipine. Symbols indicate means with S.E.M., nΩ8–13 in each group; * P∞0.05, analysis of variance for repeated measurements.

LOSARTAN, ENALAPRIL AND ARTERIAL RESPONSES

39

Fig. 2. Relaxation responses to re-addition of 1 mM Kπ after full precontraction induced by Kπ-free buffer solution in the absence (A) and presence (B) of 1 mM ouabain in isolated endothelium-denuded mesenteric arterial rings in Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), losartan-treated SHR (LSHR), and enalapril-treated SHR (ESHR). The calculated ouabain-sensitive part of the Kπ relaxation (C). Bar graphs show plasma digoxin-like immunoreactivity in study groups (D). LWKY and EWKY, losartan-treated and enalapril-treated WKY rats, respectively. Symbols indicate means with S.E.M., nΩ8–13 in each group; * P∞0.05, analysis of variance for repeated measurements; † P∞0.05, Bonferroni test.

treated spontaneously hypertensive rats. Losartan and enalapril treatments completely prevented the elevation of blood pressures in spontaneously hypertensive rats. The treatments did not affect body weights, but both losartan and enalapril decreased heart weights in Wistar-Kyoto rats and totally prevented cardiac hypertrophy in spontaneously hypertensive rats (table 1).

The presence of structural alterations in hypertensive rat arteries was indicated by increased media to lumen ratio and greater vascular preparation weight when compared with Wistar-Kyoto rats. However, no significant differences in the media cross-sectional areas were detected between the study groups. The observed remodelling of superior mesenteric arteries was effectively corrected by losartan and enal-

40

JARKKO KALLIOVALKAMA ET AL.

april treatments, i.e. both therapies reduced media to lumen ratio and vascular preparation weight in spontaneously hypertensive rats (table 1). Mesenteric arterial responses. Since the arterial responses of losartan- and enalapriltreated Wistar-Kyoto rats did not show any differences from those of untreated Wistar-Kyoto rats, the graphs of losartan- and enalapril-treated Wistar-Kyoto rats were omitted from fig. 1 and 2 for clarity reasons. In the absence of the dihydropyridine Ca2π entry blocker nifedipine, the contractile sensitivity of isolated mesenteric arterial rings induced by cumulative addition of Ca2π did not significantly differ between the study groups (fig. 1A). However, in the presence of nifedipine the responses were clearly less effectively inhibited in losartan- and enalapriltreated spontaneously hypertensive rats and Wistar-Kyoto rats than in untreated hypertensive rats (fig. 1B). Thus, the contractile response induced by the addition of Ca2π showed higher dependency on dihydropyridine-sensitive Ca2π entry in untreated hypertensive rats. The maximal wall tension induced by Ca2π cumulation (with KCl as the contractile agent; in the presence of phentolamine and atenolol) was higher in untreated hypertensive rats when compared with losartan- and enalapril-treated spontaneously hypertensive rats and untreated Wistar-Kyoto rats, and this difference was also abolished by nifedipine (table 2). Maximal wall tension and sensitivity (i.e. the pD2 values) of endothelium-denuded arterial rings to KCl were increased in untreated spontaneously hypertensive rats when compared with Wistar-Kyoto rats (table 2). Losartan and enalapril treatments decreased maximal wall tension to KCl

in spontaneously hypertensive rats, but not in Wistar-Kyoto rats. Furthermore, neither of the treatments affected the sensitivity to KCl in spontaneously hypertensive rats, while losartan, but not enalapril, diminished the sensitivity to KCl in Wistar-Kyoto rats. When the responses to KCl were induced in the presence of phentolamine and atenolol, no differences in maximal wall tension or sensitivity were found between spontaneously hypertensive rats and WistarKyoto rats (table 2). Maximal wall tension elicited by Kπ-free solution in the absence and presence of ouabain did not differ in untreated hypertensive rats and Wistar-Kyoto rats, and losartan and enalapril were without effect on these responses (table 2). After the return of potassium to the organ bath upon the Kπ-free precontractions the rate of the subsequent relaxation was faster in Wistar-Kyoto rats than in spontaneously hypertensive rats. Furthermore, both losartan and enalapril enhanced the potassium relaxation in spontaneously hypertensive rats (fig. 2A). This relaxation was effectively inhibited by the Naπ,Kπ-ATPase inhibitor ouabain in all groups, although a significant difference was still detected between untreated hypertensive rats and Wistar-Kyoto rats (fig. 2B). The calculated ouabain-sensitive part of the potassium relaxation was initially (during the first 4 min. after Kπ repletion) lower in untreated hypertensive rats when compared with Wistar-Kyoto rats. Furthermore, both losartan and enalapril treatments especially enhanced the ouabainsensitive part of the potassium relaxation in spontaneously hypertensive rats (fig. 2C). Plasma digoxin-like immunoreactivity did not differ between untreated spontaneously hypertensive rats and Wistar-Kyoto rats. Although a trend towards lower values was

Table 2. π

Contractile responses induced by calcium, KCl and K -free solution in isolated mesenteric arterial rings in the experimental groups. Variable Calcium cumulation (125 mM KCl as the contractile agent; with phentolamine and atenolol) Maximal wall tension (mN/mm) ªE ªE with nifedipine KCl pD2 (ªlog M) ªE ªE with phentolamine and atenolol Maximal wall tension (mN/mm) ªE ªE with phentolamine and atenolol Kπ-free contraction Maximal wall tension (mN/mm) ªE ªE with ouabain

WKY

LWKY

EWKY

SHR

LSHR

ESHR

2.14∫0.27 1.88∫0.26

3.87∫0.50* 2.68∫0.32*†

2.95∫0.23*† 2.27∫0.23

4.18∫0.45* 1.91∫0.35

2.72∫0.29† 2.03∫0.17

2.44∫0.21† 1.81∫0.18

1.56∫0.02 1.62∫0.02

1.50∫0.02*†‡ 1.60∫0.02

1.55∫0.03† 1.60∫0.04

1.63∫0.01* 1.67∫0.03

1.61∫0.02 1.62∫0.04

1.59∫0.02 1.62∫0.04

4.88∫0.51 2.80∫0.34

4.45∫0.48† 3.56∫0.59

5.22∫0.44† 3.07∫0.68

7.26∫0.48* 3.41∫0.26

5.14∫0.32† 2.64∫0.37

5.58∫0.67† 2.65∫0.26

3.62∫0.47 4.37∫0.96

3.37∫0.36 3.60∫0.34

4.61∫0.94 4.53∫0.66

4.77∫0.70 5.91∫0.84

3.96∫0.40 4.64∫0.24

4.77∫0.71 5.13∫0.82

Values are mean∫S.E.M., nΩ8–10. WKY, LWKY and EWKY, untreated, losartan-treated and enalapril-treated Wistar-Kyoto rats, respectively; SHR, LSHR and ESHR, untreated, losartan-treated and enalapril-treated spontaneously hypertensive rats, respectively. ªE, endothelium-denuded arterial rings. pD2 is the negative logarithm of the concentration of agent producing 50% of maximal contractile response. Concentrations of phentolamine, atenolol, ouabain and nifedipine, 1 mM, 10 mM, 1 mM and 0.5 nM, respectively. * P∞0.05 compared to the WKY group, † P∞0.05 versus SHR, ‡ P∞0.05 versus LSHR (Bonferroni test).

41

LOSARTAN, ENALAPRIL AND ARTERIAL RESPONSES Table 3. Contractile responses to 5-hydroxytryptamine in isolated mesenteric arterial rings in the experimental groups. Variable

WKY

5-Hydroxytryptamine pD2 (ªlog M) πE πE with diclofenac πE with L-NAME and diclofenac

6.48∫0.06 6.39∫0.08 6.40∫0.13

Change in pD2 value induced by (DpD2) Diclofenac ª0.10∫0.09 L-NAME 0.02∫0.15 Maximal wall tension (mN/mm) πE πE with diclofenac πE with L-NAME and diclofenac

5.20∫0.58 4.16∫0.48 5.10∫0.52

LWKY

6.43∫0.08‡ 6.07∫0.11*†‡ 6.24∫0.06†‡ ª0.36∫0.11† 0.17∫0.12 6.04∫0.39 4.30∫0.45 5.37∫0.39

EWKY

6.46∫0.09‡ 6.21∫0.05† 6.31∫0.07†‡

SHR

6.52∫0.08 6.51∫0.07 6.61∫0.12

ª0.25∫0.10 0.10∫0.03

ª0.01∫0.10 0.09∫0.11

6.51∫0.75 5.00∫0.57 5.41∫0.47

5.61∫0.46 4.29∫0.24 5.25∫0.35

LSHR

6.69∫0.02*† 6.39∫0.07 6.62∫0.09 ª0.31∫0.07† 0.24∫0.08 4.87∫0.26 4.31∫0.47 4.86∫0.32

ESHR

6.33∫0.08‡ 6.28∫0.07† 6.46∫0.05 ª0.05∫0.08‡ 0.18∫0.05 6.89∫0.40*‡ 5.47∫0.32* 6.21∫0.31‡

Values are mean∫S.E.M., nΩ8–10. WKY, LWKY and EWKY, untreated, losartan-treated and enalapril-treated Wistar-Kyoto rats, respectively; SHR, LSHR and ESHR, untreated, losartan-treated and enalapril-treated spontaneously hypertensive rats, respectively. L-NAME, NG-nitro-L-arginine methyl ester; πE, endothelium-intact arterial rings. pD2 is the negative logarithm of the concentration of agonist producing 50% of maximal contractile response. Concentrations of L-NAME and diclofenac, 0.1 mM and 3 mM, respectively. * P∞0.05 compared to the WKY group, † P∞0.05 versus SHR, ‡ P∞0.05 versus LSHR (Bonferroni test).

observed in drug-treated spontaneously hypertensive rats (PΩ0.08–0.10), neither losartan nor enalapril treatment did significantly affect plasma digoxin-like immunoreactivity in spontaneously hypertensive rats or Wistar-Kyoto rats (fig. 2D). No correlation between plasma digoxin-like immunoreactivity and potassium relaxation rate in vitro were observed. The endothelium-intact vascular rings of untreated hypertensive rats showed comparable maximal wall tension and sensitivity in response to 5-hydroxytryptamine in the absence and presence of diclofenac and L-NAME when compared with Wistar-Kyoto rats (table 3). Neither losartan nor enalapril treatment significantly affected the maximal wall tension elicited by 5-hydroxytryptamine when compared with untreated controls, while the response was higher in enalapril- than in losartan-treated spontaneously hypertensive rats, and this difference was also observed in the presence of L-NAME and diclofenac. Losartan, but not enalapril, slightly increased the sensitivity to 5-hydroxytryptamine in spontaneously hypertensive rats. Diclofenac elicited a more pronounced decrease in sensitivity to 5-hydroxytryptamine in losartan-treated spontaneously hypertensive rats than in untreated and enalapril-treated spontaneously hypertensive rat groups, while L-NAME comparably increased the sensitivity to 5-hydroxytryptamine in all groups (table 3). Discussion A characteristic morphological change in the resistance arteries of patients with essential hypertension and spontaneously hypertensive rats is inward eutrophic remodelling, i.e. increased wall to lumen ratio with unaltered media cross-sectional area (Mulvany 1999). In this study, both losartan and enalapril therapies decreased the media to lumen ratio, but were without significant effect on the media cross-

sectional area in the mesenteric artery of hypertensive rats. Although these measurements were not performed in pressurized arteries, the results agree with previous observations in small mesenteric vessels of spontaneously hypertensive rats (Rizzoni et al. 1998). Therefore, the changes in vascular structure induced by losartan and enalapril are compatible with outward eutrophic remodelling of arteries. However, the alterations of blood vessel structure following losartan and enalapril therapy in spontaneously hypertensive rats were not exclusively eutrophic, since the weight of standard sections of the superior mesenteric artery was higher in untreated spontaneously hypertensive rats than in the other groups. This suggests that vascular hypertrophy was also present in hypertensive rats, which was corrected by the present treatments. The arteries of spontaneously hypertensive rats are more sensitive to the actions of dihydropyridine calcium blockers than those of Wistar-Kyoto rats (Arvola et al. 1992; Ka¨ho¨nen et al. 1996), which was confirmed in the present study. Moreover, the proportions of voltage-dependent calcium currents are different in blood vessels of hypertensive and normotensive rats, in spontaneously hypertensive rats the L-type current is greater whereas in Wistar-Kyoto rats the T-type predominates (Rusch & Hermsmeyer 1988). Longterm angiotensin-converting enzyme inhibition in hypertensive rats has been found to suppress calcium channel agonist-induced contractions (Sada et al. 1989a) and decrease calcium-dependent tone in the aorta (Sada et al. 1989b), and reduce intracellular free calcium concentration in arterial smooth muscle (Sada et al. 1989a & 1990). In the present study, nifedipine less effectively inhibited the calcium-induced contractions in losartan- and enalapriltreated hypertensive rats and Wistar-Kyoto rats than in untreated hypertensive rats. This suggests higher calcium entry via L-type channels in the arteries of untreated hypertensive rats when compared with the other groups. Thus, losartan

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JARKKO KALLIOVALKAMA ET AL.

and enalapril treatments seem to normalize the function of voltage-dependent calcium channels in the vascular smooth muscle of spontaneously hypertensive rats. Vascular Naπ,Kπ-ATPase function was evaluated indirectly by the readdition of potassium to the organ bath upon Kπ-free medium-induced precontractions (Arvola et al. 1992). The return of potassium activates Naπ,Kπ-ATPase which repolarizes the cell membrane and initiates smooth muscle relaxation (Bonaccorsi et al. 1977). Although general smooth muscle relaxation mechanisms are also involved in this response, previous results suggest that potassium relaxation reflects Naπ,Kπ-ATPase activity in arterial smooth muscle (Arvola et al. 1992). The present results showed that potassium relaxation was slower in spontaneously hypertensive than Wistar-Kyoto rats, and enhanced in hypertensive rats by losartan and enalapril. The finding whereby potassium relaxation was inhibited by ouabain in all groups indicates the involvement of the sodium pump in this response. Moreover, the fact that especially the ouabain-sensitive part of the relaxation was augmented after the treatments suggests increased recovery rate of ionic gradients across the cell membrane via Naπ,Kπ-ATPase in hypertensive rats following losartan and enalapril therapies. Some ouabain-resistant relaxation was detected particularly in the Wistar-Kyoto rats, whereby the function of the sodium pump was not completely inhibited by ouabain, or additional mechanisms took part in the response. However, the fact that ouabain abolished the losartan- and enalaprilinduced improvement of potassium relaxation supports the notion that Naπ,Kπ-ATPase function was augmented in hypertensive rats following these treatments. Several investigations have provided evidence for elevated levels of circulating sodium pump inhibitors in hypertensive subjects (Hamlyn & Manunta 1992) and experimental animals (Wauquier et al. 1988; Doris 1994). This digitalis-like factor augments natriuresis, but it also depolarizes smooth muscle via Naπ,Kπ-ATPase inhibition, and increases calcium influx through voltage-dependent channels. Subsequently, peripheral arterial resistance is elevated (Zhu et al. 1994). The non-pharmacological treatment of hypertension has lowered plasma ouabain-like activity in hypertensive rats (Doris 1988 & 1994), and quinapril therapy has reduced plasma digoxin-like immunoreactivity in spontaneously hypertensive rats, possibly via an alteration of sodium balance (Ka¨ho¨nen et al. 1995). In the present study, however, both losartan and enalapril treatments were without significant effects on plasma digoxin-like immunoreactivity in hypertensive rats. Thus, augmented potassium relaxation after losartan and enalapril therapies could not be explained by decreased plasma digoxin-like material, but rather resulted from the lowering of blood pressure. Previously, quinaprilat has increased the availability of nitric oxide in the forearm circulation of patients with chronic heart failure, whereas enalaprilat was without effect (Hornig et al. 1998). Therefore, it seems possible that differences exist in the long-term vascular effects of enalapril and quinapril, and on the basis of the present results the influences

of these treatments on plasma digoxin-like immunoreactivity also appear to be dissimilar. It is noteworthy that the impaired potassium relaxation in arteries of untreated hypertensive rats was practically abolished by ouabain. Thus, the arteries of spontaneously hypertensive rats were probably also more sensitive to the actions of circulating sodium pump inhibitors, which in turn could result in smooth muscle depolarization, enhanced calcium entry, and elevation of peripheral arterial resistance. In previous reports losartan treatment has not affected maximal arterial contractions to KCl or 5-hydroxytryptamine, but has reduced constrictor sensitivity to 5-hydroxytryptamine in spontaneously hypertensive rats (Soltis 1993; Rodrigo et al. 1997). Moreover, various angiotensin converting enzyme inhibitor therapies have differently influenced vasoconstrictor responses in experimental hypertension, since ramipril and captopril have not affected arterial contractions in spontaneously hypertensive rats (Hutri-Ka¨ho¨nen et al. 1997; Rodrigo et al. 1997), while perindopril has reduced the pressure against which the mesenteric resistance arteries of Milan hypertensive rats could contract (Mulvany et al. 1991). In addition, quinapril treatment has consistently attenuated both the sensitivity and maximal contractile responses to 5-hydroxytryptamine and noradrenaline in arteries of spontaneously hypertensive rats (Major et al. 1993; Arvola et al. 1993; Ka¨ho¨nen et al. 1996). The deviations in the vascular actions of angiotensin converting enzyme inhibitors may result from differences in their affinity to tissue angiotensin converting enzyme and also from diverse effects on endothelial autacoid release (Hornig et al. 1998). Some differences in vasoconstrictor sensitivity following the present treatments were observed. Losartan, but not enalapril, increased the sensitivity to 5-hydroxytryptamine in hypertensive rats, and diclofenac elicited a more pronounced decrease in sensitivity to 5-hydroxytryptamine in both losartan-treated groups than in the other groups, suggesting that the products of the cyclooxygenase pathway differentially modulated the responses to 5-hydroxytryptamine in the study groups. On the other hand, L-NAME induced a corresponding shift in constrictor sensitivity to 5hydroxytryptamine in all groups, which suggests that endothelium-derived nitric oxide similarly modulated constrictor responses in all study groups. Taken together, since the antihypertensive therapies in the present study somewhat differently modulated the sensitivity to 5-hydroxytryptamine in hypertensive rats, other factors in addition to the reduction of blood pressure and inhibition of angiotensin II may have contributed to these effects, one candidate being the potentiation of the actions of bradykinin following the enalapril treatment (Minshall et al. 1997). In conclusion, long-term AT1-receptor antagonism and angiotensin-converting enzyme inhibition corrected the mesenteric arterial structural alterations, and normalized the increased dihydropyridine-sensitivity in arterial segments of spontaneously hypertensive rats. Treatment with losartan and enalapril also augmented arterial potassium

LOSARTAN, ENALAPRIL AND ARTERIAL RESPONSES

relaxation in spontaneously hypertensive rats, suggesting an enhanced function of Naπ,Kπ-ATPase, but this effect could not be attributed to changes in circulating sodium pump inhibitor concentration. Altogether the vascular actions of losartan and enalapril were very similar, and these findings can be attributed to the reduction of blood pressure and the inhibition of the actions of angiotensin II in the vasculature.

Acknowledgements This research was supported by the Medical Research Fund of Tampere University Hospital, and the Aarne Koskelo Foundation, Finland and the National Institutes of Health (DDK RO1 45538 to PAD), and by an educational grant from Merck Pharmaceutical Company, USA.

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