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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
1Tulane Hypertension and Renal Center of Excellence, and Department of Physiology, Tulane University Health Sciences Center, New Orleans, Louisiana; and 2Michael E. DeBakey Veterans Affairs Medical Center, and Departments of Medicine and Pharmacology, Baylor College of Medicine, Houston, Texas
Submitted 8 November 2004 ; accepted in final form 3 December 2004
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ABSTRACT |
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salt-sensitive hypertension; vascular tone; arterioles
Dahl/Rapp salt-sensitive (Dahl-S) rats are models of salt-induced hypertension (25). Attenuated endothelium-dependent vasodilation has been repeatedly demonstrated in blood vessels isolated from hypertensive Dahl-S rats (4, 16, 21, 34); however, the pathological basis remains uncertain. Although plasma substrate levels for nitric oxide synthesis are normal in these animals (7), salt-induced hypertension (6, 14), as well as vascular endothelial dysfunction (34), can be corrected by the administration of L-arginine, suggesting that impaired intracellular L-arginine availability may be a contributing factor. Because enhanced arginase activity has been shown to decrease nitric oxide production and promote endothelial dysfunction in various pathological conditions, we hypothesized that arginase may contribute to endothelial dysfunction in Dahl-S rats with salt-induced hypertension. To test this hypothesis, we evaluated arteriolar arginase I and II content using immunohistochemistry in Dahl-S rats on high- and low-salt diets. To examine endothelial function, we conducted experiments using skeletal muscle arterioles taken from these Dahl-S rats and examined the responses to an endothelium-dependent vasodilator and increases in luminal flow while in the presence or absence of an inhibitor of arginase or L-arginine.
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METHODS |
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Animals. Male inbred Dahl-S rats (SS/Jr, n = 40) were purchased at 5–6 wk of age (Harlan, Indianapolis, IN) and had free access to high-salt (8.0% NaCl) or low-salt (0.3% NaCl) diets (Dyets, Bethlehem, PA) and tap water for 4 wk. All procedures were approved by the institutional animal care and use committee.
Blood pressure measurements and tissue extractions. On the day of the experiment, rats were weighed and anesthetized with a single injection of Inactin (100 mg/kg ip), and blood pressure and heart rate were measured via a carotid arterial catheter as previously described (16). After stable readings were obtained, animals were heparinized (1,000 U/kg ia) and the heart, left kidney, and the gracilis anticus muscles were removed and placed into ice-cold modified Krebs buffer. Left kidney and heart wet weights were then determined.
Arginase I and II immunohistochemistry. Gracilis anticus muscles were harvested and fixed overnight (10% formalin). Specimens were embedded in paraffin, sectioned, and immunohistochemical staining for arginase I and II, which was performed with the avidin-biotin method (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA), as previously detailed (17). Briefly, sections were deparaffinized and hydrated, and endogenous peroxidase activity was quenched. After incubation with a rabbit polyclonal antibody against arginase I or II (1:200 dilution) or incubation with vehicle only for control slides, sections were treated with biotinylated anti-rabbit IgG antibody. After incubation with the ABC reagent, sections were developed with 3,3'-diaminobenzidine solution and counterstained with hematoxylin. The presence of arginase I or II immunoreactivity was indicated by a brown color.
Isolated microvessel experiments. Segments of first-order gracilis muscle arterioles were isolated by microdissection (28) and cannulated at both ends with glass micropipettes in a vessel chamber (Living Systems Instrumentation, Burlington, VT). The vessel chamber was continuously superfused with gassed buffer (14% O2-5% CO2-balanced with N2; 37°C) via a nonrecirculating system. For internal diameter measurements, the vessel chamber was mounted on a stage of an inverted microscope (Nikon TS 100-F) fitted with a charge-coupled device video camera. The camera was connected to a personal computer equipped with video dimensioning software (ImagePro Express, Media Cybernetics). With this setup, a magnified image of the arteriolar segment was performed on the computer monitor and internal diameter was measured by manually adjusting guides superimposed by the video dimensioning software. The software collected images at 1 frame/s that were stored as digital video files for further analysis and documentation.
For experiments with no luminal flow, the proximal micropipette was connected to a pressure servo-controller (Living Systems Instrumentation) and the distal micropipette to a closed stopcock to achieve and maintain 80 mmHg constant luminal pressure with no flow. After a 60-min stabilization period, the arginase inhibitor (100 µmol/l BEC), the arginase and nitric oxide synthase substrate (1 mmol/L L-arginine), or vehicle was included in the superfusion buffer 20 min before the experiment. This treatment regime was continued throughout the experiment. After the pretreatment period, increasing concentrations of the endothelium-dependent vasodilator, acetylcholine (1 nmol/l to 3 µmol/l) were added to the superfusion buffer. Each concentration was tested for 5 min, internal diameter was recorded every minute, and the average of the last two measurements was used to determine the response.
To study flow-induced dilation, both the proximal and distal micropipettes were connected to pressure servo-controllers and an inline microflowmeter (Living Systems Instrumentation). The arginase inhibitor (100 µmol/l BEC), the arginase and nitric oxide synthase substrate (1 mmol/L L-arginine), or vehicle was included in the luminal perfusion buffer. During a 60-min stabilization period, both proximal and distal pressures were adjusted to 80 mmHg with no luminal flow. During the experiments, proximal and distal pressures were adjusted equally in opposite directions to maintain the midline pressure at 80 mmHg and establish graded levels of luminal flow (0–50 µl/min in 5 µl/min increments). Each flow was tested for 5 min, internal diameter was recorded every minute, and the average of the last two measurements was used to determine the response.
Statistics. All data are expressed as means ± SE. Vascular response data were analyzed by ANOVA using a computer statistical package (Sigma Stat 3.0). When significant differences were observed, orthogonal contrasts were performed as a post hoc analysis (27). All other data were analyzed by t-tests. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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max: 36 ± 8 µm, n = 5, vs. high-salt max: 16 ± 4 µm, n = 7; P < 0.05, where max is maximum change). Acute in vitro pretreatment with an inhibitor of arginase, 100 µmol/l BEC, enhanced maximal responses and abolished the difference between high- and low-salt arterioles (low-salt max: 57 ± 2 µm, n = 6, vs. high-salt max: 49 ± 3 µm, n = 6) (Fig. 2, middle). Similarly, acute in vitro pretreatment with the arginase and nitric oxide synthase substrate, 1 mmol/l L-arginine, enhanced maximal responses in both groups and abolished the difference between high- and low-salt arterioles (low-salt max: 54 ± 3 µm, n = 6, vs. high-salt max: 48 ± 8 µm, n = 6) (Fig. 2, bottom).
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max: 20 ± 1 µm, n = 7, vs. high-salt max: –1 ± 1 µm, n = 6; P < 0.05) (Fig. 3, top). Acute in vitro pretreatment with an inhibitor of arginase, 100 µmol/l BEC, restored flow-induced responses in high-salt arterioles and abolished the difference between high- and low-salt groups (low-salt max: 22 ± 3 µm, n = 5, vs. high-salt max: 19 ± 1 µm, n = 4) (Fig. 3, middle). Similarly, acute in vitro pretreatment with the arginase and nitric oxide synthase substrate, 1 mmol/l L-arginine, restored flow-induced dilation in high-salt arterioles and abolished the difference between high- and low-salt groups (low-salt max: 22 ± 1 µm, n = 4, vs. high-salt max: 20 ± 1 µm, n = 4) (Fig. 3, bottom).
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DISCUSSION |
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Arginase is the central enzyme in the urea cycle, metabolizing L-arginine to L-ornithine and urea. Two distinct isoforms of arginase, arginase I and II, have been identified (9, 29). These isozymes possess similar enzymatic properties, but differ with regard to their tissue distribution and subcellular localization (15). Type I arginase is a cytosolic enzyme that is highly expressed in the liver, whereas arginase II is a mitochondrial isoform that is found predominantly in extrahepatic tissue. Previous studies have documented the presence of arginase I and II in cultured vascular smooth muscle and endothelial cells as well as the expression of arginase I in the aorta and coronary microvasculature (2, 10, 13, 20, 30). Our present findings extend this work by demonstrating the expression of arginase I and II in first-order gracilis muscle arterioles of Dahl-S rats. Moreover, we show that salt-induced hypertension in these animals enhances the arteriolar expression of both arginase isoforms. Interestingly, increases in vascular arginase activity have also been detected in DOCA-salt hypertensive rats (26) and in pigs with aortic coarctation (33), suggesting that upregulation of arginase is a general feature of salt-sensitive forms of hypertension.
The Dahl-S rat is a genetic model of salt-induced hypertension (25) that exhibits cardiac hypertrophy (12) and renal injury accompanied by an increase in renal weight (11). After 4 wk of a high-salt diet, mean arterial pressure and kidney and heart weights were significantly increased in Dahl-S rats compared with the low-salt group. We and others have previously shown that Dahl-S rats with salt-induced hypertension develop vascular endothelial dysfunction (4, 16, 21, 34) but do not display impaired responsiveness to an endothelium-independent activator of soluble guanylate cyclase (16). We again found that gracilis muscle arterioles isolated from high-salt Dahl-S rats exhibit attenuated endothelium-dependent vasodilator responses to acetylcholine compared with low-salt Dahl-S animals. Muscarinic agonists, such as acetylcholine, are widely used to assess nitric oxide-dependent endothelial function (16, 21, 28, 34). An alternative means for assessing endothelial function is to generate nitric oxide in response to increased shear forces along the vascular endothelium (24). Our group previously reported (18) that in skeletal muscle arterioles isolated from normotensive male Sprague-Dawley rats flow-induced dilation could be completely abolished by nitric oxide synthase inhibition. We found that the arterioles from high-salt Dahl-S rats fail to dilate in response to flow. To test whether elevated arginase expression contributes to the impairment in endothelial function in arterioles from high-salt animals, we employed the arginase inhibitor BEC. This boronic acid-based L-cysteine analog is a highly potent and specific competitive inhibitor of both arginase I and II and has no direct effect on nitric oxide synthase (19). We found that acute in vitro pretreatment with the arginase inhibitor BEC augments acetylcholine and flow-induced dilation in arterioles from both low- and high-salt groups. Furthermore, BEC restores acetylcholine and flow-mediated dilation in high-salt arterioles to levels observed in low-salt vessels. These findings suggest that elevated arginase expression contributes to arteriolar nitric oxide dysfunction in Dahl-S rats with salt-induced hypertension. It was further shown that constitutive arginase expression may counteract nitric oxide-mediated vasodilatory function in microvessels during normotensive states. Interestingly, increased arginase activity has recently been demonstrated to impair nitric oxide-mediated dilation in coronary arterioles following ischemia-reperfusion (13), in aortic vessels from old animals (2), and in coronary arterioles of pigs with aortic coarctation hypertension (33). Thus arginase may trigger endothelial dysfunction in various pathological conditions.
Our finding that arginase inhibition increases endothelium-dependent vascular relaxation suggests that arginase may regulate L-arginine availability for endothelial nitric oxide synthase. Although the affinity of L-arginine is much higher for purified nitric oxide synthase (Km of 
2–20 µmol/l) than for arginase (Km of 2–20 mmol/l), the maximum activity of arginase is more than 1,000 times that of nitric oxide synthase, suggesting similar rates of substrate utilization at physiological L-arginine concentrations (31). Consistent with a role for arginase in depleting substrate for endothelial nitric oxide synthase, we found that the acute exogenous administration of L-arginine mimics the actions of BEC. L-Arginine enhances acetylcholine and flow-induced dilation in arterioles from low- and high-salt groups and normalizes the impaired endothelium-dependent relaxation in arterioles from high-salt animals. Competition between arginase and nitric oxide synthase has also been described in cultured endothelial cells and in other blood vessels (2, 5, 8, 13, 20, 32), further highlighting the potential importance of such an interaction in the vasculature. Interestingly, increased microvascular arginase expression and the subsequent depletion of intracellular L-arginine may also explain the ability of chronic oral L-arginine supplementation to improve endothelial dysfunction in Dahl-S rats (34). Furthermore, arginase-mediated reduction in intracellular L-arginine stores may also serve to sensitize endothelial cells to the endogenous nitric oxide synthase inhibitor, NG-dimethyl-L-arginine, which is elevated in hypertensive Dahl-S rats (22). Previous reports suggested that limited L-arginine availability may promote uncoupling of endothelial nitric oxide synthase, which results in oxygen free radical formation rather than nitric oxide (1). Because oxygen free radicals can inactivate nitric oxide, the possibility exists that increased vascular arginase activity and the resulting intracellular L-arginine depletion may also decrease nitric oxide bioavailability. Thus it appears that increased vascular arginase expression may promote endothelial dysfunction via various mechanisms.
In summary, we found that arginase I and II are expressed in Dahl-S rat skeletal muscle arterioles and that the expression of both arginase isoforms is increased with salt-induced hypertension. In addition, acute in vitro pretreatment with an arginase inhibitor restores endothelium-dependent vasodilator responses and abolishes the differences between high- and low-salt arterioles. Similarly, we found that acute in vitro pretreatment with the arginase and nitric oxide synthase substrate L-arginine restores endothelium-dependent responses and eliminates the differences between the two salt groups. These data suggest that enhanced vascular arginase activity contributes to endothelial dysfunction in Dahl-S rats with salt-induced hypertension. Because skeletal muscle arterioles represent a substantial portion of total peripheral resistance, they are major determinants of blood pressure. Hence, the possibility exists that arteriolar endothelial dysfunction contributes to hypertension in Dahl-S rats on high-salt diets. Therefore, our present study might provide additional insights into the well-demonstrated blood pressure-lowering effects of L-arginine in hypertensive Dahl-S rats (6, 7, 14).
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FOOTNOTES |
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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.
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) or high-salt (
) diets. Arterioles were acutely pretreated in vitro with vehicle (top, n = 5 for low salt, n = 7 for high salt), with an inhibitor of arginase [100 µmol/l S-(2-boronoethyl)-L-cysteine (BEC)] (middle, n = 6 for low salt, n = 6 for high salt), or with 1 mmol/l L-arginine (bottom, n = 6 for low salt, n = 6 for high salt) for 20 min before experiments. Data are expressed as means ± SE of change in diameter. *P < 0.05 relative to low-salt group. Brackets indicate concentration.