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Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study was designed
to test the hypothesis that homocysteine (Hcys) reduces intracellular
nitric oxide (NO) concentrations ([NO]i) and stimulates
superoxide (O
risk factor of cardiovascular diseases; endothelium-derived relaxing factor, transnitrosation, reactive oxygen species, renal circulation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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HYPERHOMOCYSTEINEMIA (hHcys) is recognized as a novel independent risk factor of thrombosis and arteriosclerosis involving coronary, cerebral, and peripheral arteries (3, 8-10, 30). Homocysteine (Hcys), a sulfur-containing amino acid, is not present in our regular diet and is primarily formed from methionine by demethylation in a variety of tissues or cells, and L-Hcys is the primary active form within the cells. On the basis of observations in patients with an inborn metabolic error (homocystinuria) and in animal models with experimental hHcys, Dr. McCully formulated the "homocysteine theory" of atherosclerosis to speculate that deranged Hcys regulation might cause cardiovascular diseases in the general population (25, 28). Recently, numerous clinical and epidemiological studies have demonstrated a positive correlation between plasma Hcys levels and cardiovascular diseases. It has been reported that plasma total Hcys levels are increased in many patients with essential hypertension, stroke, atherosclerosis, or end-stage renal disease (2, 8-10, 15). hHcys is also considered as a new crucial element in the pathogenesis of uremic cardiovascular complications (12).
Despite intensive clinical studies demonstrating the association of hHcys and cardiovascular diseases, the mechanisms by which hHcys produces cardiovascular dysfunction, atherosclerosis, and end-stage organ damage have not been fully elucidated. There is accumulating evidence indicating that endothelial injury or dysfunction is one of the important early pathological changes in the development of atherothrombotic vascular disease and some other end-stage organ damage (2, 8-10, 15). In this regard, animals with chronic hHcys exhibited an impaired endothelium-dependent vasodilator response (34). It has been suggested that Hcys produces endothelial dysfunction by decreasing NO production (23, 36). However, this conclusion was drawn based on only results obtained using pharmacological interventions or indirect measurements of NO metabolism (11, 14, 17, 28, 32, 35). In addition, oxidative stress or lipid peroxidation has been reported to be involved in the development of atherosclerosis or thrombosis in hHcys, but little is known about the direct effects of Hcys on the production of reactive oxygen species in the intact endothelium (7, 8).
The present study was designed to test the hypothesis that Hcys reduces
intracellular nitric oxide (NO) concentrations ([NO]i) and stimulates superoxide (O
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation of small renal arteries. Male Sprague-Dawley rats weighing between 250 and 300 g were anesthetized with pentobarbital sodium (80 mg/kg body wt ip), and the kidneys were rapidly removed and kept in ice-cold HEPES-buffered physiological saline solution (PSS) of the following composition (in mM): 140 NaCl, 4.7 KCl, 1.6 CaCl2, 1.17 MgSO4, 1.18 NaH2PO4, 5.5 glucose, and 10 HEPES; pH 7.4. Small renal arteries (250-300 µm inner diameter) were carefully dissected on ice and transferred to a 35-mm Sylgard-coated dissecting dish containing ice-cold PSS. The arterial segment was cut open along its longitudinal axis, and pinned onto the dish with lumen side upward. Care was taken not to disrupt the endothelium.
In an additional group of rats, the aorta below the left renal artery was isolated and cannulated. After the aorta was ligated at a site above the right renal artery, the kidneys were flushed with 10 ml ice-cold PSS after 60-ml air perfusion to remove the endothelium of the renal arteries. The small renal arteries were then dissected to measure agonist-induced NO production. These experiments were performed to confirm that NO was derived from the renal arterial endothelium (1, 19).Measurement of [NO] within the endothelium of small renal arteries. A novel fluorescent NO indicator, DAF-2DA, which was recently developed by Kojima et al. (19), was used to measure [NO] within the endothelial cells of freshly isolated small renal arteries. DAF-2DA can readily enter the cells and be hydrolyzed by cytosolic esterases to DAF-2, which is trapped inside the cells. In the presence of NO and oxygen, the relatively nonfluorescent DAF-2 is converted into the highly green fluorescent triazole form, DAF-2T. Thus the increases in DAF-2T fluorescence represent the elevation of [NO]. After a 1-h equilibrium period, the arterial segment was incubated with DAF-2DA (10 µM, Calbiochem) in 1 ml PSS at room temperature for 30 min. The segment was then rinsed three times with PSS, and the dish was mounted on the stage of an epifluorescence microscope (Nicon E600) equipped with a ×20 objective and 490-nm excitation and 535-nm emission filters. Digital images were acquired and analyzed using a personal computer-controlled digital charge-coupled device (CCD) camera (Roper Scientific RTE/CCD-1300-Y/HS) by MetaMorph image analysis software (Universal Imaging) as previously described (39).
To determine the agonist-induced NO production in the arterial endothelium, A-23187 (2 µM) or bradykinin (BK; 2 µM) was added to the bath solution to activate NO synthase (NOS). To study the effect of Hcys on the endothelial NO response to these agonists, the arteries were preincubated with L-Hcys (10-40 µM) for 30 min before the addition of A-23187 or BK. In another group of renal arteries, N
-nitro-L-arginine
methyl ester (L-NAME; 10 µM) was used to block the
A-23187-induced NO release. NO fluorescence was measured every 5 min in the same area of the endothelial layer. NO levels are expressed as integrated average fluorescence intensities within a
certain tubular planar area (in mm2). To determine the role
of the sulfhydryl group in reducing NO concentrations in endothelial
cells, an oxidized form of Hcys, homocystine (40 µM), was used to
examine whether removal of the sulfhydryl group from Hcys eliminates
its effect on endothelial NO in this arterial preparation. Moreover,
the thiol analogs of Hcys, L-cysteine and reduced or
oxidazed glutathione (GSH or GSSH; 40 µM), were used to examine
whether other thiol compounds have an effect on the NO response similar
to Hcys.
Measurement of [O
Activity of NOS.
NOS activity was determined by measuring the conversion rate of
[3H]arginine to [3H]citrulline
(5) using an isotopic NOS detection kit (Calbiochem) according to the manufacturer's instructions. Briefly, homogenates prepared from small renal arteries were incubated in 50 µl of reaction mixture containing (in mM) 25 Tris · HCl (pH 7.4), 0.6 CaCl2, 1 
-NADPH, 0.003 tetrahydrobiopterin
(BH4), 0.001 FAD, 0.001 FMN, and 0.005 cold
L-arginine and 1.0 µCi [3H]arginine in the
absence or presence of L-Hcys. In this reaction mixture, a
high concentration of Ca2+ was used to maximize the NOS
activity. After incubation for 60 min at 37°C, the reaction was
terminated by the addition of 400 µl of ice-cold stop buffer
containing 50 mM HEPES (pH 5.5) and 5 mM EDTA. Equilibrated cation
exchange resin was added to the samples, and they were then applied to
spin columns. After centrifugation, the eluate (containing
[3H]citrulline) was collected, and the radioactivity was
determined with a liquid scintillation counter. To determine the effect
of L-Hcys on NOS activity, L-Hcys (100 µM)
was added to the reaction mixture. EGTA (1 mM) was used as a positive
control for NOS inhibition. In these experiments, the formation rate of
[3H]citrulline represents NOS activity, which is
expressed as femtomoles per milligram of protein per minute.
Determination of the direct reaction of L-Hcys with NO. Because Hcys contains a sulfhydryl group that can serve as an accepter of transnitrosation, we hypothesized that Hcys may directly bind to and trap NO, thereby reducing [NO]i. To test this hypothesis, NO release from PAPA-NONOate (100 µM) was determined in the presence or absence of L-Hcys. During the incubation of PAPA-NONOate with DAF-2, the fluorescence intensity was monitored every 5 min for 30 min using a fluorescence spectrometric microplate reader equipped with a 490-nm excitation and a 510- to 560-nm emission filter. To test the effect of Hcys on [NO] in this reaction system with PAPA-NONOate, L-Hcys (10-40 µM) was added to the reaction mixture, and DAF-2 fluorescence was continuously monitored for 30 min. In an additional group of experiments, oxyhemoglobin (3 µM) was used to trap NO as a positive control.
Statistics. Data are presented as means ± SE. The significance of differences in mean values between and within multiple groups was examined using an ANOVA for repeated measures followed by a Duncan's multiple-range test (SigmaStat). P < 0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Blockade of the A-23187-induced increase in endothelial NO
fluorescence by L-NAME.
As shown in Fig. 1A, the
addition of A-23187 (2 µM) to the bath solution produced a marked
increase in endothelial green NO fluorescence of small renal arteries.
In the presence of L-NAME, the A-23187-induced increase in
NO green fluorescence was significantly attenuated, suggesting that
L-NAME is capable of blocking the A-23187-induced increase
in NO within the intact endothelium of these freshly isolated renal
arteries. Figure 1B summarizes A-23187-induced alterations
of [NO] measured by DAF-2T fluorescence intensity in the absence or
presence of L-NAME (n = 6). A-23187
produced a significant increase in [NO] within the endothelium of
renal arteries. In the presence of L-NAME, the
A-23187-induced increase in [NO] was completely blocked. To confirm
that the NO fluorescence in these arteries is within the endothelium,
the arteries were denuded, and the A-23187 response of NO was then
examined. In the denuded renal arteries, there was no detectable
A-23187-induced increase in DAF-2T fluorescence.
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Effects of L-Hcys on the A-23187-induced NO
increase in renal arterial endothelium.
L-Hcys was added to the incubation bath to determine
the effect of Hcys on the A-23187-induced increase in endothelial
[NO]. As shown in Fig. 2A,
the A-23187-induced [NO] increase in the endothelium of the renal
artery was significantly attenuated by L-Hcys (40 µM). The results of these experiments are summarized in Fig.
2B (n = 7). Consistent with the results in
Fig. 1, A-23187 significantly increased [NO] in the renal arterial
endothelium. L-Hcys blocked the A-23187-induced increase in
[NO] in a concentration-dependent manner with a 78% blockade of this
NO response at 40 µM. L-Hcys at 10 µM did not
significantly attenuate the A-23187-induced NO increase in this
preparation.
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Effects of L-Hcys on the bradykinin-induced
[NO] increase in renal arterial endothelium.
Figure 4 presents the results
obtained from these experiments. Incubation of the arteries with BK (2 µM) induced a significant NO increase within endothelial cells. In
the presence of L-Hcys (40 µM), the BK-induced increase
in endothelial [NO] was markedly inhibited. Figure 4A
presents typical fluorescence microscopic images showing the BK-induced
increase in [NO] in the renal arterial endothelium in the absence or
presence of L-Hcys. As summarized in Fig. 4B,
L-Hcys attenuated the BK-induced increase in [NO] by 58%
(n = 6).
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Effects of L-Hcys on NOS activity in renal arterial
homogenates.
To address whether Hcys decreases [NO] in renal arteries
through inhibition of NOS, we examined the effects of
L-Hcys on NOS activity by measurement of the conversion
rate of L-[3H]arginine to
L-[3H]citrulline. As shown in Fig.
5, the formation rate of
[3H]citrulline in the homogenates prepared from renal
arteries was found to be 59.7 ± 12 fmol · mg
protein
1 · min1. The addition of
EGTA (1 mM) to decrease Ca2+ concentrations in the reaction
mixture largely inhibited the formation of
[3H]citrulline, to 21.6 fmol · mg
protein1 · min1. However,
L-Hcys even at a high concentration of 100 µM had no significant effect on the activity of NOS in the arterial homogenates (n = 6).
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Trapping effect of L-Hcys on NO in solution.
With the use of a microtiter plate reader with a fluorescence
spectrometer, PAPA-NONOate was demonstrated to time dependently release
NO, as monitored by the DAF-2T fluorescence increase. This
time-dependent NO increase in the solution was abolished by
oxyhemoglobin, a classical NO trapping or scavenging reagent. Similar
to the effect of oxyhemoglobin, the addition of 20 or 40 µM
L-Hcys significantly blocked the NO increase in solution with PAPA-NONOate (n = 6). L-Hcys at 10 µM had no significant effect on NO concentrations in the PAPA-NONOate
solution (Fig. 6).
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Effects of L-Hcys on
[O
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we directly measured [NO]i in the endothelium of small renal arteries using DAF-2 fluorescence microscopic imaging analysis and monitored the production of NO within renal arterial endothelial cells in response to the endothelium-dependent vasodilators A-23187 and BK. In the intact endothelium of freshly dissected small renal arteries loaded with DAF-2DA, both A-23187 and BK were found to stimulate the production of a strong green fluorescence, which represents increases in [NO] within endothelial cells (19). The NOS inhibitor L-NAME or the removal of the endothelium completely blocked A-23187- and BK-induced increases in [NO] in this preparation, suggesting that detected NO increases in response to both compounds are derived from the endothelium of these arteries.
In the last decade, numerous studies have been performed to examine the regulation of NO production in the arterial endothelium and to explore the mechanisms of NO-mediated endothelial dysfunction under different pathological conditions, such as hypertension, atherosclerosis, and myocardial ischemia and reperfusion (16, 37). In the kidney, endothelium-derived NO has been found to play a critical role in the regulation of renal vascular tone and renal function (11, 41, 42). However, most of those studies used pharmacological interventions to block or enhance NOS activity and then observed the changes in endothelial function such as endothelium-dependent vasodilation or endothelium-dependent alterations of blood perfusion in different vascular beds (16, 37). There were few studies to address these issues by directly measuring endothelial NO. The present study used DAF-2DA, a novel cell-permeable fluorescent indicator of NO, and dynamically monitored the NO response in the intact endothelium of freshly dissected small renal arteries to endothelium-dependent vasodilators. This preparation provides a valuable model for studying the regulation of NO production and its physiological or pathological significance in the renal circulation.
With the use of this direct measurement of NO in the intact endothelium of freshly isolated small renal arteries, we examined the effects of Hcys on the NO response of these small arteries to A-23187 and BK. It was found that L-Hcys at concentrations of 20 and 40 µM markedly attenuated A-23187- or BK-induced increases in [NO] within renal arterial endothelial cells. To our knowledge, this provides the first direct evidence that Hcys decreases [NO] in the renal arterial endothelium and inhibits the endothelial NO response to A-23187 and BK. These results support the view that hHcys may produce endothelial dysfunction through NO-mediated mechanisms (20, 34, 36). In previous studies, Hcys has been reported to impair endothelium-dependent vasodilation (20). In animals with chronic hHcys, acetylcholine (ACh)-induced, NO-mediated vasodilation was significantly blunted (21, 34). With the use of isolated arterial preparations, the addition of L-Hcys to the bath solution produced a significant concentration-dependent impairment of the relaxation response to both ACh and a Ca2+ ionophore, A-23187 (20, 23). Taken together, these results indicate that the impairment of NO-mediated endothelial function may be one of the important mechanisms mediating the detrimental effects of elevated plasma Hcys levels (17, 32, 35). Endothelial dysfunction may induce the growth of arterial smooth muscle cells, ultimately resulting in alterations of the extracellular matrix and sclerotic plaques on the artery wall (2-4, 8, 15, 26a-26c).
However, it seems that the Hcys-induced decrease in the NO response of
endothelial cells does not necessarily predict the occurrence of
arteriosclerosis, because the NO response of the arterial endothelium
was also found to be abolished by other thiol compounds such as
cysteine and glutathione. These compounds contain a free sulfhydryl
group and are generally not considered as sclerotic factors. It remains
unknown why elevations of plasma Hcys but not cysteine or glutathione
produce arterioslcerosis, despite the fact that they all can decrease
intracellular NO levels in the endothelium. In the present study, we
examined the effects of Hcys and its thiol analogs on
O
To explore the mechanisms by which Hcys reduces [NO], we determined the effects of L-Hcys on NOS activity in renal arterial homogenates. By measuring the conversion rate of L-arginine to L-citrulline, we surprisingly demonstrated that L-Hcys had no effect on the conversion of L-arginine to L-citrulline, suggesting that Hcys does not inhibit NOS activity to reduce [NO] in the renal arteries. Because Hcys contains a sulfhydryl group that may bind to NO to form nitrosohomocysteine and thereby decrease NO levels within the endothelium, we tested the hypothesis that Hcys serves as an acceptor of transnitrosation to trap NO and thereby decreases free [NO] within cells. With the use of a microtiter plate reader with a fluorescence spectrometer, the NO donor PAPA-NONOate was found to produce a time-dependent increase in [NO]. Similar to the effect of a classical NO scavenger, oxyhemoglobin, the addition of L-Hcys to the reaction mixture completely abolished the increase in NO fluorescence induced by the NO donor. These results strongly indicate that Hcys may decrease NO levels in endothelial cells through its direct trapping effect on NO molecules. However, these results do not exclude the possibility that Hcys may indirectly inhibit NOS activity in vivo during hHcys. Recently, it has been reported that NOS uncoupling due to BH4 conversion to dihydrobiopterin by oxidative stress may result in a decrease of NO production (18). As discussed above, hHcys has been found to increase local oxidative stress in arterial wall, which may result in NOS uncoupling, decreasing NO production. Because we determined NOS activity under a condition with standard cofactor concentrations, this indirect effect of Hcys on NOS activity cannot be detected. Therefore, our results importantly suggest that Hcys had no direct effect on NOS activity and that NO trapping through its sulfhydryl group represents one of the mechanisms mediating the Hcys-induced reduction of endothelial NO levels.
In summary, the present study directly detected the production of NO in
the intact endothelium of small renal arteries in response to
endothelium-dependent vasodilators, A-23187 and BK. L-Hcys
was found to decrease [NO] in the arterial endothelium through a
direct trapping effect on NO. We conclude that Hcys may reduce
[NO]i, abolish the NO release response of the vascular endothelium to stimulators, and consequently result in endothelial or
vascular dysfunction. This reduction of endothelial NO levels was not a
specific effect of L-Hcys to induce arteriosclerosis, however, because nonsclerotic thiol compounds such as
L-cysteine and glutathione also decreased NO levels in
these endothelial cells. L-Hcys, but not
L-cysteine and glutathione, stimulated O
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54927 and American Heart Association Grant 96007310.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A.-P. Zou, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: azou{at}post.its.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.
May 23, 2002;10.1152/ajpheart.00680.2001
Received 31 July 2001; accepted in final form 20 May 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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