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J Physiol (2003), 548.3, pp. 893-906
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.034116

Angiotensin II-induced modulation of endothelium-dependent relaxation in rabbit mesenteric resistance arteries

Takeo Itoh*, Junko Kajikuri*, Toyohiro Tada†, Yoshikatsu Suzuki‡ and Yoshio Mabuchi§

	ABSTRACT

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The role of local endogenous angiotensin II (Ang II) in endothelial function in resistance arteries was investigated using rabbit mesenteric resistance arteries. First, the presence of immunoreactive Ang II together with Ang II type-1 receptor (AT₁R) and angiotensin converting enzyme (ACE) was confirmed in these arteries. In endothelium-intact strips, the AT₁R-blocker olmesartan (1 µM) and the ACE-inhibitor temocaprilat (1 µM) each enhanced the ACh (0.03 µM)-induced relaxation during the contraction induced by noradrenaline (NA, 10 µM). Similar effects were obtained using CV-11974 (another AT₁R blocker) and enalaprilat (another ACE inhibitor). The nitric-oxide-synthase inhibitor N^G-nitro-L-arginine (L-NNA) abolished the above effect of olmesartan. In endothelium-denuded strips, olmesartan enhanced the relaxation induced by the NO donor NOC-7 (10 nM). Olmesartan had no effect on cGMP production (1) in endothelium-intact strips (in the absence or presence of ACh) or (2) in endothelium-denuded strips (in the absence or presence of NOC-7). In -escin-skinned strips, 8-bromoguanosine 3',5' cyclic monophosphate (8-Br-cGMP, 0.01-1 µM) concentration dependently inhibited the contractions induced (a) by 0.3 µM Ca²⁺ in the presence of NA+GTP and (b) by 0.2 µM Ca²⁺+GTPS. Olmesartan significantly enhanced, while Ang II (0.1 nM) significantly inhibited, the 8-Br-cGMP-induced relaxation. We propose the novel hypothesis that in these arteries, Ang II localized within smooth muscle cells activates AT₁Rs and inhibits ACh-induced, endothelium-dependent relaxation at least partly by inhibiting the action of cGMP on these cells.

(Resubmitted 9 October 2002; accepted after revision 20 February 2003; first published online 21 March 2003)
Corresponding author T. Itoh: Department of Cellular and Molecular Pharmacology, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan. Email: titoh{at}med.nagoya-cu.ac.jp

	INTRODUCTION

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Vascular endothelial cells release vasorelaxing factors (such as nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor (EDHF)) and these, by virtue of their important roles in the regulation of vascular tone, smooth muscle cell growth, vascular permeability and blood coagulation, help to maintain cardiovascular homeostasis (Moncada et al. 1991; Kuriyama et al. 1998). It has been suggested that a decrease in the roles played by these endothelium-derived relaxing factors may lead to serious cardiovascular disease, such as hypertension, congestive heart failure, acute myocardial infarction or atherosclerosis (Ohara et al. 1993; Harrison, 1997; Onaka et al. 1998).

Angiotensin II (Ang II) is produced both systemically (via the classical, renal renin-angiotensin system (RAS)) and locally (via tissue RAS (local RAS, Dzau, 1989; Touyz & Schiffrin, 2000)). Cells located in the vascular wall, such as endothelial cells and smooth muscle cells, are able to synthesize their own tissue-based components of the local RAS (Campbell, 1987; Haller et al. 1996; Saris et al. 2000). It has been found that administering an inhibitor of angiotensin-converting enzyme (ACE) ameliorates the deleterious effects of elevated Ang II levels in patients with cardiovascular diseases by bringing about an improvement in their endothelial function (Mancini et al. 1996). This suggests a significant pathophysiological role for Ang II: namely, a downregulation of the function of the endothelium (Griendling et al. 1994; Harrison, 1997; Touyz & Schiffrin, 2000). In mammalian cells, the effects of Ang II are mediated via at least two high-affinity plasma membrane receptors, Ang II type-1 (AT₁) and type-2 (AT₂) receptors (Murphy et al. 1991; Sasaki et al. 1991; Kambayashi et al. 1993; Mukoyama et al. 1993). The AT₁ receptors (AT₁Rs) are the ones thought to be responsible for the Ang II-mediated vascular effects, such as endothelial dysfunction (Touyz & Schiffrin, 2000). Recently, a potent and selective AT₁R blocker was developed and the results of basic as well as clinical studies using AT₁R blockade support the pathophysiological role of Ang II as a causative factor in endothelial dysfunction (Onaka et al. 1998; Prasad et al. 2000).

Exogenously applied Ang II upregulates the components of NAD(P)H oxidases in cultured smooth muscle cells and increases the generation of the superoxide anion (Griendling et al. 1994; Harrison, 1997). The superoxide anion quickly binds to and degrades NO, leading to a downregulation of the function of endothelium-derived NO (Daemen et al. 1991). So far, this mechanism has been thought of as being responsible for the Ang II-induced dysfunction of endothelium-derived NO under pathophysiological conditions. However, it has recently been found that the arterial relaxation induced by sodium nitroprusside (an NO donor), admittedly at a very low concentration, was not modified in patients with essential hypertension and atherosclerosis following chronic treatment with an ACE inhibitor (Prasad et al. 2000; Schiffrin et al. 2000). Furthermore, in mesenteric resistance arteries from spontaneously hypertensive rats, the ACE inhibitor enalapril had no effect on the endothelium-derived, NO-mediated relaxation, although it did enhance the EDHF-mediated membrane hyperpolarization in the smooth muscle cells (Onaka et al. 1998). Thus, the role played by superoxide anion in Ang II-induced endothelial dysfunction in resistance arteries remains unclear. A functional role for local RAS in isolated arteries has recently been suggested by the finding that the Ang II generated by local RAS modulates Ca²⁺ mobilization in smooth muscle cells in the rat tail artery (Asada et al. 1999). However, no role for endogenous Ang II in isolated arteries was found in a study of mesenteric resistance arteries obtained from spontaneously hypertensive rats (Onaka et al. 1998). Thus, it is still unclear what physiological role might be played by the Ang II generated by local RAS in endothelium-dependent relaxation in resistance arteries.

To attempt to clarify the unresolved issues mentioned above, we first investigated whether Ang II is or is not generated by local RAS in isolated rabbit mesenteric resistance arteries. Next, we tried pharmacologically to characterize the role played by endogenous Ang II in endothelium-dependent relaxation by performing experiments using either the recently developed potent AT₁R-blocker olmesartan (active form of olmesartan medoxomil, 2,3-dihydroxy-2-butenyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imidazole-5-carboxylate, cyclic 2, 3-carbonate; Mizuno et al. 1995) or CV-11974 (active form of candesartan, the most potent AT₁R blocker known; Eto et al. 2002) and other experiments using either enalaprilat (well-known classical ACE inhibitor) or temocaprilat (a more potent ACE inhibitor; Oizumi et al. 1988). Finally, we investigated the mechanism underlying the effect of Ang II by observing the effect of olmesartan on the amount of cGMP produced (a) in the absence or presence of ACh (in endothelium-intact strips) or (b) in the absence or presence of the NO donor NOC-7 [3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine] (in endothelium-denuded strips). We also observed the effect of olmesartan and that of exogenously applied Ang II on the relaxation induced by 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP, a phosphodiesterase-resistant cGMP analogue) in -escin-skinned strips.

	METHODS

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Tissue preparation

Male Japan White albino rabbits (supplied by Kitayama Labes, Ina, Japan), weighing 1.9 to 2.5 kg were anaesthetized by injection of pentobarbitone sodium (40 mg kg^-1 given I.V.), then killed by exsanguination. The protocols used conformed with guidelines on the conduct of animal experiments issued by Nagoya City University Medical School and were approved by the Committee on the Ethics of Animal Experiments of Nagoya City University Medical School. The third and fourth branches of the mesenteric artery distributing to the region of the ileum (diameter, approximately 80-120 µm) were excised immediately, then cleaned by removal of connective tissue. After each artery had been cut open along its long axis using small scissors, circularly cut strips were carefully prepared, as described previously, so as not to damage the endothelium (Yamashita et al. 1999). In some strips, the endothelium was carefully removed by gentle rubbing of the internal surface of the vessel using small pieces of razor blade, satisfactory ablation of the endothelium being pharmacologically verified by the absence of a relaxing effect when ACh (3 µM) was applied during the contraction induced by noradrenaline (NA), as described previously (Itoh et al. 1992; Yamashita et al. 1999).

Recording of mechanical responses

Circularly cut strips (0.2-0.3 mm long, 0.10-0.15 mm wide, 0.02-0.03 mm thick) were prepared for tension measurement. A strip of mesenteric artery was placed in a chamber with a capacity of 0.3 ml and superfused with Krebs solution at a flow rate of about 2 ml min^-1. Both ends of the preparation were fixed using fine silk threads to allow isometric tension to be recorded via a strain-gauge transducer (AE801; SensoNor a.s, Horten, Norway). A resting tension of 2-3 mg was applied so as to obtain a maximum contraction to 128 mM K⁺. Each preparation was allowed to equilibrate for 1 to 2 h before the start of the experiment.

Endothelium-dependent relaxation was induced by an application of ACh (0.03-0.3 µM) during the contraction induced by NA. Unless otherwise stated, the concentration of NA used was 10 µM and propranolol (a non-selective -receptor antagonist, 3 µM) was co-applied with the NA to prevent it activating -receptors. For this set of experiments, the preparations were first contracted with NA + propranolol and, after a steady-state contraction had been attained (at 6 min after the NA application), ACh (0.03-0.3 µM) was cumulatively applied from low to high concentration (for 2 min at each concentration) during the ongoing NA-induced contraction. Following removal of the ACh for 1 min, the NA + propranolol was washed out to produce relaxation. This protocol was repeated as required at 30 min intervals.

When the effects of olmesartan or CV-11974 or those of temocaprilat or enalaprilat were to be examined, the appropriate agent was used at a concentration of 1 µM (since this is sufficient to inhibit AT₁Rs and ACE, respectively: for AT₁Rs, Mizuno et al. 1995; Eto et al. 2002; and for ACE, Oizumi et al. 1988; Eto et al. 2002). The effects of these agents on the NA-induced contraction were observed by examining the changes they induced in the tonic tension developed after an application of 10 µM NA (i.e. just before the application of the first concentration of ACh). The effect of each agent on the ACh-induced relaxation was observed by examining the change it induced in the maximum relaxation attained during a 2-min application of a given concentration of ACh.

The roles that might be played by endothelium-derived NO and prostaglandins in the action of olmesartan or temocaprilat were examined in endothelium-intact strips treated with N^G-nitro-L-arginine (L-NNA, an inhibitor of NO synthase) and L-NNA + diclofenac (an inhibitor of cyclo-oxygenase), respectively. To this end, after records of the responses to NA (10 µM) and ACh (0.03 µM) had been obtained, the strips were treated either with L-NNA (0.1 mM) or with L-NNA (0.1 mM) + diclofenac (3 µM) for 60 min. Then NA (0.5 µM) was applied and after 6 min, ACh (0.03 µM) was applied (for 2 min) in the continued presence of NA. Finally, olmesartan (1 µM) or temocaprilat (1 µM) was applied for 160 min, and the protocol described above was repeated in the continued presence of the relevant drug.

The effect of olmesartan (1 µM) on the NO-induced relaxation in smooth muscle was examined by observing its effect on the relaxation induced by NOC-7 (NO donor; Hrabie et al. 1993) in strips denuded of endothelium. A similar experimental protocol was used when the effect of this agent on the ACh-induced relaxation was to be examined in endothelium-intact strips.

Permeabilized smooth muscle

Endothelium-denuded muscle strips (0.2-0.3 mm long, 0.07- 0.10 mm wide, 0.02-0.03 mm thick) were permeabilized by a 30 min application of -escin (30 µM). This was applied in a relaxing solution containing the calcium ionophore A23187 (3 µM, to avoid spurious effects due to Ca²⁺ release from intracellular storage sites in the skinned muscle; Itoh et al. 1992). To prevent deterioration of the Ca²⁺-induced contraction, 0.1 µM calmodulin was present throughout the experiment. All experiments were performed at 25 °C. The amplitude of the contraction induced by 0.3 µM Ca²⁺ was normalized with respect to that induced by 10 µM Ca²⁺ in one and the same strip. To observe the concentration-dependent effect of 8-Br-cGMP on the contraction induced by Ca²⁺ (0.3 µM) in the presence of NA, 0.3 µM Ca²⁺ together with GTP (30 µM) was first applied to produce a contraction, then NA (10 µM) was applied in the presence of 0.3 µM Ca²⁺ + GTP. Finally, various concentrations of 8-Br-cGMP (0.01-10 µM) were cumulatively applied from low to high in the presence of 0.3 µM Ca²⁺ + GTP + NA. When the effect of olmesartan was to be examined on the above action of 8-Br-cGMP in skinned smooth muscle, olmesartan (1 µM) was applied for 4 h in Krebs solution (before skinning) and was present throughout the experiment (i.e. during and after skinning).

The effect of Ang II (0.1 nM) on the action of 8-Br-cGMP was examined in the presence of 0.2 µM Ca²⁺ plus 30 µM GTPS. To this end, GTPS was first applied in a relaxing solution, then 0.2 µM Ca²⁺ was applied in the presence of GTPS. Finally, various concentrations of 8-Br-cGMP (0.01-10 µM) were cumulatively applied from low to high in the presence of 0.2 µM Ca²⁺ + GTPS. When used, Ang II (0.1 nM) was applied together with GTPS in relaxing solution and was present throughout the experiment.

Assay for cGMP

After equilibration for 2 h in Krebs solution, strips with or without endothelium were further incubated for 4 h in the absence or presence of olmesartan (1 µM). Either ACh (final concentration, 0.1 or 1 µM) in the case of endothelium-intact strips, or NOC-7 (0.1 or 1 µM) in the case of endothelium-denuded strips was then added for 5 min, the reaction being halted by soaking the strips in ice-cold 8 % trichloroacetic acid. In some endothelium-intact strips, L-NNA (0.1 mM) was used for pre-treatment for 1 h before the application of ACh and was present thereafter. Strips were homogenized in a solution containing trichloroacetic acid in a glass homogenizer, the homogenate was centrifuged and the pellet used for the protein assay, which was a modified Lowry assay procedure (DC Protein Assay Kit; Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as standard. The supernatant fraction was treated with water-saturated ether three times and assayed for cGMP using an enzyme-immunoassay kit (Amersham Pharmacia Biotech, Tokyo, Japan).

Western blot analysis

Endothelium-intact strips were homogenized in a solution containing 62.5 mM Tris-HCl (pH 6.8), 10 % glycerol and 2 % SDS. After centrifugation, proteins were quantified in the supernatant. Protein samples (45 µg) were heated for 5 min at 100 °C in sample buffer (62.5 mM Tris-HCl, 10 % glycerol, 2 % SDS, 5 % 2-mercaptoethanol and 0.0025 % bromophenol blue), electrophoresed on SDS-gradient polyacrylamide gel (4-20 % for AT₁Rs and 2-15 % for ACE), then transferred to nitrocellulose membranes. The membranes were rinsed with phosphate-buffered saline (PBS) solution (10 mM Na₂HPO₄ and 250 mM NaCl, pH 7.6), then incubated with a primary antibody overnight at 4 °C. Following a washout with PBS solution, the membranes were incubated for 1 h at room temperature with a secondary antibody together with 1 % BSA. A polyclonal antibody against AT₁Rs (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a monoclonal antibody against ACE (1:100 dilution; QED Bioscience Inc., San Diego, CA, USA) were used as primary antibody, with peroxidase-conjugated IgG being used as the secondary antibody (1:1000 dilution). The signals from the immunoreactive bands were detected by means of an enhanced-chemiluminescence-detection system (SuperSignal, West Pico, Pierce, IL, USA) using Hyperfilm (Amersham Pharmacia Biotech, Buckinghamshire, UK). The density of the protein was measured by densitometric scanning as described previously (Asano et al. 2001).

Histochemical examination

Segments of mesenteric arteries were fixed in methacarn fixative (absolute methanol combined with chloroform and acetic acid) for 3 h, dehydrated in ethanol and embedded in paraffin. Sections were then cut at 3 µm thickness for immunohistochemistry and haematoxylin-eosin staining. After deparaffinizing with xylene, followed by a wash with PBS, the sections were treated with 0.3 % hydrogen peroxide in methanol for 30 min to inactivate endogenous peroxidase and then incubated overnight at 4 °C either with guinea-pig anti-Ang II serum as the primary antibody (1:100 dilution; Peninsula Laboratories Inc., San Carlos, CA, USA) or with a monoclonal antibody against the smooth-muscle-specific isoform of myosin heavy chain SM2 (1:300 dilution; Yamasa Corporation, Chiba, Japan). The control for the specificity of the Ang II antibody consisted of its pre-incubation with Ang II peptide (0.1 mM) for 1 h at 37 °C in PBS followed by incubation of the sections with the mixture overnight at 4 °C. The sections were rinsed and incubated sequentially with secondary antibody and the streptavidin-biotin-peroxidase complex, using a peroxidase-labelled streptavidin-biotin complex system (Histofine SAB-PO kit; Nichirei, Tokyo, Japan). The secondary antibodies used in procedures involving Ang II antibody and SM2 antibody were goat biotinylated anti-guinea-pig IgG antibody (1:400 dilution; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and goat biotinylated anti-mouse IgG antibody (1:400 dilution; Dako, Tokyo, Japan), respectively. After the sections had been washed with PBS, the peroxidase reaction was developed by incubation in a 0.02 % solution of 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co., St Louis, MO, USA) containing 0.003 % hydrogen peroxide and 10 mM sodium azide. Haematoxylin was used as the counterstain. For histological studies, sections from each artery were stained with haematoxylin and eosin. Immuno-staining was examined using a CCD camera (C6790; Hamamatsu Photonics, Hamamatsu, Japan) fitted to a microscope (ECLIPSE TE300, 40 oil-immersion objective lens, NA 1.3; Nikon, Tokyo, Japan). The images were captured using commercial software (AquaCosmos; Hamamatsu Photonics, Hamamatsu, Japan). These experiments were performed in such a way that control and test images were captured under the same conditions.

Solutions

The ionic composition of the Krebs solution was as follows (mM): Na⁺ 137.4; K⁺ 5.9; Mg²⁺ 1.2; Ca²⁺ 2.6; HCO₃^- 15.5; H₂PO₄^- 1.2; Cl^- 134; glucose 11.5. High-K⁺ solution (128 mM) was prepared by isosmotically replacing sodium chloride with potassium chloride. All the solutions used in the present experiments contained guanethidine (5 µM, to prevent effects due to release of sympathetic transmitters). The solutions were bubbled with 95 % O₂ and 5 % CO₂ and the pH was adjusted to 7.3-7.4 using NaOH and HCl.

The relaxing solution used in the skinned-muscle experiments contained (mM): 4 EGTA, 87 potassium methanesulphonate (KMS), 5.1 Mg(MS)₂, 5.2 ATP, 5 creatine phosphate, 20 Pipes, and 3 µM A23187 and 0.1 µM calmodulin. The pH was adjusted to 7.1 at 25 °C using KOH and the ionic strength was standardized at 0.18 M by changing the amount of KMS added. The free-Ca²⁺ concentration was calculated as described previously (Itoh et al. 1986).

Drugs

The drugs used in the current experiments were as follows: noradrenaline (NA), 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), histamine, -escin, calmodulin, diclofenac sodium and enalaprilat-HCl (Sigma Chemical Co.), N^G-nitro-L-arginine (L-NNA), bradykinin and Hoe 140 (Peptide Institute Inc., Osaka, Japan), A23187 (free acid; Calbiochem, La Jolla, CA, USA), GTP and GTPS (Bohringer Mannheim, Mannheim, Germany), 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7; Dojindo, Kumamoto, Japan), ACh-HCl (Daiichi Pharmaceutical, Tokyo, Japan) and guanethidine (Tokyo Kasei, Tokyo, Japan). Olmesartan and temocaprilat were kindly provided by Sankyo Pharmaceutical Co. (Tokyo, Japan), CV-11974 by Takeda Pharmaceutical Co. (Osaka, Japan) and famotidine by Yamanouchi Pharmaceutical Co. (Tokyo, Japan). Olmesartan and temocaprilat were dissolved in 2.5 % NaHCO₃ (as a 50 mM stock solution), CV-11974 in 5 % Na₂CO₃ (as a 10 mM stock solution) and NOC-7 in 0.1 M NaOH (as a 10 mM stock solution). All the stock solutions were stored at -80 °C and used within a week. These drugs were diluted in Krebs solution to the required final concentrations immediately before use. A23187 was dissolved in DMSO (as a 10 mM stock solution) and diluted in a relaxing solution. All other drugs were dissolved in ultra-pure Milli-Q water (Japan Millipore Corp., Tokyo, Japan). At their final concentration in Krebs solution (less than 0.1 %), none of the solvents had any noticeable effect on muscle contraction or relaxation.

Statistical analysis

The EC₅₀ values for the relaxant actions of ACh and 8-Br-cGMP were obtained by fitting the data points for each strip by a non-linear least-squares method using software (Kaleida graph; Synergy Software, PA, USA) written for the Macintosh Computer (Apple Co.). All results are expressed as the mean ± S.E.M. The n values represent the number of strips. A two-way repeated-measures ANOVA (followed by Scheffé's post hoc F test) or Student's paired or unpaired t test with an F test were used for statistical analysis. The level of significance was set at P < 0.05.

	RESULTS

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Immunohistochemical study

In rabbit mesenteric resistance arteries, diffuse staining for angiotensin II (Ang II) was detected in media and adventitia regions in each vessel examined (n = 5; Fig. 1Aa). In some preparations (two out of five preparations from five animals), faint staining of the endothelium was also noted. When Ang II peptide (0.1 mM) was applied with the primary antibody against Ang II, the staining was almost abolished under identical conditions, indicating the specificity of this particular primary Ang II antibody for Ang II (n = 5; Fig. 1Ab), as noted previously (Kohler et al. 1997; Alliot et al. 1999). As shown in Fig. 1Ac, the Ang II staining in the media region appeared similar in distribution to that of the staining seen when we used a monoclonal antibody against the smooth-muscle-specific isoform of myosin heavy chain SM2 (Aikawa et al. 1993).

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Figure 1. Immunostaining for angiotensin II (Ang II) together with Western blot analysis of angiotensin-converting enzyme (ACE) and Ang II type-1 receptor (AT1R) Aa, immunostaining against Ang II (brown) can be seen in the vascular wall. Ab, specificity was verified by co-application of Ang II peptide (0.1 mM) and its primary antibody. Ac, immunofluorescence staining of the smooth-muscle-specific isoform of myosin heavy chain SM2. Similar observations were made in other sections (obtained from 4 preparations from 4 other animals). B, Western blot analysis of ACE and AT1R. Homogenates of rabbit mesenteric arteries were separated by electrophoresis (SDS-gradient polyacrylamide gels). The positions of the molecular mass markers are shown. Similar observations were made in three other experiments.

By Western blot analysis, immunoreactive proteins against angiotensin-converting enzyme (ACE) and angiotensin type-1 receptor (AT₁R) were identified in these arteries (n = 4; Fig. 1B). The apparent molecular mass of ACE was estimated to be ~190 kDa and that of AT₁R ~50 kDa. These values are roughly the same as those reported previously in vascular cells (for ACE, ~170 kDa; Papapetropoulos et al. 1996 and for AT₁R, 41~61 kDa; Touyz & Schiffrin, 2000; de Gasparo et al. 2000).

Effects of Ang II inhibitors in endothelium-intact strips

ACh produces an endothelium-dependent relaxation in rabbit mesenteric resistance arteries (Yamashita et al. 1999). In the course of a 160 min application, the AT₁R-blocker olmesartan (1 µM) significantly enhanced the ACh (0.03 µM)-induced relaxation (n = 5; P < 0.05) but had no effect on the NA (10 µM)-induced tension (P > 0.05; Fig. 2). Following a washout of the olmesartan, the ACh (0.03 µM)-induced relaxation was progressively increased, as a function of time, with a concomitant decrease in the NA-induced tension (until the end of the experimental period at 290 min, n = 5; for ACh and NA, P < 0.01) (Fig. 2). At time (t) = 160 min, the effect of olmesartan on the ACh (0.03 µM)-induced relaxation was concentration dependent: by 1.46 ± 0.08 times control at 0.03 µM (n = 4, P < 0.05), by 1.78 ± 0.04 times at 0.1 µM (P < 0.01) and by 2.11 ± 0.21 times at 1 µM (P < 0.01). When a longer application of olmesartan (1 µM) was used (for 290 min), its effects at 290 min were not significantly different from those seen at the same time point when this agent was applied for 160 min followed by a 130 min washout (NA-induced contraction: n = 4, P > 0.1; ACh-induced relaxation, P > 0.1). At t = 160 min, CV-11974 (1 µM, another AT₁R blocker) had effects similar to those of olmesartan: it significantly enhanced the ACh (0.03 µM)-induced relaxation (n = 4, P < 0.05) without changing the NA (10 µM)-induced tension (P > 0.05; Fig. 3A).

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Figure 2. Effects of olmesartan (AT1R blocker) and temocaprilat (ACE inhibitor) on noradrenaline (NA)-induced contraction and ACh-induced relaxation in endothelium-intact strips A, effects of olmesartan (1 µM) and temocaprilat (1 µM) on NA (10 µM)-induced contraction. Olmesartan or temocaprilat was applied as indicated by the bar. Time-matched NA-induced control response (i.e. without either inhibitor) is included as the 'No drug' control. B, effects of olmesartan and temocaprilat on ACh-induced relaxation. Ba, 0.03 µM ACh. Bb, 0.1 µM ACh. Bc, 0.3 µM ACh. The time-matched ACh response (i.e. without either inhibitor) at each concentration is included as the 'No drug' control. Data are shown as means and S.E.M. * P < 0.05, ** P < 0.01 vs. 'No drug' control (two-way repeated-measures ANOVA and Scheffé's F test).

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Figure 3. Effects of CV-11974 (AT1R blocker) and enalaprilat (ACE inhibitor) on NA-induced contraction and ACh-induced relaxation, and influence of NG-nitro-L-arginine (L-NNA) over the effect of the AT1R blocker olmesartan A, effects of CV-11974 (1 µM) and enalaprilat (1 µM) on NA (10 µM)-induced contraction and ACh (0.03 µM)-induced relaxation in endothelium-intact strips. After records had been obtained of the control responses to NA and ACh (labelled 'No drug'), one of the agents was pre-treated for 160 min and the NA and ACh responses were observed in its presence. Data are shown as means and S.E.M. * P < 0.05 vs. 'No drug' control (Student's unpaired t test). B, effects of olmesartan (1 µM) on the contraction induced by NA (0.5 µM) and the ACh (0.03 µM)-induced relaxation of this contraction in endothelium-intact strips in the presence of L-NNA (0.1 mM). The ACh (0.03 µM)-induced relaxation was first recorded during the contraction induced by 10 µM NA. Next, the strips were treated with L-NNA for 60 min and then the ACh (0.03 µM)-induced relaxation was observed during the contraction induced by 0.5 µM NA. Subsequently, olmesartan (1 µM) was applied for 160 min in the presence of L-NNA and finally the ACh (0.03 µM)-induced relaxation was observed during the contraction induced by 0.5 µM NA in the presence of L-NNA + olmesartan. Data are shown as means and S.E.M. There were no statistically significant differences in panel B (Student's paired t test).

By the end of a 160 min application, the ACE inhibitor temocaprilat (1 µM) had significantly enhanced the ACh (0.03 µM)-induced relaxation (n = 5; P < 0.01) without changing the NA-induced tension (P > 0.05). Following a washout of the temocaprilat, the ACh (0.03 µM)-induced relaxation was progressively increased, as a function of time, with a concomitant decrease in the NA-induced tension (until the end of the experimental period at 290 min, n = 5; for ACh and NA, P < 0.01; Fig. 2). At t = 160 min, enalaprilat (1 µM, another ACE inhibitor) had similar effects to temocaprilat: it enhanced the ACh (0.03 µM)-induced relaxation (n = 4, P < 0.05) without changing the NA (10 µM)-induced tension (P > 0.05; Fig. 3A and B).

When the effect of a co-application of olmesartan (1 µM) and temocaprilat (1 µM) on the ACh (0.03 µM)-induced relaxation during the NA (10 µM)-induced contraction was examined at t = 160 min, the effect was not significantly different from that obtained when either agent was applied alone (n = 4; P > 0.1 in each case).

Effect of L-NNA on the action of Ang II inhibitors in endothelium-intact strips

In endothelium-intact strips, L-NNA (0.1 mM) enhanced the contraction induced by 10 µM NA by 1.67 ± 0.13 times control (n = 5; P < 0.05) and, in the presence of L-NNA, 0.5 µM NA produced a tension (27.5 ± 2.6 mg) comparable to that induced by 10 µM NA before the application of L-NNA (28.5 ± 2.2 mg, P > 0.1; Fig. 3Ba). We therefore examined the effect of L-NNA on the action of olmesartan (at 160 min after the latter's application) on the ACh (0.03 µM)-induced relaxation during the contraction induced by 0.5 µM NA. In the presence of L-NNA, olmesartan (1 µM) did not significantly modify the ACh-induced relaxation (n = 5, P > 0.1; Fig. 3Bb). Similarly, L-NNA abolished the enhancement by temocaprilat (1 µM, at 160 min after its application) of the ACh (0.03 µM)-induced relaxation during the contraction induced by 0.5 µM NA (n = 5, P > 0.1). Diclofenac (3 µM) did not modify the effect of olmesartan (1 µM, at the end of a 160 min application) on the ACh (0.03 µM)-induced relaxation during the NA (0.5 µM)-induced contraction in the presence of L-NNA (n = 4; P > 0.1) (data not shown).

Effect of olmesartan in endothelium-denuded strips

In strips denuded of their endothelium, the NO donor NOC-7 (1-100 nM) produced a concentration-dependent relaxation on the contraction induced by NA (10 µM). By the end of a 160 min application, olmesartan (1 µM) had significantly enhanced the NOC-7 (10 nM)-induced relaxation (n = 5; P < 0.01) without significantly changing the NA-induced tension (P > 0.05; Fig. 4). Following a washout of olmesartan, the NOC-7 (10 nM)-induced relaxation was progressively increased, as a function of time, with a concomitant decrease in the NA-induced tension (until the end of the experimental period at 290 min, n = 5; P < 0.01). Furthermore, olmesartan (1 µM, at 160 min after its application) also enhanced the NOC-7 (10 nM)-induced relaxation (1.78 ± 0.28 times control, n = 4, P < 0.05) when 10 µM histamine (instead of NA) was used to produce a contraction in the presence of famotidine (3 µM, an H₂ receptor blocker). At this time point, olmesartan did not significant modify the histamine-induced tension (P > 0.05). Following a washout of the olmesartan for 130 min, the NOC-7 (10 nM)-induced relaxation was progressively increased, as a function of time, with a concomitant decrease in the histamine-induced tension (36.8 ± 3.7 mg and 19.8 ± 3.4 mg before olmesartan application and at 290 min from the start of the application, respectively, n = 5; P < 0.05).

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Figure 4. Effects of olmesartan on NA-induced contraction and NOC-7-induced relaxation in endothelium-denuded strips A, time-dependent changes in NA (10 µM)-induced tension in the absence (Control, labelled 'No drug') and presence of olmesartan (1 µM). Olmesartan was applied as indicated by the bar. B, effect of olmesartan on NOC-7-induced relaxation. Ba, 1 nM NOC-7. Bb, 10 nM NOC-7. Bc, 100 nM NOC-7. Data are shown as means and S.E.M. * P < 0.05, ** P < 0.01 vs. 'No drug' control (two-way repeated-measures ANOVA and Scheffé's F test).

Effect of bradykinin in endothelium-intact strips

Bradykinin, at 0.1 µM, produces a near-maximal endothelium-dependent relaxation through an activation of B₂ receptors in arteries from various animal species (Vanhoutte et al. 1995). However, bradykinin (0.1 µM) did not produce a significant relaxation of the NA (10 µM)-induced contraction in endothelium-intact strips from the rabbit mesenteric artery (0.3 ± 0.3 % inhibition, n = 5). Even at 0.3 µM, this peptide produced only a marginal relaxation (6.8 ± 3.1 % inhibition, n = 5; P > 0.05). Neither Hoe 140 (a B₂ receptor antagonist, 0.1 µM) nor bradykinin (0.1 µM) modified the concentration-dependent relaxation to ACh (0.03-0.3 µM) of the NA (10 µM)-induced contraction in endothelium-intact strips. The EC₅₀ values for the relaxation induced by ACh were 47.2 ± 11.3 nM and 44.5 ± 7.9 nM, respectively, in the absence and presence of Hoe 140 (n = 5; P > 0.5) and 62.6 ± 11.0 nM and 71.0 ± 29.9 nM, respectively, in the absence and presence of bradykinin (n = 5; P > 0.5).

Effects of olmesartan on cGMP production

Under basal conditions, the concentration of cGMP was 115 ± 17 fmol (mg protein)^-1 in endothelium-intact strips (n = 10) and 51 ± 11 fmol (mg protein)^-1 in endothelium-denuded strips (n = 8; P < 0.05). ACh (0.1 and 1 µM) concentration dependently increased the production of cGMP in endothelium-intact strips (at 0.1 µM, 564 ± 60 fmol (mg protein)^-1 and at 1 µM, 889 ± 41 fmol (mg protein)^-1, n = 5; P < 0.001 in each case vs. the basal condition). L-NNA (0.1 mM) greatly reduced the concentration of cGMP whether applied in the absence or in the presence of 0.1 µM ACh (n = 4; P < 0.05; Fig. 5A). By contrast, olmesartan (1 µM) had no effect on the concentration of cGMP in the presence or absence of 0.1 µM ACh in endothelium-intact strips. In endothelium-denuded strips, NOC-7 (0.1 and 1 µM) concentration dependently increased the production of cGMP (at 0.1 µM, 247 ± 34 fmol (mg protein)^-1 and at 1 µM, 552 ± 34 fmol (mg protein)^-1, n = 4; P < 0.01 in each case vs. the basal condition). Olmesartan had no effect whether applied in the absence or in the presence of 0.1 µM NOC-7 (n = 4; P > 0.5 in each case; Fig. 5B).

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Figure 5. Effect of olmesartan on production of cGMP A, effects of olmesartan (1 µM) and L-NNA (0.1 mM) on cGMP production in the absence and presence of ACh (0.1 µM) in endothelium-intact strips. B, effect of olmesartan in the absence and presence of NOC-7 (100 nM) in endothelium-denuded strips. Data are shown as means and S.E.M. ** P < 0.01 vs. 'ACh (-)' or 'NOC-7 (-)' (Student's unpaired t test).

Effects of olmesartan and Ang II on cGMP-induced relaxation

In -escin-skinned strips, Ca²⁺ concentrations over 0.1 µM produced a contraction, with the maximum being obtained at 10 µM Ca²⁺ (Itoh et al. 1986). In the presence of 30 µM GTP: (a) the amplitude of the contraction induced by 0.3 µM Ca²⁺ was 0.11 ± 0.05 times that induced by 10 µM Ca²⁺ (n = 8) and (b) NA (10 µM) enhanced the Ca²⁺ (0.3 µM)-induced contraction (1.79 ± 0.13 times that seen with 0.3 µM Ca²⁺ + GTP, n = 8). Olmesartan had no effect on the contraction induced by 0.3 µM Ca²⁺ + GTP (relative tension of 0.11 ± 0.06 when normalized with respect to that induced by 10 µM Ca²⁺, n = 8; P > 0.5) but it attenuated the NA-induced enhancement of the Ca²⁺-induced contraction (1.36 ± 0.08 times that induced by 0.3 µM Ca²⁺ + GTP, n = 8; P < 0.05; Fig. 6A).

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Figure 6. Effect of olmesartan on the relaxation induced by 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) in -escin-skinned strips A, actual tracings (obtained in the absence or presence of 1 µM olmesartan) of the concentration-dependent effect of 8-Br-cGMP on the contraction induced by 0.3 µM Ca2+ + GTP (30 µM) followed by NA (10 µM) in -escin-skinned strips. After the response to 10 µM Ca2+ had been recorded, 0.3 µM Ca2+ with GTP was applied for 5 min. NA (10 µM) was then added and finally 8-Br-cGMP (0.01-10 µM, open bars) was cumulatively applied in the presence of 0.3 µM Ca2+ + NA + GTP. B, summary of the effect of 8-Br-cGMP (in the absence or presence of 1 µM olmesartan) on the 0.3 µM Ca2+-induced contraction in the presence of NA + GTP in -escin-skinned strips. Data are shown as means ± S.E.M. * P < 0.05, ** P < 0.01 vs. 'Olmesartan (-)' (two-way repeated-measures ANOVA and Scheffé's F test).

During the contraction induced by 0.3 µM Ca²⁺ + GTP with NA, 8-Br-cGMP (0.01-10 µM) produced a concentration-dependent relaxation. Olmesartan significantly enhanced the 8-Br-cGMP-induced relaxation without changing its EC₅₀ value (0.25 ± 0.05 µM in non-olmesartan-treated strips and 0.15 ± 0.06 µM in olmesartan-treated strips, n = 8; P > 0.1; Fig. 6B).

GTPS enhances the contraction induced by low concentrations of Ca²⁺ in skinned smooth muscle of the rabbit mesenteric artery (Fujiwara et al.1989). We studied the effect of Ang II (0.1 nM) on the concentration-dependent relaxation induced by 8-Br-cGMP of the contraction induced by 0.2 µM Ca²⁺ in the presence of GTPS (30 µM) in -escin-skinned strips (Fig. 7A). The relative amplitude of the contraction induced by 0.2 µM Ca²⁺ + GTPS was 0.31 ± 0.05 (n = 4) when normalized with respect to that induced by 10 µM Ca²⁺. Ang II (0.1 nM) failed to produce a contraction in a relaxing solution containing GTPS (relative tension = 0.01 ± 0.00, n = 8) and it did not modify the amplitude of the contraction induced by 0.2 µM Ca²⁺ + GTPS (relative tension of 0.29 ± 0.04, n = 4; P > 0.5).

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Figure 7. Effect of Ang II on the relaxation induced by 8-Br-cGMP in -escin-skinned strips A, actual tracings (obtained in the absence or presence of 0.1 nM Ang II) of the concentration-dependent effect of 8-Br-cGMP on the contraction induced by 0.2 µM Ca2+ in the presence of GTPS (30 µM) in -escin-skinned strips. After the response to 10 µM Ca2+ had been recorded (not shown), GTPS with or without Ang II (0.1 nM) was applied for 10 min in a relaxing solution and 0.2 µM Ca2+ was then applied as shown. Finally, 8-Br-cGMP (0.01-10 µM) was cumulatively applied in the presence of 0.2 µM Ca2+ + GTPS with or without Ang II. B, summary of the effects of 8-Br-cGMP on Ca2+-induced contraction in the presence of GTPS with or without Ang II in -escin-skinned strips. Data are shown as means and S.E.M. The error bars are within the symbols. * P < 0.05, ** P < 0.01 vs. 'Angiotensin II (-)' (two-way repeated-measures ANOVA and Scheffé's F test).

In the presence of 0.2 µM Ca²⁺ + GTPS, 8-Br-cGMP (0.01- 10 µM) produced a concentration-dependent relaxation and Ang II (0.1 nM) significantly inhibited the 8-Br-cGMP-induced relaxation with a rightward shift in the concentration-response relationship (Fig. 7B). The EC₅₀ values for 8-Br-cGMP were 69.3 ± 4.2 nM (n = 4) and 80.3 ± 3.5 nM (n = 4; P < 0.01) in the absence and presence of Ang II, respectively.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

In this study, immunoreactive angiotensin II (Ang II) was found to be localized to the media and adventitia regions in rabbit mesenteric resistance arteries, as is also the case in human coronary artery and rat aorta (Haller et al. 1996; Ohishi et al. 1999) The distribution of Ang II in the media region appeared similar to that of the smooth-muscle-specific isoform of myosin heavy chain SM2, suggesting that Ang II may be present in the smooth muscle cells. Furthermore, the presence of immunoreactive angiotensin-converting enzyme (ACE) as well as Ang-II type-1 receptor (AT₁R) was also identified in these arteries. These results suggest that a local rennin-angiotensin system (RAS) may operate in rabbit mesenteric resistance arteries. In our subsequent experiments, the main findings were as follows. AT₁R blockers (olmesartan and CV-11974) and ACE inhibitors (enalaprilat and temocaprilat) had similar effects: each drug enhanced the ACh-induced, endothelium-dependent relaxation during the contraction induced by NA. The actions of the AT₁R blockers as well as those of the ACE inhibitors in endothelium-intact strips were abolished by L-NNA (an inhibitor of NO synthase) but the effect of L-NNA was not modified by co-application of diclofenac (a cyclo-oxygenase inhibitor). These results suggest that Ang II localized within smooth muscle cells activates AT₁Rs and plays a significant role in rabbit mesenteric resistance arteries by inhibiting the ACh-induced relaxation mediated by endothelium-derived NO.

Role of Ang II in NO-induced relaxation

It has been suggested that ACE inhibitors enhance agonist-induced, endothelium-dependent relaxation due (1) to an inhibition of the action of Ang II (by inhibiting its synthesis) and (2) to an increase in the action by which bradykinin releases endothelium-derived relaxing factors (via an inhibition of its breakdown) (Touyz & Schiffrin, 2000; Tabibiazar et al. 2001). In addition, in dog coronary arteries and hypertensive-rat aorta Ang II activates AT₂ receptors and increases the release of endothelium-derived NO (an action mediated by bradykinin), thus enhancing the production of cGMP (Seyedi et al. 1995; Gohlke et al. 1998). These findings suggest that in some blood vessels, bradykinin plays a significant role in the enhancement of the ACh-induced endothelium-dependent relaxation that is seen not only with ACE inhibitors but also with AT₁R blockers. In the present experiments, however, bradykinin (0.1 µM) did not produce an endothelium-dependent relaxation and neither bradykinin (0.1 µM) nor Hoe 140 (0.1 µM, a B₂ receptor antagonist) modified the ACh-induced endothelium-dependent relaxation, indicating that bradykinin is unlikely to be a mediator of the effects of AT₁R blockers and ACE inhibitors in rabbit mesenteric resistance arteries. Furthermore, we found that during the contraction induced by NA, AT₁R blockade enhanced both the endothelium-dependent NO-mediated relaxation induced by ACh and the endothelium-independent relaxation induced by NOC-7 (an NO donor, Hrabie et al. 1993). An enhancement of the NOC-7-induced relaxation by AT₁R blockade was also observed when the contraction was induced by H₁-receptor activation. These results suggest that in rabbit mesenteric resistance arteries, the action of NO in smooth muscle cells is enhanced by AT₁R blockade.

There are several possibilities for the mechanism underlying the Ang II-induced inhibition of NO-mediated smooth muscle relaxation. In aortae obtained from rat and rabbit, Ang II increases the production of superoxide anion, which rapidly binds to and inactivates NO (Rajagopalan et al. 1988; Pagano et al. 1997; Wang et al. 1999). Thus, if an AT₁R blocker attenuates superoxide anion production, such an agent would be expected to enhance NO-induced relaxation. However, in the present experiments AT₁R blockade had no effect on the amount of cGMP produced (1) in the absence or presence of ACh (in endothelium-intact strips) or (2) in the absence or presence of NOC-7 (in endothelium-denuded strips), even though the cellular concentration of cGMP would be expected to increase if AT₁R blockade ameliorated the NO breakdown induced by the superoxide anion. Hence, an inhibition of superoxide anion production by AT₁R blockers is an unlikely explanation for their observed effects. Most importantly, we found that AT₁R blockade enhanced the relaxation induced by cGMP (0.01-10 µM) during the Ca²⁺-induced contraction in -escin-skinned smooth muscle. Conversely, Ang II (0.1 nM) decreased the sensitivity shown by the skinned muscle to cGMP in the presence of Ca²⁺. In accordance with these findings, it was recently noted that that chronic administration of an ACE inhibitor tended to improve nitroglycerine-induced vasodilatation in the brachial artery in patients with coronary heart disease (Anderson et al. 2000). Taken together, all these pieces of evidence suggest that in the rabbit mesenteric resistance artery, AT₁R blockade enhances the relaxation response to cGMP in the smooth muscle cells and thereby enhances the relaxation mediated by endothelium-derived NO.

Role played by Ang II in agonist-induced contraction

In addition to their enhancing action on NO-mediated relaxation (described above), the AT₁R blocker olmesartan and the ACE-inhibitor temocaprilat each attenuated the NA-induced contraction in both endothelium-intact and -denuded strips, although this action became apparent only after a long latency (the agent needed to be present for over 160 min). The inhibition was larger in endothelium-intact strips than in endothelium-denuded strips, while in endothelium-intact strips L-NNA roughly halved the effect of the AT₁R blocker (from ~60 % inhibition to ~30 % inhibition), an effect of the same extent as that seen in endothelium-denuded strips (authors' unpublished observation). Furthermore, olmesartan attenuated the contraction induced by H₁-receptor activation when this agent had been present for over 160 min. Moreover, in -escin-skinned strips this AT₁R blocker inhibited the NA-induced enhancement of the Ca²⁺-contraction without changing the Ca²⁺-contraction itself. These results suggest that blocking AT₁Rs inhibits agonist-induced smooth muscle contractions by enhancing the function of endothelium-derived NO as well as by inhibiting the mechanism responsible for the agonist-induced smooth muscle contraction itself. In other words, Ang II localized within the vascular wall in the rabbit mesenteric artery may enhance the NA-induced contraction via both an inhibition of the function of endothelium-derived NO and an increase in agonist-induced myofilament Ca²⁺ sensitivity.

Agonists increase the Ca²⁺ sensitivity of vascular smooth muscle cells (agonist-induced myofilament Ca²⁺ sensitization, Somlyo & Somlyo, 1994; Kuriyama et al. 1998). In various types of vascular smooth muscle cells, Ang II activates protein kinase C (PKC), which in turn increases the myofilament Ca²⁺ sensitivity by phosphorylating CPI-17 (Touyz & Schiffrin, 2000; Woodsome et al. 2001). Ang II is also known to activate tyrosine kinase, which may be involved in agonist-induced myofilament Ca²⁺ sensitization in the rabbit mesenteric artery (Sasaki et al. 1998; Touyz & Schiffrin, 2000). Furthermore, it is well established that agonists enhance the myofilament Ca²⁺ sensitivity through an action of rho kinase in vascular smooth muscle (Somlyo & Somlyo, 1994; Uehata et al. 1997). Thus, although at present the mechanism underlying the effect of AT₁R blockade on the NA-induced increase in myofilament Ca²⁺ sensitivity remains unknown, a change in the mutual relationships among these kinases in the signal-transduction cascade may well be the mechanism responsible in the smooth muscle of the rabbit mesenteric resistance artery.

Ang II enhances sympathetic nerve activity via AT₁Rs and thus increases the release of NA from the nerve endings (Touyz & Schiffrin, 2000). However, it seems unlikely that this was a factor in our results since all the solutions used contained 5 µM guanethidine (which is sufficient to inhibit the release of NA from the sympathetic endings in the rabbit mesenteric artery: Mishima et al. 1984).

In the present experiments, the time required for the actions of AT₁R blockers and ACE inhibitors to reach significance was quite long (over 160 min) and the effects continued even after a 2-h washout of these agents. It should be mentioned that Ang II is present in the cytosol (Anderson et al. 1993; Haller et al. 1996) and, when injected intracellularly, it increases [Ca²⁺]_i in cultured smooth muscle cells derived from the rat aorta, a response that is inhibited by a co-injected AT₁R blocker (Haller et al. 1996). Furthermore, it has been suggested that endocytosis of membrane-bound polypeptide receptors may play an active role in intracellular signalling (Griendling et al. 1987; Jans, 1994). Thus, if the site of action of olmesartan is an AT₁R located within the cell, the slow time course we observed might reflect the length of time required for this agent to pass into the cell. This remains to be clarified.

The physiological role of the Ang II generated by local RAS in resistance arteries remains uncertain. Asada et al. (1999) recently suggested that Ang II generated by a local RAS might mobilize [Ca²⁺]_i in the smooth muscle cells of the isolated rat tail artery under resting conditions. We now propose a novel hypothesis: that in the rabbit mesenteric resistance artery, the Ang II generated by the local RAS enhances agonist-induced contraction, due in part to an enhancement of agonist-induced myofilament Ca²⁺ sensitization and also to a downregulation of cGMP-mediated relaxation. The precise mechanism remains to be clarified in future work.

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Acknowledgements

We thank Dr R. J. Timms for a critical reading of the manuscript. We also thank Professor H. Soji, Drs T. Yamamoto and N. Kusama and Mr T. Kamiya for their cooperation in this study. This work was partly supported by a Grant-In-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

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