Br. J. Pharmacol. (1992), 106, 157-165

C Macmillan Press Ltd,

1992

Contractions induced by potassium-free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats 'Pertti Arvola, Ilkka Pdrsti, Pauli Vuorinen, Anu Pekki & Heikki Vapaatalo Department of Biomedical Sciences, University of Tampere, P.O. Box 607, SF-33101, Tampere, Finland 1 Vascular contractions induced by K+-free solution and relaxation responses following the return of K+ to the organ bath were studied in mesenteric arterial rings from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) with particular focus on the role of vascular adrenergic nerve-endings and endothelium. 2 In endothelium-denuded rings the omission of K+ from the incubation medium resulted in gradual contractions, the rate of which was slower in SHR than WKY. Nifedipine (1 ALM) inhibited the contractions more effectively in SHR than WKY. 3 Adrenergic denervation in vitro with 6-hydroxydopamine reduced the contractions induced by the K+-free medium in endothelium-denuded rings. The remaining contractions after denervation were markedly greater in SHR than WKY. 4 The presence of intact vascular endothelium attenuated the K+-free contractions in both strains, the attenuation being smaller in SHR than WKY. N0-nitro-L-arginine methyl ester (L-NAME, 0.1 mM) and methylene blue (10 fiM), but not indomethacin (10 JM), abolished the attenuating effect of endothelium on the K+-free contractions. L-Arginine (1 mM) reversed the effect of L-NAME in WKY but not in SHR. 5 The re-addition of K+ after full K+-free contractions dose-dependently relaxed the rings. The rate of this K+-induced relaxation was significantly slower in SHR than WKY at all K+ concentrations (0.15.9 mM) studied, whether the endothelium or functioning adrenergic nerve-endings were present or not. Ouabain (1 mM) totally inhibited the K+ relaxation in SHR but only partially in WKY. 6 Vascular smooth muscle contractions induced by high concentrations of potassium were comparable between the strains. The EC50 for noradrenaline-induced contractions was lower in SHR than WKY, but the maximal forces did not differ significantly. 7 In conclusion, the contractile response in K+-free solution more clearly differentiates vascular rings from SHR and WKY than the responses induced by the classical contractile agents noradrenaline and high concentrations of potassium. The depressant effect of the presence of intact endothelium on the K+-free contractions, which was smaller in SHR than WKY, is mediated via the endothelium-derived relaxing factor. Neurotransmitter release from vascular adrenergic nerve-endings participates less in the K+-free contractile response in SHR than WKY. Moreover, the contractile response is more dependent on calcium entry through nifedipine-sensitive calcium channels in SHR than WKY. The greater K+-free contractions of denervated endothelium-denuded rings and the reduced K+ relaxation rate in SHR when compared to WKY suggest increased cell membrane permeability and decreased activity of vascular Na+, K+-ATPase, respectively, in this type of genetic hypertension. Keywords: Adrenergic nerve-endings; endothelium; potassium; potassium-free contraction; potassium relaxation; spontaneously hypertensive rat; vascular smooth muscle; Wistar-Kyoto rats

Introduction Increased responses to the classical contractile agents noradrenaline and high concentrations of KCl and decreased relaxation have been described in arterial smooth muscle of spontaneously hypertensive rats (SHR) (Field & Soltis, 1985). However, the findings in different investigations have been somewhat inconsistent, and not all studies have been able to demonstrate increased sensitivity to vasoconstrictor agents in experimental forms of hypertension (see Bohr & Webb, 1984). The controversial results and rather small changes between hypertensive and normotensive blood vessels suggest that the study of vasoconstrictor sensitivity alone does not reveal all alterations in arterial smooth muscle function associated with hypertension. Bohr et al. (1988) have suggested that a generalized membrane defect is associated with genetic hypertension. In-

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creased membrane permeability both in vascular smooth muscle (Hopp et al., 1986) and in non-vascular cells (Tamura et al., 1985; Furspan et al., 1989) has been demonstrated in hypertensive animals. The contractile response induced by inhibition of vascular Na', K+-ATPase with K+-free medium has commonly been used as a tool to study smooth muscle permeability to ions in both hypertensive and normotensive rats (Bohr et al., 1988; Lamb et al., 1988). However, since these investigations involved endothelium-intact preparations, different endothelial influences on the contractile responses in hypertensive and normotensive blood vessels may have affected the results. In addition, the participation of adrenergic nerve-endings in vascular contractions induced by N', K+-ATPase inhibition via endogenously released noradrenaline (NA) (Vanhoutte & Lorenz, 1984) may alter responses differently in hypertensive and normotensive arteries. Vascular endothelium has a basal release of the endothelium-derived relaxing factor (EDRF), which stimulates

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soluble guanylate cyclase and elevates guanosine 3': 5'-cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (Furchgott & Vanhoutte, 1989), and is thus capable of controlling vascular tone (Pique et al., 1989; Kelm & Schrader, 1990; Vane et al., 1990). EDRF has been identified as nitric oxide (Moncada et al., 1991) or a closely related substance (Myers et al., 1990) synthesized from L-arginine, and the synthesis can be inhibited with arginine analogues such as NG-nitro-L-arginine methyl ester (L-NAME) (Ishii et al., 1990). We have previously found that the contractile response induced by K+-free solution and the K+ relaxation clearly differentiate vascular smooth muscle of hypertensive and normotensive rats (Porsti et al., 1992). The aim of the present study was to investigate in greater detail the vascular smooth muscle responses induced by alterations in organ bath K+ concentration in SHR and their normotensive controls Wistar-Kyoto rats (WKY). Special attention was paid to the modulatory role of adrenergic nerve-endings and endothelium.

Methods

Animals Male SHR of the Okamoto-Aoki strain and WKY (M0llegaard's Breeding Centre, Ejby, Denmark) were used at the age of 22-25 weeks (weight 370-410 g in SHR, 370-430 g in WKY). The rats were housed four animals a cage in a room maintained at 220C and illuminated from 06 h 00 min to 18 h 00 min and had free access to drinking fluid (tap water) and food pellets (Ewos, Sodertalje, Sweden). The systolic blood pressures of the unanaesthetized rats were measured by the tail cuff method at 280C (Model 129 Blood Pressure Meter; IITC Inc., Woodland Hills, Ca., U.S.A.). The experimental design of the study was approved by the Animal Experimentation Committee of the University of Tampere.

Vascular responses in vitro The animals were decapitated and the mesenteric arteries excised and cleaned of connective tissue. Three successive sections (3 mm in length) of the artery from each animal were cut, beginning 1 cm distally from the mesenteric arteryaorta junction. The vascular endothelium was removed by gently rubbing with a scuffed injection needle. The mesenteric arterial rings were placed between stainless steel hooks and mounted in an organ bath chamber in physiological salt solution (PSS) (pH 7.4) of the following composition (mM): NaCl 119.0, NaHCO3 25.0, glucose 1 1.1, CaCl2 1.6, KCI 4.7, KH2PO4 1.2, MgSO4 1.2, and aerated with 95% 02: 5% CO2. The rings were equilibrated for 1 h at 37°C with a resting tension of 1.5 g. The force of contraction was measured with an isometric force-displacement transducer (Grass FT03) and registered on a polygraph (Model 7 E Polygraph; Grass Instrument Co., Quincy, Ma., U.S.A.). The absence of endothelium was confirmed by the lack of relaxation response to 1 laM acetylcholine (ACh) in vascular rings precontracted with 1 tiM NA. If any relaxation was observed, the endothelium was further rubbed. After a 30 min stabilization period, cumulative concentration-response curves for NA and KCl were determined. In solutions containing high concentrations of K+ (20-125 mM), NaCI was substituted with KCl on an equimolar basis. After another 30 min of stabilization, K+ was omitted from the PSS. The K+-free buffer solution (pH 7.4) was prepared by substituting KH2PO4 and KCI with NaH2PO4 and NaCl, respectively, on an equimolar basis. The baths were rinsed with fresh K+-free solution every 20min. The K+-free solution resulted in a gradual contraction in all vascular rings. After the contraction had reached a plateau, a

single dose of K+ was re-added to the baths and the subsequent relaxation registered. The K+-free contraction and K+ relaxation procedure was also performed in endothelium-denuded rings after chemical adrenergic denervation and in the presence of ouabain (1 mM), and in endothelium-intact rings as such and in the presence of the competitive EDRF synthesis inhibitor LNAME (0.1 mM) with or without L-arginine (1 mM). The effect of soluble guanylate cyclase inhibition by methylene blue (101iM) on K+-free contractions was studied in endothelium-intact rings. The cyclo-oxygenase inhibitor, indomethacin (10,JM), was used to examine the possible effects of endothelial prostacyclin on K+-free contractions. The effect of the Ca2" antagonist, nifedipine (1 ,UM), on contractions was investigated in endothelium-denuded rings. The endothelium-denuded rings were denervated in vitro by exposing them to a buffer-free solution containing 1.2 mM 6-hydroxydopamine, and vigorously gassing them with nitrogen for 15 min, followed by a 2 h recovery period in normal PSS (Aprigliano & Hermsmeyer, 1976). L-NAME, L-arginine and indomethacin were added to the baths 15 min before, and the other compounds simultaneously with the introduction of K+-free solution. To test the dependence of K+-free contractions on organ bath Ca2" and Na+ concentrations, further experiments with endothelium-denuded mesenteric arterial rings were made with the K+-free, Ca2"-free solution (Ca2" omitted from K+-free solution) and the K+-free, low Na+ solution (Na+ 25 mM, NaCl substituted with LiCl on an equimolar basis). Some endothelium-denuded rings from SHR and WKY were also exposed to 1 mM ouabain in normal PSS. In an additional series, concentration-response curves for ACh-induced relaxations were determined cumulatively in 1 IM NA-precontracted mesenteric arterial rings with intact endothelium.

Morphological studies To test the adequacy of our mechanical endothelium denudation, both endothelium-denuded and -intact mesenteric arterial rings from WKY were prepared for electron microscopy. The rings were fixed in 2% glutaraldehyde at 4°C 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 (LX112 Resin, Ladd, Burlington, Vt., U.S.A.). Ultrathin sections were stained with 4% uranyl acetate and 0.2% lead citrate and examined under a Jeol JEM 1200 electron microscope (Jeol Ltd., Tokyo, Japan) at 60 kV accelerating voltage.

Drugs The following drugs were used: acetylcholine chloride, 6hydroxydopamine, indomethacin, NG-nitro-L-arginine methyl ester hydrochloride, ouabain octahydrate (Sigma Chemical Co., St. Louis, Mo., U.S.A.), L-arginine, methylene blue (E. Merck AG, Darmstadt, Germany), nifedipine (Orion Pharmaceutical Co., Espoo, Finland) and (-)-noradrenaline Lhydrogentartrate (Fluka Chemie AG, Buchs SG, Switzerland). The stock solutions were dissolved in distilled water, with the exception of indomethacin (in 1.5 mM Na2CO3), nifedipine (in 50% ethanol), 6-hydroxydopamine and ouabain (in PSS). All solutions were freshly prepared and protected from light.

Analysis of results EC50 for NA and KCI in each ring was calculated from the plots of the concentration of agonist versus the percentage of maximal contraction response. ICm for ACh-induced relaxations was calculated correspondingly. Contractile responses in K+-free solution were normalized by comparing them to the maximal response induced by 125 mM KCl. After readdition of K+ the greatest reduction in smooth muscle

POTASSIUM AND VASCULAR RESPONSES IN SHR AND WKY

contractile force during a 1 min period was considered the maximal relaxation rate, which was normalized by comparing it to the maximal 125 mM KCl-induced contraction, or by relating it to the pre-existing maximal K+-free contraction or tissue dry weight. Statistical analysis was carried out by one-way analysis of variance (ANOVA) supported by Bonferroni confidence intervals. Differences were considered significant when P