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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 12, 2001 as doi:10.1096/fj.00-0681fje. |
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Department of Medical Physiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA
2Correspondence: Department of Medical Physiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA. E-mail: LKUO{at}tamu.edu
SPECIFIC AIMS
Arginase shares a common substrate, L-arginine, with nitric oxide synthase (NOS). It is speculated that arginase, if it is expressed in the endothelium, might play a pivotal role in the regulation of NO-mediated vasodilation by reducing the L-arginine availability to NOS. In the present study, we tried to determine whether arginase is expressed and active in coronary arterioles and to demonstrate whether endothelial arginase can influence NO production and play a functional role in regulating NO-mediated dilation of coronary microvessels. We determined the expression and cellular localization of arginase in porcine coronary microvessels (50–200 µm) by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and studied NO production and vasodilation of isolated coronary arterioles (60–110 µm) with and without arginase inhibition.
PRINCIPAL FINDINGS
1. Arginase mRNA and protein are constitutively expressed in
coronary arterioles
We determined whether arginase mRNA is present in the
isolated coronary arterioles using RT-PCR. Arginase I (131 bp), but not
arginase II (360 bp), transcript was detected in coronary arterioles
after PCR (Fig. 1
). Positive controls for the arginase I and II transcripts were revealed
in liver and kidney tissues, respectively.
|
To determine the cellular localization of arginase I in the coronary arteriole, vessels were subjected to immunohistochemical study. The background level of staining was determined in an isolated vessel treated with the nonimmune mouse serum, secondary antibody, and fluorescein avidin D. In the presence of anti-arginase I monoclonal antibody, high levels of immunostaining, as represented by a pseudo-color spectral display, were detected in both the endothelial and smooth muscle cells.
2. Arginase inhibitor 
-difluoromethylornithine (DFMO)
specifically potentiates endothelium-dependent, NO-mediated coronary
arteriolar dilation
The putative role of endothelial arginase in modulating
vasodilation was examined in isolated and pressurized coronary
arterioles. These vessels developed basal tone, i.e., 64 ± 1% of
maximal diameter (132±3 µm) at 60 cm H2O
intraluminal pressure and dilated in a concentration-dependent manner
to adenosine (Fig. 2A
) and serotonin (Fig. 2B
). In the presence of NOS
inhibitor L-NMMA (10 µM), the dilations of vessels to adenosine (Fig. 2A
) and serotonin (Fig. 2B
) were significantly
inhibited. In another set of experiments, the vessels were pretreated
with the arginase inhibitor DFMO (0.4 mM), and the dilations to
serotonin and adenosine were re-examined. In contrast to L-NMMA, DFMO
potentiated vasodilations in response to adenosine (Fig. 2A
)
and serotonin (Fig. 2B
). The DFMO-enhanced vasodilation was
not observed in the presence of L-NMMA (Fig. 2)
or after endothelial removal, suggesting a regulatory role of
endothelial arginase in the NO-mediated response. The altered
vasodilations to adenosine and serotonin were not due to the
nonspecific effects of L-NMMA or DFMO on vascular
smooth muscle function because these two inhibitors, administered
either independently or simultaneously, did not affect vasodilation to
endothelium-independent vasodilator sodium nitroprusside.
|
To determine the effect of DFMO on arginase and NOS, these two enzyme activities were assessed in the coronary arteriolar lysates in the presence and absence of DFMO. In the absence of DFMO, arginase activity was 206 ± 14 nmol urea/g protein and NOS activity was 156 ± 27 nmol nitrite/g protein; in the presence of DFMO, arginase activity was significantly reduced to 41 ± 6 nmol urea/g protein without affecting NOS activity (149±35 nmol nitrite/g protein). These results demonstrated the efficacy and specificity of DFMO in inhibiting arginase activity.
3. DFMO enhances NO production
Because L-arginine is a common substrate for NOS and
arginase, it is speculated that inhibition of the arginase pathway in
the endothelium, and thus increased arginine availability, would
enhance the amount of NO released in response to adenosine and
serotonin. To support this contention, coronary arteriolar production
of NO in response to adenosine and serotonin in the presence and
absence of NOS and arginase inhibitors was examined. In the absence of
agonists, only a small amount of NO was produced from coronary
arterioles (68±18 nmol nitrite/g protein). However, NO production was
significantly increased about threefold by adding serotonin and
adenosine to the vessels. These agonist-stimulated NO productions were
inhibited by L-NMMA. Conversely, NO production in response
to adenosine and to serotonin was significantly enhanced about twofold
by arginase inhibitor DFMO. DFMO did not alter NO production in the
absence of agonists. To examine whether increased
L-arginine availability can enhance the NO-mediated
vasodilatory responses, coronary arteriolar dilations to adenosine (1
µM), serotonin (0.1 µM), and sodium nitroprusside (1 µM) were
examined before and after intraluminal incubation with
L-arginine (3 mM) or D-arginine (3 mM). It was
found that L-arginine, but not D-arginine,
treatment significantly increased the vasodilations to adenosine and
serotonin. In contrast, L-arginine had no effect on the
vasodilation in response to sodium nitroprusside.
CONCLUSIONS AND SIGNIFICANCE
It is well established that vascular endothelial cells produce NO from L-arginine via a constitutive NOS. The regulated control of endothelial NOS allows for the maintenance of vascular tone and normal blood pressure and for the inhibition of platelet adherence and aggregation. In addition, activation of endothelial NOS plays an important role in the relaxation of vascular smooth muscle to certain endogenous agonists such as serotonin and adenosine, as demonstrated in the present study. Besides NOS, arginase is another major L-arginine consuming enzyme, which converts L-arginine to ornithine and urea. Arginase is expressed most abundantly in the liver for ammonia detoxification via the urea cycle. Although recent studies have indicated that some extrahepatic tissues/cells that do not possess complete urea cycle enzymes also express arginase, the biological role of this extrahepatic arginase remains unclear. Intravenous administration of arginase can cause constriction of cerebral arterioles and enhance platelet aggregation in vivo. These findings implicate that exogenous arginase may influence endothelial function through attenuation of NO production. However, it is unclear whether the endogenous arginase can play a counteracting role in the regulation of NOS activity, and thus NO production, for vasoregulation. Furthermore, although two different arginase isoforms (arginase I and II) have been cloned, their relative role in NO-mediated vasoregulation has not been investigated. In the present study, we documented that arginase inhibitor DFMO significantly enhanced endothelium-dependent NO-mediated coronary arteriolar dilations to adenosine and serotonin, but did not alter the dilation to an endothelium-independent vasodilator, sodium nitroprusside. These results suggest that inhibition of arginase specifically augments the NO-mediated vasodilatory pathway at the upstream of smooth muscle cells, since the relaxation of vascular smooth muscle to an NO donor (i.e., sodium nitroprusside) was not compromised by DFMO. It appears that the arginase that resides in the endothelium contributes to the regulation of NO-mediated vasodilation (see Scheme 1). This is supported by evidence that the stimulated NO production from coronary arterioles was significantly increased by DFMO and because the effect of DFMO was observed only in the vessels with an intact endothelium. The enhanced NO-mediated vasodilation by DFMO is unlikely to be a result of increased NOS activity because this inhibitor did not alter either resting NO production or NOS enzyme activity.
|
It is speculated that the enhanced NO production by DFMO might be a result of increased L-arginine availability to NOS. If this is the case, we would expect to observe an enhanced NO-mediated vasodilation by increasing the supply of L-arginine. Indeed, exogenous administration of L-arginine, but not D-arginine, to arterioles specifically enhanced NO-mediated vasodilation to adenosine and serotonin. This L-arginine-dependent NO response has also been observed in various microvascular beds, including human coronary microvessels. These results indicate that L-arginine can be a limiting factor for stimulated NO synthesis in the microvessels. The arginase expressed in the endothelium might compete with NOS for their common substrate L-arginine and thus influence NO production (see Scheme 1 ).
At least two distinct isoforms of arginase have been identified: arginase I in the liver and arginase II in extrahepatic tissues. Although arginase I is generally regarded as the hepatic isoform, recent studies have shown that other tissues also express arginase I mRNA and protein. However, the expression and cellular localization of arginase isoforms have not yet been revealed in intact blood vessels. Our present findings using RT-PCR showed that arginase I, but not arginase II, mRNA was expressed in the coronary arterioles. The cellular localization of arginase I, as detected by immunohistochemistry, appears to be in both the endothelium and vascular smooth muscle. These results demonstrate a constitutive expression of arginase I in the intact blood vessel and physically support a functional role of endothelial arginase in modulating NO-mediated dilation of coronary arterioles.
The present findings are important because many studies have demonstrated that vascular dysfunction associated with hypercholesterolemia, diabetes, and hypertension can be related to the deficiency of L-arginine availability for NO production in endothelial cells. However, the endogenous regulation of the L-arginine pool for NOS remains elusive. Our results not only demonstrate a role of arginase in modulating NO-mediated vascular function, but also provide a speculation of its contribution to the vascular dysfunction during disease states. This contention is supported by recent evidence that arginase activity can be increased by diabetes and hypertension and induced by inflammatory stimuli and certain cytokines in various cell types. It is conceivable that the NO-mediated biological function can be compromised if an up-regulation of arginase occurs under these pathophysiological conditions. Further elucidation of mechanisms involved in the regulation of NO production by arginase under physiological and pathophysiological conditions is imperative in the microcirculation because these vessels are the primary regulators of blood flow in the tissue.
In conclusion, the present study provides the first evidence, at both the molecular and functional levels, for a biological role of arginase in the coronary microcirculation. Specifically, we demonstrated that coronary arteriolar endothelial cells express arginase. Inhibition of its activity enhances the stimulated NO production and increases arteriolar dilation. It appears that arginase is an endogenous competitor of NOS for their common substrate L-arginine and thus plays a counteracting role in NO-mediated vasodilatory function.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0681fje ; to cite this
article, use FASEB J. (March 12, 2001)
10.1096/fj.00-0681fje 
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X174-DNA marker.
P < 0.05 control and DFMO vs.
L-NMMA and L-NMMA+DFMO.