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Am J Physiol Heart Circ Physiol 287: H545-H552, 2004. First published March 25, 2004; doi:10.1152/ajpheart.01098.2003
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Chronic AT1 receptor blockade alters mechanisms mediating responses to hypoxia in rat skeletal muscle resistance arteries

Shane A. Phillips, Ines Drenjancevic-Peric, Jefferson C. Frisbee, and Julian H. Lombard

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Submitted 1 December 2003 ; accepted in final form 15 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The goal of this study was to determine the effect of angiotensin type 1 (AT1) receptor antagonism on vasodilator responses in isolated skeletal muscle resistance arteries. Normotensive Sprague-Dawley rats were fed normal rat chow with the AT1 receptor antagonist losartan (1mg/ml) in the drinking water for 7 days and compared with untreated control rats. Changes in the diameter of isolated resistance arteries supplying the gracilis muscle were assessed with a video micrometer. Arteriolar responses to acetylcholine, iloprost, and sodium nitroprusside were unaffected by losartan administration, whereas dilation to reduced PO2 was converted into a constriction. Hypoxia-induced constriction of vessels from losartan-treated rats was inhibited by endothelium removal or indomethacin (1 µM). Blockade of the PGH2-thromboxane A2 receptor with SQ-29548 (10 µM), thromboxane synthase inhibition with dazoxiben (10 µM), or the addition of the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL, 100 µM) converted hypoxic vasoconstriction to a dilation that was blocked by inhibiting nitric oxide synthase with N{omega}-nitro-L-arginine methyl ester (100 µM). These data suggest that AT1 receptor activation has an important role in maintaining the vascular release of prostaglandins responsible for mediating hypoxic dilation in skeletal muscle microvessels.

angiotensin II; angiotensin II receptors; vascular smooth muscle; endothelium; microcirculation

INCREASING EVIDENCE suggests that the renin-angiotensin system (RAS) is critical for maintaining arteriolar structure and vascular reactivity. For example, rats placed on a high-salt (HS) diet exhibit microvascular rarefaction (i.e., decreased microvessel density), alterations in the ultrastructure of individual microvessels, and a reduced relaxation to a variety of vasodilator stimuli, all of which are independent of elevations in blood pressure (10, 11, 15). Microvascular rarefaction and impaired relaxation to vasodilator stimuli developing in animals on HS diet appear to be due to the suppression of ANG II that occurs in response to a HS diet (11, 18, 27).

Previous studies from our laboratory indicate that the impaired relaxation of skeletal muscle resistance arteries to ACh, hypoxia, and the stable prostacyclin analog iloprost in vessels of animals on a HS diet is restored by maintaining circulating levels of angiotensin with a low-dose ANG II infusion (5 ng·kg–1·min–1) (18, 27, 28). Other studies have demonstrated that the protective effect of ANG II infusion to maintain vasodilator reactivity in animals receiving a HS diet is abolished by coinfusion of the angiotensin type 1 (AT1) receptor antagonist losartan. The results of these studies indicate that the action of ANG II to maintain arterial relaxation in response to these vasodilator stimuli is mediated via the AT1 receptor subtype in animals on a HS diet (28).

Taken together, these experimental observations may have particular clinical importance because many patients are administered angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers to treat cardiovascular diseases such as hypertension. Although others have evaluated the effect of ACE inhibition on responses to dilator stimuli during nonpathological conditions (5, 8), few studies have investigated the effects of AT1 receptor blockade alone on vascular relaxation. The purpose of the present study was to test the hypothesis that blockade of the AT1 receptor alters vasodilator responses during normal physiological conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Age-matched, male Sprague-Dawley rats (Harlan Teklad; Madison, WI) weighing 250–400 g at the time of use were used for all experiments. All rats were given drinking water [±losartan (1 mg/ml, Merck)] ad libitum for 1 wk. An additional group of rats on normal drinking water was given intravenous infusions of losartan (20 µg·kg–1·min–1) or isotonic saline for 1 wk. All rats were housed in an animal care facility at the Medical College of Wisconsin, which is approved by the American Association for the Accreditation of Laboratory Animal Care, and all protocols were approved by the Medical College of Wisconsin Animal Care Committee. Rats were weighed and anesthetized with an injection of pentobarbital sodium (50 mg/kg ip). After the induction of anesthesia, a carotid artery and jugular vein were cannulated with polyethylene tubing for arterial pressure measurement and ANG II infusion to verify the effectiveness of AT1 receptor blockade, respectively.

Chronic animal preparation. For studies employing an intravenous infusion of losartan, age-matched, male Sprague-Dawley rats were anesthetized with an injection of a 78:22 mixture of ketamine-acepromazine (1 mg/kg im). Saline-filled catheters were inserted into a femoral artery and vein and tunneled subcutaneously to the midscapular region, where they were externalized, encased in a stainless steel spring, and attached to a swivel to allow ambulatory measurement of mean arterial pressure (MAP) in the conscious animal as well as a continuous intravenous infusion of losartan (27). The catheters consisted of a 60-cm length of clear vinyl tubing (Tygon S-54-HL; Akron, OH) coupled to a 5-cm segment of small-caliber tubing (Critchley Electrical Products; Auburn, New South Wales, Australia). All rats were allowed to recover for 5 days before drug infusion or blood pressure measurement. Infusion of sterile isotonic saline was started 2–3 days after surgery to maintain catheter patency and confirm that the volume of infusion did not change blood pressure. One group of rats was then infused with losartan (20 µg·kg–1·min–1) or continued with saline infusion for 1 wk before studies of vascular reactivity in the isolated vessels.

Preparation of isolated arteries. For studies of isolated vessels, the small muscular branch of the femoral artery supplying the gracilis muscle was located using a dissecting microscope (Leica; Buffalo, NY) and carefully isolated from surrounding parenchymal tissue, as previously described (4). After a 30-min equilibration period in situ, the vessel was carefully excised and immediately immersed in warmed (37°C) physiological salt solution (PSS) bubbled with 21% O2-5% CO2-74% N2. The PSS used in these experiments had the following ionic composition (in mM): 119.0 NaCl, 4.7 KCl, 1.6 CaCl2, 1.18 NaH2PO4, 1.17 MgSO4, 24.0 NaHCO3, 5.5 dextrose, and 0.03 EDTA. The arteries were carefully handled by the adhering connective tissue only to avoid damage to the endothelium and smooth muscle layers.

The proximal and distal ends of the artery were cannulated and secured to glass micropipettes (100–150 µm, FHC; Brunswick, ME) using 10-0 nylon sutures in a superfusion-perfusion chamber (4, 17). The inflow pipette was connected to a reservoir perfusion system that allowed the intraluminal pressure and luminal gas concentrations to be controlled. Vessel diameter was measured using television microscopy and a video micrometer. Any artery that did not exhibit significant levels of active tone (as evidenced by a substantial increase in resting diameter upon exposure to Ca2+-free PSS) was not used in the study.

In another series of experiments, the effect of endothelial removal on the response of the vessels to simultaneous superfusion and perfusion with 0% O2 was determined. In these experiments, the endothelium was removed by perfusing the lumen with a 3- to 5-ml bolus of air, as previously described (4).

Verification of losartan efficacy. In these studies, rats received losartan in the drinking water (1 mg/ml) or via intravenous infusion (20 µg·kg–1·min–1), whereas control animals received normal drinking water ad libitum or infusion of the saline vehicle. Effective blockade of angiotensin receptors was confirmed by determining the ability of the losartan treatment to block the pressor response to an intravenous bolus injection of 25 ng/kg ANG II.

Response to reduced PO2. After the initial control period in PSS equilibrated with 21% O2, the response of gracilis arteries to reduced PO2 was assessed before and after endothelium removal and before and during application of the following pharmacological agents via the superfusate and perfusate: 1) the cyclooxygenase inhibitor indomethacin (1 µM); 2) the PGH2-thromboxane A2 (TXA2) receptor inhibitor SQ-29548 (10 µM, BioMol); 3) the thromboxane synthase inhibitor dazoxiben (10 µM); and 4) the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL, 100 µM, Sigma). In a separate series of experiments, responses of vessels to reduced PO2 were assessed before and during the combined application of SQ-29548 and indomethacin or SQ-29548 and the nitric oxide (NO) synthase (NOS) inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME; 100 µM). PO2 reduction was achieved by simultaneous perfusion and superfusion of the arteries for 25 min with PSS equilibrated with a 0% O2-5% CO2-95% N2 gas mixture, as previously described (4). Under these conditions, control values for PO2 during 21% O2 perfusion and superfusion are ~ 140 Torr, whereas equilibration of the PSS reservoirs with 0% O2 reduces both the luminal and extraluminal PO2 to ~35–45 Torr (4). After exposure of the vessels to hypoxia, the perfusate and superfusate were reequilibrated with 21% O2 for 20 min, and recovery from reduced PO2 was verified by measuring vessel diameter at the end of the reequilibration period in 21% O2.

Response to vasodilator agonists and Ca2+-free solution. Responses of the arteries to the stable prostacyclin analog iloprost (10 pg/ml), the endothelium-dependent vasodilator ACh (1 µM), and the NO donor sodium nitroprusside (1 µM) were also assessed in each group of rats. When these drugs were administered, the superfusion was briefly stopped in the bath, the artery was pressurized by clamping the outflow pipette, and an appropriate amount of drug was added to the PSS to achieve the final desired concentration. Vessel diameter was monitored continuously, and the maximum change in steady-state diameter was determined following addition of the dilator agent to the tissue bath. After the response of the arteries to the various vasodilator stimuli were determined, the vessels were maximally dilated with Ca2+-free relaxing solution containing the following constituents (in mM): 92.0 NaCl, 4.7 KCl, 1.17 MgSO4·7H2O, 20.0 MgCl·6H2O, 1.8 NaH2PO4, 24.0 NaHCO3, 0.026 EDTA, 2.0 EGTA, and 5.5 dextrose.

TXA2 and prostacyclin release during hypoxia. Isolated saphenous and gracilis arteries from three rats treated with normal water or losartan were pooled and immediately equilibrated in 2 ml PSS equilibrated with a 21% O2-5% CO2-74% N2 gas mixture maintained at 37 °C for 1 h, as previously described (18). The vessels were then equilibrated under control conditions (21% O2) for 1 h in 2 ml PSS, followed by a reduction in O2 concentration to 5% O2 for 1 h, to effectively lower bath PO2 to 35–45 mmHg. After each 1-h equilibration period, 2 ml of PSS was removed from the incubation chamber and immediately snap frozen in liquid N2.

Release of prostacyclin and TXA2 during different O2 conditions was assessed in the Physiology Department Biochemical Assay Core Facility at the Medical College of Wisconsin. These metabolites were evaluated with the use of commercially available enzyme immunoassay kits purchased by Cayman Chemical (Ann Arbor, MI). Prostacyclin and TXA2 release were simultaneously evaluated by measuring the stable PGI2 metabolite 6-keto-prostaglandin F1{alpha} (PGF1{alpha}) and the stable TXA2 metabolite TXB2 in the incubation medium.

Statistical analysis. All data are expressed as means ± SE. A paired Student's t-test was used to assess the response of vessels to a single reduction of perfusate/superfusate oxygen concentration or to a single dose of an agonist. To compare multiple treatment groups receiving a single dose of a drug, one-way ANOVA was used. Differences in the means after one-way ANOVA were determined with post hoc analysis using a Student-Newman-Keuls test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General characteristics of experimental groups.

Table 1 summarizes data describing the general characteristics of rats treated with either normal drinking water or losartan for 1 wk.Losartan treatment had no effect on body weight or vessel diameter. MAP was significantly lower in rats receiving the orally active AT1 receptor blocker losartan than in the untreated controls. MAP tended to be reduced in rats treated with an intravenous infusion of losartan (MAP: 111.8 ± 5.7 mmHg; n = 5) for 1 wk versus saline-infused controls (MAP: 127.5 ± 9.0 mmHg; n = 4), although this difference was not significant.


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  		Table 1. Characteristics of rats on standard chow and receiving either normal drinking water (control) or losartan (1 mg/ml) in the drinking water

 
Response to ACh in control and losartan-treated animals.

The dilation of gracilis arteries in response to ACh was unaffected by AT1 receptor blockade with losartan (Fig. 1). Responses to ACh were endothelium and NOS dependent in both the control and losartan-treated animals, because both endothelium denudation and NOS inhibition with L-NAME (100 µM) completely eliminated ACh-induced dilation in both groups (Fig. 1).



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  		Fig. 1. Response to 1 µM ACh in gracilis arteries of rats administered normal drinking water or losartan-treated drinking water (1 mg/ml) for 1 wk (n = 16–22). Responses in each group are compared before and after nitric oxide synthase (NOS) inhibition with N{omega}-nitro-L-arginine methyl ester (L-NAME; 100 µM, n = 5–6) or before and after removal of the endothelium (n = 5–6). PSS, physiological salt solution. Data are presented as mean changes (in µm) ± SE from the diameter measured before the application of ACh. *Significant difference from the response of intact untreated vessels, P < 0.05.

 
Effect of losartan treatment on responses to reduced PO2.

Figure 2 summarizes the responses of skeletal muscle resistance arteries to reduced PO2 in control rats and in rats receiving oral losartan for 1 wk. Vessels from control rats demonstrated a significant increase in diameter in response to reduced PO2, as previously reported for isolated skeletal muscle resistance arteries (4, 6, 27). However, arteries from losartan-treated rats exhibited a large paradoxical constriction in response to the simultaneous reduction in perfusate and superfusate PO2 (Fig. 2A). Arterial dilation in control rats and the paradoxical constriction of arteries from losartan-treated rats during exposure to reduced PO2 were both eliminated by endothelial denudation, indicating that the endothelium is required for both responses (Fig. 2B).



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  		Fig. 2. A: responses to simultaneous reduction of perfusate and superfusate O2 concentration from 21% O2 to 0% O2 in isolated gracilis arteries (endothelium intact) of rats on normal salt diet receiving either normal drinking water (n = 15) or losartan in the drinking water (1 mg/ml) for 1 wk (n = 21). B: responses of isolated gracilis arteries from control and losartan-treated rats to hypoxia before and after endothelium removal (n = 6). Data are represented as means ± SE. *Significant difference from response of vessels from untreated control animals receiving normal drinking water, P < 0.05.

 
In the control animals, dilation of the vessels in response to hypoxia was not significantly different in the presence [change in internal diameter ({Delta}ID) =16.6 ± 1.45, n = 5] or absence ({Delta}ID = 13.4 ± 0.23, n = 16) of L-NAME, and hypoxic constriction of arteries from losartan-treated animals was unaffected by NOS inhibition with L-NAME [{Delta}ID = –12 ± 0.58 (n = 6) in the presence of L-NAME and –12.7 ± 0.27 (n = 22) in the absence of L-NAME].

Effect of indomethacin, PGH2-TXA2 receptor blockade, and thromboxane synthesis inhibition on responses of arteries to reduced PO2.

Figure 3 compares the effect of cycloxygenase inhibition with indomethacin (1 µM) on the responses to reduced PO2 in isolated gracilis arteries of animals on normal drinking water and animals receiving losartan in the drinking water. Indomethacin eliminated both the hypoxic dilation in vessels from control animals and the paradoxical vasoconstriction in response to reduced PO2 in vessels from animals treated with losartan.



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  		Fig. 3. Effect of indomethacin (1 µM) on the response to simultaneous reduction of superfusate and perfusate O2 concentration from 21% O2 to 0% O2 in isolated skeletal muscle resistance arteries of normotensive rats receiving normal drinking water or oral losartan (n = 5–6). Data are represented as means ± SE. *Significant difference from control animals on normal drinking water, P < 0.05.

 
The effect of the blockade of PGH2-TxA2 receptors with SQ-29548 (10 µM) on the response of gracilis arteries to reduced PO2 is summarized in Fig. 4. In the losartan-treated group, pharmacological blockade of these receptors converted the paradoxical constriction of the vessels in response to reduced PO2 into a vasodilator response. In the untreated control group, dilation of vessels in response to reduced PO2 was unaffected by SQ-29548. In another series of experiments, inhibition of thromboxane synthase activity with dazoxiben (10 µM) also reversed hypoxic constriction of isolated gracilis arteries from losartan-treated animals but did not affect vasodilation in response to reduced PO2 of vessels from control animals (Fig. 4B).



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  		Fig. 4. Effect of 10 µM SQ-29548 (A) or 10 µM dazoxiben (B) on responses to simultaneous reductions in superfusate and perfusate O2 concentration from 21% O2 to 0% O2 in isolated skeletal muscle resistance arteries of normotensive rats receiving normal drinking water (n = 5) or losartan (n = 7). Data are represented as means ± SE. *Significant difference from response of vessels from untreated controls on normal drinking water, P < 0.05.

 
Effect of L-NAME and indomethacin on the restored relaxation in response to reduced PO2 in losartan-treated rats.

Figure 5 summarizes the combined effect of NOS inhibition with L-NAME (100 µM) and the PGH2-TXA2 receptor antagonist SQ-29548 on the responses of gracilis arteries to reduced PO2 in losartan-treated animals. In these studies, SQ-29548 converted the paradoxical constriction to reduced PO2 into a hypoxic dilation, as described in Fig. 4A. Simultaneous inhibition of NOS and blockade of PGH2-TXA2 receptors eliminated the restored dilation in response to hypoxia in losartan-treated animals (Fig. 5). In contrast, inhibition of cyclooxygenase with indomethacin (1 µM) had no effect on the restoration of hypoxic dilation by SQ-29548 in arteries from losartan-treated rats.



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  		Fig. 5. Responses to simultaneous reductions in superfusate and perfusate O2 concentration from 21% O2 to 0% O2 in isolated skeletal muscle resistance arteries from rats maintained on normal drinking water or losartan (replotted from Fig. 2). In vessels of losartan-treated animals, responses to reduced PO2 were monitored in the presence of dazoxiben (10 µM, n = 6; replotted from Fig. 4B), SQ-29549 (10 µM, n = 7; replotted from Fig. 4A), simultaneous exposure to SQ-29548 (10 µM) and L-NAME (100 µM, n = 6), or simultaneous exposure to SQ-29548 and indomethacin (1 µM, n = 3). Data are represented as means ± SE. *Significant difference from response of vessels from untreated controls on normal drinking water, P < 0.05.

 
Effect of tempol on responses of arteries to reduced PO2.

Figure 6 compares the effect of the superoxide dismutase mimetic tempol (100 µM) on the responses to reduced PO2 in gracilis arteries from animals on normal drinking water and those receiving oral losartan. Perfusion and superfusion with tempol had no effect on the ability of the arteries to dilate in response to simultaneous reductions in perfusate/superfusate PO2 in control animals. However, TEMPOL converted the hypoxic vasoconstrictor response into a dilator response in arteries from animals receiving losartan in the drinking water for 1 wk.



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  		Fig. 6. Effect of the superoxide dismutase mimetic TEMPOL (100 µM) on responses to simultaneous reductions in superfusate and perfusate O2 concentration from 21% O2 to 0% O2 in isolated skeletal muscle resistance arteries of normotensive rats receiving normal drinking water (n = 4) or losartan (n = 6). Data are represented as mean changes in diameter (in µm) ± SE. *Significant difference from response of vessels from untreated controls on normal drinking water, P < 0.05.

 
Effect of losartan on responses of isolated resistance arteries to iloprost and sodium nitroprusside.

There was no difference in the response of vessels from control and losartan-treated animals to the stable prostacyclin analog iloprost (10 pg/ml) or the NO donor sodium nitroprusside (1 µM) in these studies. Vessels from control (n = 6) and losartan-treated (n = 8) animals dilated in response to both iloprost ({Delta}ID = 13.5 ± 1.8 in controls vs. {Delta}ID = 9.6 ± 1.0 in the losartan-treated group) and SNP ({Delta}ID = 28.7 ± 1.2, n = 8, in controls vs. {Delta}ID = 23.2 ± 0.9 in losartan-treated rats).

Effect of intravenous infusion of losartan on vasodilator responses of isolated resistance arteries.

Figure 7 summarizes the responses of isolated gracilis arteries to ACh (1 µM), simultaneous reductions in superfusate/perfusate PO2, and iloprost (10 pg/ml) in animals receiving normal drinking water, oral administration of losartan for 1 wk (1 mg/ml; replotted from Fig. 2), or an intravenous infusion of losartan (20 µg·kg–1·min–1) for 1 wk. In these studies, rats on normal drinking water exhibited a significant dilation in response to ACh, acute hypoxia, and iloprost, as reported in previous studies (4, 15). Arteries from animals receiving either an oral or intravenous infusion of losartan for 1 wk demonstrated similar responses to various dilator stimuli, i.e., dilation in response to ACh and iloprost, and a paradoxical constriction in response to reduced PO2. In rats treated with oral losartan (Fig. 4A) and rats receiving an intravenous infusion of losartan, the paradoxical constriction in response to reduced PO2 was converted to a dilation (9.7 ± 2.17 µm, n = 4) in the presence of the PGH2-TXA2 receptor antagonist SQ-29548.



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  		Fig. 7. Responses to ACh (1 µM), reduced PO2, and the stable prostacyclin analog iloprost (10 pg/ml) in isolated skeletal muscle resistance arteries of rats on normal drinking water (n = 16–22) or oral losartan (n = 16–22, 1 mg/ml) or receiving intravenous infusion of losartan (n = 5, 20 µg·kg–1·min–1). Data are expressed as mean changes in diameter (in µm) ± SE from the pretreatment control diameter. *Significant difference from the response of untreated vessels from control animals on normal drinking water, P < 0.05.

 
Effect of losartan on TXA2 and prostacyclin production during hypoxia.

Figure 8 summarizes the effects of reduced PO2 on TXA2 and prostacyclin (PGI2) release in skeletal muscle arteries from rats receiving oral losartan or normal drinking water for 1 wk. The release of TXA2 and PGI2 was assessed by the measurement of their stable metabolites, TXB2 and PGF1{alpha}, respectively. The production of TXA2 and PGI2 was evaluated during reductions of O2 concentration from 21% O2 to 5% O2, which reduces the PO2 in the bath from ~140 mmHg to ~35–45 mmHg. This level of PO2 reduction for measurement of PGI2 and TXA2 production is comparable to the reductions in PO2 obtained during isolated vessel experiments (16). In the present study, there was a significant increase in PGI2 release during hypoxia in vessels from control animals, as reported in previous studies (Fig. 8A). However, arterial PGI2 production was significantly reduced during hypoxia in vessels from losartan treated animals compared with controls (Fig. 8A).



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  		Fig. 8. Effect of reduced PO2 on prostacyclin (PGF1{alpha}; A) and thromboxane B2 (as an indicator of thromoboxane A2) (TXB2; B) production in skeletal muscle arteries from control and losartan-treated rats. Data are expressed as mean changes in production ± SE during the reduction of O2 concentration from 21% O2 to 5% O2. *Significant difference between vessels from losartan-treated (n = 6) vs. control rats (n = 7) drinking normal water, P < 0.05; #significant change in metabolite production during equilibration with 5% O2 vs. 21% O2 in the same experimental group, P < 0.05.

 
TXA2 production was significantly reduced in skeletal muscle arteries of control animals drinking normal water, consistent with previous studies of cerebral arteries from animals on a low-salt diet (Fig. 8B). In contrast, there was no change in the production of TXA2 in response to reduction of the O2 concentration from from 21% to 5% O2 concentration in vessels from animals treated with losartan for 1 wk (Fig. 8B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Recent studies have elucidated the importance of normal circulating ANG II and the subsequent activation of AT1 receptors on the maintenance of normal vasodilator responses in microvessels and resistance arteries of normotensive rats (5, 8, 18, 27, 28). Impairments in vessel responses to vasodilator stimuli in animals on a HS diet are caused by the suppression of circulating ANG II and altered signaling pathways mediating hypoxic and ACh-induced vascular relaxation (7, 26, 27). Importantly, the restoration of vasodilator responses during ANG II infusion in rats on a HS diet can be prevented by coinfusing the AT1 receptor antagonist losartan, suggesting that AT1 receptor activation may maintain normal vascular relaxation mechanisms (28).

Blockade of the RAS is an important component of the treatment regimen for patients with hypertension and other cardiovascular diseases (9, 21). Because some studies suggest that chronic RAS blockade improves endothelial dysfunction in patients (12, 22) and that ANG II is an important regulator of signaling mechanisms that are important for peripheral vascular function (1, 5, 27, 28), the role of the AT1 receptor in the maintenance of normal vascular function is of critical importance. The goal of the present study was to determine the effect of AT1 receptor antagonism on the responses to vasodilator stimuli in isolated skeletal muscle resistance arteries of normotensive animals on a normal salt diet to gain insight into the role of ANG II in maintaining vascular relaxation mechanisms under normal physiological conditions.

Effect of losartan on responses to ACh. ACh-induced dilation of resistance arteries is impaired after exposure to an elevated dietary salt intake (15, 18, 26, 27). As previously noted, previous studies have demonstrated that AT1 receptor activation mediates the protective effect of a low-dose ANG II infusion to restore dilator responses to ACh in vessels from animals on a HS diet (28). The alterations in NOS-dependent vascular relaxation mechanisms in rats during ANG II suppression with a HS diet may be caused by endothelial NOS (eNOS) uncoupling and/or alterations in the ACh-dependent signaling pathway upstream from NOS activation (26, 31). However, other studies suggest that chronic infusion of ANG II enhances calcium-dependent NOS activity in the microcirculation, whereas eNOS protein expression is augmented in other organs (20).

In the present study, isolated gracilis arteries from rats fed normal drinking water and from losartan-treated rats on standard rat chow both exhibited vasodilation in response to ACh (Figs. 1 and 7), as previously reported (15, 27). Responses to ACh in both groups were completely eliminated after removal of the endothelium (Fig. 1) and during NOS inhibition with L-NAME. There was no difference in the ability of vessels from losartan-treated animals to dilate in response to the NO donor sodium nitroprusside (see RESULTS). Taken together, these data argue against the possibility that losartan alters the release and/or sensitivity of vessels to the primary mediators of ACh-induced vasodilation, as reported in other experimental models (13, 30). These results also suggest that the loss of AT1 receptor activation may not alter the expression or activity of eNOS, because the mechanisms of vascular relaxation to ACh under normal physiological conditions seem unaltered by losartan treatment.

Effect of losartan on hypoxic dilation. Previous studies have demonstrated that the impairment of hypoxia-induced dilation of skeletal muscle resistance arteries and cerebral arteries of normotensive animals during an elevated dietary salt intake is also due to a reduction in the interaction between circulating ANG II and the AT1 receptor (28). In the present experiments, isolated resistance arteries of untreated control animals on a normal salt diet exhibited a significant dilation in response to simultaneous reductions in perfusate and superfusate PO2 to 35–45 mmHg, as previously reported (4, 15). In contrast, gracilis arteries from rats treated with losartan, either by oral administration or via intravenous infusion, demonstrated a paradoxical constriction when exposed to low PO2 (Figs. 2 and 7).

The hypoxic constriction of vessels from losartan-treated animals could be due to several factors, including the liberation of vasoconstrictor substances from the endothelium, an intrinsic alteration in the vascular smooth muscle cells that affects their response to reduced PO2, or an altered response of the smooth muscle cells to the prostacyclin that is released from the endothelium in response to reduced PO2 in the rat gracilis artery (4). The present study indicates that the hypoxic constriction of gracilis arteries from losartan-treated animals is due to a vasoconstrictor substance released from the endothelium, because removal of the endothelium eliminated both the paradoxical constriction in response to reduced PO2 in arteries of losartan- treated rats (Fig. 2B) and hypoxic dilation of arteries from nontreated controls (Fig. 2B).

Contribution of cyclooxygenase products to responses to reduced PO2 in arteries of losartan-treated animals. Although a number of studies have demonstrated that an increased release of cyclooxygenase products is responsible for hypoxic dilation in skeletal muscle and cerebral resistance arteries (4, 6, 16, 19), other studies have demonstrated that the constriction of arteries from rats on a HS diet (18) and during hypertension (2, 25) is mediated by an enhanced synthesis and/or release of TXA2. Taken together with data demonstrating that continuous low-dose ANG II infusion restores hypoxic dilation in animals on a HS diet, the results of these studies indicate that normal circulating ANG II levels may be important to maintain the generation of vasodilator products of cyclooxygenase.

The present study provides insight into the contribution of metabolites of the cyclooxygenase pathway of arachidonic acid metabolism to the paradoxical constriction observed during exposure to reduced PO2 in arteries of losartan-treated animals. In these studies, hypoxic constriction of arteries from losartan-treated rats was blocked by indomethacin (Fig. 3), indicating that a cyclooxygenase-derived constrictor product was responsible for the altered response to reduced PO2 in those animals. The results of the present study also demonstrate that synthesis and/or release of potential mediators of hypoxia-induced dilation are altered by the administration of losartan (Figs. 2A and 8). Previous studies have determined that cyclooxygenase-dependent increases in PGI2 and reductions in TXA2 contribute to the dilation of resistance arteries in response to reduced PO2 (6, 18). In this study, the likely source of PGI2 and TXA2 is the endothelium, because cyclooxygenase-1 and -2 expression is up to 20 times greater in the endothelium than in smooth muscle cells (3) and because removal of the endothelium eliminated the constrictor response to hypoxia in the losartan-treated animals (Fig. 2B).

Role of altered PGI2 and TXA2 release in contributing to altered responses to hypoxia in losartan-treated animals. Important to the present study is the observation that PGH2-TXA2 receptor activation contributes to the loss of hypoxia-induced dilation during oral and intravenous administration of losartan, because treatment of the vessels with SQ-29548 converted hypoxic constriction into a dilation (Fig. 4A). Consistent with previous studies, there was a significant reduction in TXA2 release during hypoxia in arteries from control animals (Fig. 8). However, it is likely that TXA2 is the constrictor substance that promotes the paradoxical constriction of the arteries during exposure to hypoxia in losartan-treated rats, because hypoxic constriction of these vessels was reversed in the presence of the thromboxane synthase inhibitor dazoxiben (Fig. 4B). Supporting the hypothesis that TXA2 release promotes hypoxic vasoconstriction in arteries from losartan-treated rats are data suggesting that the reduction in the release of TXA2 release during reduced PO2 was significantly less during exposure of these vessels to hypoxia (Fig. 8).

It is also likely that altered PGI2 release contributes to the impaired responses of gracilis arteries to hypoxia in losartan-treated rats. Consistent with previous studies of rats on normal salt diets, release of the vasodilator prostaglandin PGI2 from skeletal muscle resistance arteries of control animals increased in response to reduced PO2 (Fig. 8A). However, there was a significant reduction in the release of PGI2 during exposure to hypoxia in skeletal muscle arteries from losartan-treated rats (Fig. 8B). The hypothesis that AT1 receptor antagonism alters PGI2 production during hypoxia is further supported by the experiments indicating that NO, rather than PGI2, is the likely mediator responsible for the restored hypoxic vasodilation in vessels of losartan-treated rats during blockade of the PGH2-TXA2 receptor with SQ-29548 (Fig. 5). In those studies, the restored dilation to hypoxia was eliminated during simultaneous exposure to SQ-29548 and L-NAME but was unaffected by combined exposure of vessels to SQ-29548 and indomethacin (Fig. 5). Finally, in contrast to vascular responses in rats on a HS diet (15, 18, 27), it is unlikely that reduced responsiveness to PGI2 contributes to the constrictor response to hypoxia during AT1 receptor blockade because there was no difference in the ability of gracilis arteries to dilate in response to the stable prostacyclin analog iloprost.

Possible role of superoxide anion formation on responses to reduced PO2 in resistance arteries of losartan-treated animals. Previous studies have determined that reactive oxygen species may alter endothelium-dependent vascular responses in the microcirculation and aorta during situations that suppress circulating levels of ANG II in rats (i.e., elevated dietary salt intake) (14, 31). Superoxide (O2–) is inactivated by NO to form peroxynitrite, a possible mediator of protein nitrosylation in vascular cells (29, 32). Because O2– formation may be elevated during hypoxia in the skeletal muscle and mesenteric microcirculation and may alter prostaglandin production in vascular cells (23, 24, 29), the superoxide dismutase mimetic tempol was used to determine whether elevated superoxide may contribute to altered responses to hypoxia during AT1 receptor blockade in losartan-treated rats. In these studies, acute administration of tempol to the vessels had no effect on vasodilation in response to reduced PO2 from control rats (Fig. 6). However, hypoxic constriction of arteries from losartan-treated animals was reversed and converted to a dilation when tempol was added to the perfusion and superfusion solutions (Fig. 6). These results indicate that prolonged AT1 receptor antagonism may alter the generation and/or scavenging of O2– during hypoxia in rat gracilis arteries.

In summary, the present study demonstrates that treatment of normotensive rats with the AT1 receptor blocker losartan dramatically alters the responses of skeletal muscle resistance arteries to acute reductions in PO2. The paradoxical constriction produced by hypoxia after 1 wk of losartan treatment seems to be due to alterations in the release of the cyclooxygenase-dependent constrictor TXA2 and the vasodilator prostaglandin PGI2 from the endothelium. These observations suggest that AT1 receptor activation promotes the formation cyclooxygenase-dependent products of arachidonic acid metabolism that result in vascular relaxation in response to hypoxia. The present studies also indicate that prolonged AT1 receptor blockade leads to increased levels of superoxide during exposure to hypoxia (and possibly during resting conditions) as well, which play a role in contributing to the loss of hypoxic dilation of the vessels during AT1 receptor blockade. The dramatic reduction in the relaxation of skeletal muscle resistance arteries in response to hypoxia during AT1 receptor blockade could alter vascular and hemodynamic responses to physiological perturbations such as systemic hypoxia, exercise, and hemorrhage.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by the National Heart, Lung, and Blood Institute Grants HL-29587, HL-65289, and HL-72920.


   ACKNOWLEDGMENTS
 
We thank Brian Corson and Anne Ansley for excellent technical assistance in the measurement of protacyclin and thromboxane release. The authors are grateful for the gracious gift of losartan from Merck (Rahway, NJ) and Schering (Berlin, Germany) and Pfizer (UK) for the generous gifts of iloprost and dazoxiben, respectively.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Lombard, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: jlombard{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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