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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 283:684 –691, 1997
Vol. 283, No. 2
Printed in U.S.A.
Plasma and Vascular Tissue Arginine Are Decreased in
Diabetes: Acute Arginine Supplementation Restores
Endothelium-Dependent Relaxation by Augmenting cGMP
Production1
GALEN M. PIEPER and LYNN A. DONDLINGER
Department of Transplant Surgery, Medical College of Wisconsin, Milwaukee Wisconsin
Accepted for publication July 23, 1997
Chronic experimental diabetes mellitus produces impairment of endothelium-dependent relaxation (see reviews:
Pieper and Gross, 1991; Cohen 1993; Kamata et al., 1992).
This defect has been confirmed in type I (Johnstone et al.,
1993; McNally et al., 1994) and type II (McVeigh et al., 1992)
diabetic patients. There is growing evidence from experimental models that alterations in several independent pathways
may contribute to impaired endothelium-dependent relaxation of diabetic blood vessels and that a single factor may be
inadequate to uniformly explain dysfunctional relaxation in
all instances and at various stages of the disease. These
factors include: 1) co-release of an endothelium-derived constricting factor derived from the cyclooxygenase pathway
(Tesfamariam et al., 1989; Mayhan, 1989); 2) increase in
protein kinase C (Pelligrino et al., 1994); 3) increased quenching of NO by advanced glycosylation end-products (Bucala et
al., 1991); 4) decreased NO activity because of interaction
with increased production of oxygen radicals (Pieper et al.,
Received for publication May 2, 1997.
1
This work was supported by grant HL47072 from the National Institutes
of Health, Heart and Lung Institute.
hibit cyclooxygenase activity and potassium channel activity,
respectively. Acetylcholine-stimulated cGMP generation (which
was blocked by L-nitroarginine) was diminished in diabetic rings
compared with control rings. L-Arginine restored cGMP in diabetic rings (with but not without endothelium) to levels similar to
control rings. L-Arginine did not alter cGMP generated by nitroglycerin. Incubation with L-arginine had no effect on acetylcholine-stimulated cGMP generation in control rings (with and
without endothelium). These data suggest a potential intracellular substrate deficiency in nitric oxide production by diabetic
endothelium which can be overcome acutely in vitro by provision of substrate for nitric oxide synthase.
1992,1996a; Diederich et al., 1994; Tesfamariam and Cohen,
1992); and 5) abnormal NO synthase activity caused by inadequate co-factor (Pieper, 1997). One factor that has received scant attention is the possibility that substrate supply
for efficient NO production by NO synthase in the diabetic
endothelium may be compromised.
NO or a closely related molecule accounts for a major
portion of the endothelium-dependent relaxation produced in
a variety of blood vessels. NO is synthesized in endothelial
cells by enzymatic conversion by NO synthase of the precursor amino acid, ARG, to form NO plus citrulline. Thus, disease states in which ARG levels are reduced may produce
inadequate levels of NO and impaired endothelium-dependent relaxation. In this regard, the plasma concentration of
ARG has decreased in experimental diabetic animals (Mans
et al., 1987) as well as in diabetic patients (Grill et al., 1992;
Hagenfeldt et al., 1989).
In previous studies from our laboratory, we observed that
L-ARG given acutely in vitro (Pieper and Peltier, 1995; Pieper
et al., 1995) or in vivo (Pieper et al., 1996b) restored endothelium-dependent relaxation to ACH in diabetic rat aorta
ABBREVIATIONS: NO, nitric oxide; NE, norepinephrine; L-NA, L-nitroarginine; L-ARG, L-arginine; D-ARG, D-arginine; ACH, acetylcholine; TEA,
tetraethylammonium; NTG, nitroglycerin.
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ABSTRACT
Arginine is a precursor amino acid for the synthesis of nitric
oxide by nitric oxide synthase. A defect in arginine supply could
regulate nitric oxide-mediated, endothelium-dependent relaxation. In this study, we evaluated the effect of supplementation
with L-arginine given in vitro on both functional relaxation and
cGMP generation in response to acetylcholine in the streptozotocin-induced diabetic rat aorta. The concentration of arginine in plasma and aortic tissue were both decreased by diabetes. Acute incubation in vitro with L-arginine augmented the
impaired relaxation to acetylcholine in diabetic rings although
not altering relaxation in control rings. L-Arginine also enhanced
relaxation to acetylcholine in diabetic rings incubated in the
presence of either indomethacin or tetraethylammonium to in-
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Arginine Augments NO in Diabetes
Materials and Methods
Adult male Sprague-Dawley (Sasco, Inc., Madison, WI) rats (approximately 90 days of age) were anesthetized with an intraperitoneal injection of 100 mg/kg Ketaset. Diabetes was induced in anesthetized animals by an intravenous tail-vein injection of
streptozotocin (55 mg/kg in 0.1 M citrate buffer, pH 4.5). A drop of
tail blood was obtained at 1 week after administration of streptozotocin to verify hyperglycemia by use of a glucometer. Diabetic and
age-matched control rats were housed for 2 months before experiments were performed.
On the day of experimentation, rats were anesthetized with 65
mg/kg sodium pentobarbital. Descending thoracic and abdominal
aortae were carefully isolated, removed and placed in 4°C Krebs’
bicarbonate buffer. The aortic segments were carefully cleaned of fat
and loose connective tissue and sectioned into 3-mm-long rings. In all
instances, care was taken to avoid stretching and contact with the
luminal surface to avoid damage to the endothelium during isolation.
Isolated vascular ring experiments. Thoracic aortic rings were
suspended between parallel hooks in 10-ml tissue baths which were
thermoregulated at 37°C in a Krebs-Henseleit medium as described
previously (Pieper and Peltier, 1995; Pieper et al., 1995). The medium was gassed with 95% O2-5% CO2 to maintain pH at 7.4. Aortic
rings were equilibrated and resting tension was set at an optimal
level of 2.0 g for both control and diabetic rings based on lengthtension studies. Changes in isometric tension were recorded on a
Gould TA6000 (Gould Instruments, Oxnard, CA) recorder via Radnoti (Radnoti Instruments, Monrovia, CA) force-displacement transducers. At the completion of each experiment, the rings were blotted
dry and weighed, and the lengths were measured to calculate tension
as normalized for cross-sectional areas based on the formula: crosssectional area (mm2) 5 weight (mg) 3 [length (mm) 3 density] with
the density of vascular tissue being 1.05 mg/mm3 (Abebe et al., 1990).
Individual vascular function protocols. Each ring was exposed to increasing concentrations of NE to generate concentrationresponse curves. Stock solutions of NE contained 20 nM ascorbate to
prevent autooxidation. After generating NE contraction-response
curves, each ring was serially washed to base-line tension and equilibrated. Rings were then contracted with a submaximal NE concentration which elicited approximately 70% of the maximum response.
The NE concentration was usually 1 mM but was varied, if necessary,
to achieve equieffective agonist activity.
At the plateau of contraction, relaxation responses to cumulative
concentrations of the endothelium-dependent vasodilator, ACH. We
have previously shown that relaxation to ACH is virtually blocked in
both control and diabetic rat aorta by NO synthase inhibitors (Pieper
and Peltier, 1995; Pieper et al., 1995). For these studies, the rings
were challenged once with ACH to verify that responses between
individual rings from the same animal were similar. Rings were then
washed serially, reequilibrated and incubated with 3 mM L-ARG for
45 min. Rings were then contracted with NE followed by a second
ACH challenge. With this technique, we have previously shown
similar responses between first and second challenges in both control
and diabetic aortic rings (Pieper and Peltier, 1995; Pieper et al.,
1995). This technique allows any potential differences in intravessel
reactivity to be minimized by verification before exposure to various
drug interventions. The experiments with L-ARG were also conducted in the presence of 10 mM indomethacin or 1 mM TEA to
eliminate the possibility that ARG produced enhanced relaxation via
a cyclooxygenase-dependent or K-channel-dependent pathway, respectively.
Amino acid analysis. At the time of experimentation, plasma for
arginine analysis was taken from a random subgroup of control and
diabetic rats. All samples were extracted 1:1 in 35% (wt/vol) sulfosalicylic acid dihydrate. After mixing and centrifugation, the supernatant was mixed 1:1 with lithium-D buffer before analysis.
Abdominal aorta was taken from a subset of animals in which the
descending thoracic aorta was used for functional analysis. The fresh
vascular tissue was homogenized in ground-glass microhomogenizing tubes in 800 ml of 0.5 N perchloric acid and centrifuged. An
aliquot of supernatant (700 ml) was mixed with 196 ml 2 M potassium
carbonate, centrifuged and frozen at 220°C for amino acid analysis.
Plasma and tissue arginine (including ARG) were examined with a
Beckman 6300 amino acid analyzer (Palo Alto, CA).
cGMP studies. Thoracic aortic rings from rats not used for functional studies were equilibrated in phosphate-buffered saline for 45
min. Rings with endothelium were either exposed to the following:
100 nM NE alone for 10 min plus 10 mM ACH (in the absence or
presence of 100 mM L-NA and in rings with or without endothelium),
or NE plus ACH in rings pretreated for 45 min with 3 mM D-ARG or
L-ARG (in rings with or without endothelium). The reaction was
terminated 1 min after the addition of buffer control or ACH by
freezing with liquid nitrogen and homogenizing in trichloroacetic
acid followed by extraction in water-saturated ether. In a few se-
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without altering responses to nitroglycerin. These results are
similar to the improved endothelium-dependent relaxation
elicited by L-ARG in experimental models of cardiomyopathy
(Mayhan and Rubinstein, 1992), chronic hypoxia (Eddahibi et
al., 1992; Carville et al., 1993), balloon-induced endothelial
injury (Hamon et al., 1994; Tarry and Makhoul, 1994), hypercholesterolemia/atherosclerosis (Cooke et al., 1991; Kuo et
al., 1992; Böger et al., 1995) and hypertension (Kitazono et
al., 1996) and in hypercholesterolemia in human patients
(Creager et al., 1992; Clarkson et al., 1996).
In all of these disease models, the mechanism for the
improved relaxation induced by ARG treatment was assumed to be caused specifically by enhanced NO-mediated
and enhanced cGMP-dependent production. Despite these
assumptions, no one has specifically addressed this assumption in any of the models listed above, including diabetes. We
believe these assumptions require further examination for
several reasons. First, NO-mediated relaxation has been
shown to act via cGMP-independent pathways and to elicit
relaxation via K1-sensitive channel activation (Cohen and
Vanhoutte, 1995). In some instances ARG analogs inhibit
dilation mediated by certain K1-sensitive vasodilators (Kontos and Wei, 1996). Thus, it is possible that ARG may enhance relaxation via a K1-sensitive mechanism. Furthermore, there is even some question whether ARG exclusively
enhances relaxation via NO because ARG analogs that inhibit NO synthase also blunt prostaglandin-mediated relaxation (Koller et al., 1993) and indomethacin blocks L-ARGenhanced dilation in hypertensive rats (Riedel et al., 1995).
Because of these new uncertainties, we reexamined the
hypothesis that L-ARG treatment of diabetic blood vessels
increases endothelium-dependent relaxation via a NO/
cGMP-dependent pathway. First, we sought to determine
whether reduced plasma ARG levels in diabetes leads to
frank reductions in vascular tissue stores of ARG because
this had not been evaluated previously. Second, we determined whether this was associated with decreased NO production as assessed by both functional relaxation and cGMP
generation. Third, we performed additional experiments in
the presence of indomethacin or TEA to circumvent potential
pathways of enhanced L-ARG-induced relaxation via prostanoid or K1-sensitive pathways. Finally, we sought to determine whether the improvement by L-ARG supplementation in endothelium-dependent relaxation of diabetic blood
vessels could be explained by an actual improvement in NO/
cGMP production.
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Vol. 283
lected experiments, 100 mM L-NA was given during the last 20 min
of incubation to block NO synthase. For control purposes, the effects
of L-ARG were also studied in diabetic rings that were stimulated
with 30 mM nitroglycerin for 2 min before terminating the reaction.
cGMP was determined by radioimmunoassay with a commercial
diagnostic kit (PerSeptive Diagnostics, Cambridge, MA).
Statistical analysis. Data are expressed as the mean 6 S.E.M.
Data were analyzed by analysis of variance followed by Fisher’s
projected least-squares difference test for multiple mean comparisons or unpaired t test for comparisons of two group means or paired
t test for comparisons of two group means in a pretest/posttest
format. A value of P , .05 was set to denote statistical significance.
Results
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Body weight and blood analysis. A total of 128 rats
were used for this study. Body weight of control rats (total
n 5 60) increased from an initial weight of 387 6 5 g to 532 6
8 g at the completion of the study. In contrast, diabetic rats
(total n 5 68) weighed 388 6 5 g initially and 347 6 9 g at the
end of the study. Blood glucose levels of diabetic animals
were significantly increased (P , .001) above those of control
animals at 1 week (i.e., 370 6 9 vs. 80 6 4 mg/dl) and at the
end of the study (i.e., 385 6 9 vs. 83 6 5 mg/dl).
Diabetes produced a significant decrease in the plasma
concentration of ARG (fig. 1, upper panel). In addition to
diminished plasma ARG concentration in diabetic animals,
the ARG content of aortic tissue was diminished significantly
by diabetes (fig. 1, lower panel). Incubation of diabetic aortic
segments with 3 mM L-ARG increased tissue arginine content by approximately 20-fold (i.e., to 10.6 6 0.2 nmol/mg
tissue, n 5 4 determinations).
Vascular relaxation. Initial studies were performed in
which the mean relaxation response to ACH for all rings was
averaged for each animal and the mean for each group calculated. ACH produced a concentration-dependent relaxation in both control and diabetic aortic rings (fig. 2). The
relaxation produced in diabetic rings was impaired relative
to rings from age-matched control animals. The addition of
L-ARG did not alter the relaxation to ACH in control rings
(fig. 2, upper panel), but significantly improved the attenuated relaxation observed in diabetic rings (fig. 2, lower panel). The relaxation to ACH in L-ARG-treated diabetic rings
was not different from that observed in untreated control or
L-ARG-treated control rings. For example maximal relaxation was significantly improved by L-ARG compared with
untreated diabetic rings (i.e., 86 6 3% and 67 6 3%, respectively; P , .01) which was not significantly different from
untreated and treated control rings (i.e., 91 6 3% and 94 6
6%, respectively). Furthermore, treatment with L-ARG restored the pD2 for ACH in diabetic rings (untreated diabetic,
5.46 6 0.17; L-ARG-treated diabetic, 6.25 6 0.18, P , .01) to
a value which was not statistically different from control
values (untreated control, 6.45 6 0.10; L-ARG-treated control, 6.61 6 0.22, not significant).
In the presence of indomethacin, responses to ACH were
still impaired by diabetes and pair-matched rings incubated
in the presence of L-ARG also augmented relaxation to ACH
(fig. 3) in diabetic rings (lower panel) but not control rings
(upper panel). For example maximal relaxation was significantly improved by L-ARG compared with untreated diabetic
rings (i.e., 93 6 2% and 66 6 6%, respectively; P , .01) which
was not significantly different from untreated and treated
Fig. 1. Decrease in the plasma ARG (n 5 6 –7) concentration and
tissue ARG (n 5 5 each) content in diabetic rats. *P , .01 and **P , .01
vs. control rats.
control rings (i.e., 97 6 3% and 101 6 6%, respectively).
Treatment with L-ARG also did not alter pD2 for ACH in
indomethacin-treated control rings (i.e., 6.86 6 0.12 and
6.94 6 0.06 for rings without or with L-ARG, respectively) but
did significantly change (P , .01) the pD2 for ACH in indomethacin-treated diabetic rings (i.e., 5.97 6 0.26 vs. 6.55 6
0.14 for rings without and with L-ARG, respectively).
In the presence of TEA, L-ARG enhanced relaxation to
ACH in diabetic rings (fig. 4, lower panel). For example,
maximum relaxation to ACH was 64 6 6% vs. 88 6 2% (P ,
.01) and the pD2 for ACH was 5.46 6 0.30 vs. 6.29 6 0.10 (P ,
.01) for rings without or with L-ARG, respectively. There was
no significant change in either sensitivity to ACH in control
rings by L-ARG (i.e., pD2 5 6.50 6 0.10 and 6.72 6 0.07 for
rings without or with L-ARG, respectively, P 5 .16) or max-
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Arginine Augments NO in Diabetes
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Fig. 2. Concentration-dependent relaxation to ACH in aortic rings of
control and diabetic rats in the absence (n 5 25 and 33 for control and
diabetic, respectively) or presence (n 5 15 and 25 for control and
diabetic, respectively) of 3 mM L-ARG. **P , .01 and ***P , .001 vs.
control rats.
imum relaxation (98 6 5% and 100 6 1% for rings without or
with L-ARG, respectively) (fig. 4, upper panel).
cGMP studies. cGMP was significantly increased by ACH
in control rings with endothelium compared with rings without endothelium or in rings pretreated with L-NA (fig. 5). The
ACH-stimulated cGMP production was significantly attenuated in diabetic compared to control rings. Incubation with
L-ARG did not alter ACH-stimulated cGMP production in
control rings with or without endothelium (fig. 5). In contrast, L-ARG potentiated ACH-stimulated cGMP production
in diabetic rings with endothelium but not in diabetic rings
Fig. 3. Effects of addition of L-ARG on relaxation to ACH in aortic rings
of control (n 5 5 each) and diabetic (n 5 7 each) rats pretreated with
indomethacin. *P , .05 and **P , .01 vs. untreated.
without endothelium (fig. 5). D-ARG did not alter cGMP
production in response to ACH in diabetic rings (not shown).
Furthermore, L-ARG did not alter NTG-stimulated cGMP
production in diabetic rings (i.e., 4.8 6 0.4 and 5.0 6 0.6
pmol/mg protein, with and without L-ARG, respectively, n 5
4 pair-matched rings each).
Discussion
In the intact cell, various factors can potentially regulate
efficient NO production by NO synthase. In the present
study, we provide evidence that a decrease in supply of ARG
might regulate the full expression of NO release by diabetic
endothelium. Accordingly, we show for the first time that
both plasma and vascular tissue ARG concentrations are
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Vol. 283
Fig. 4. Effects of addition of L-ARG on relaxation to ACH in aortic rings
of control (n 5 8 each) and diabetic (n 5 8 each) rats pretreated with
TEA. *P , .05 and **P , .01 vs. untreated.
decreased in diabetes and that this decrease is associated
with both diminished ACH-mediated relaxation and cGMP
production, a measure of NO production. Acute addition of
L-ARG but not D-ARG restored relaxation and cGMP production similar to that observed in untreated and ARG-treated
control blood vessels.
Although decreases in plasma ARG concentrations have
been reported in experimental diabetic animals (Mans et al.,
1987) and human diabetic patients (Grill et al., 1992; Hagenfeldt et al., 1989), it was not known previously whether
decreases in plasma ARG concentration would be reflected in
actual reductions in the ARG content of vascular tissue. This
was particularly uncertain because of one report that ARG
transport was actually enhanced in hepatocytes of diabetic
rats (Handlogten and Kilberg, 1984); however, the liver is an
important organ for the utilization of various amino acids for
gluconeogenesis, etc.
In the endothelial cell, L-ARG is usually transported via
the system y1 transporter (Greene et al., 1993; Bussolati et
al., 1993). Previous estimates indicate that the Km for L-ARG
uptake in cultured bovine pulmonary artery endothelial cells
was 300 mM (Greene et al., 1993). In contrast, a more recent
study with human umbilical vein endothelial cells revealed a
Km for L-ARG transport closer to 100 mM and that the Km did
not change in gestational diabetes (Sobrevia et al., 1995).
In the present study, we report that plasma ARG concentration in streptozotocin-induced diabetic rats decreased
from 190 to 65 mM. It is not likely that streptozotocin produces a generalized decrease in plasma amino acids because
several other amino acids are either unchanged or actually
increased (Pieper and Peltier, 1995; Pieper et al., 1995,
1996b) and decreases in plasma ARG but not other amino
acids have been observed in diabetic patients (Grill et al.,
1992). Because the plasma ARG concentration is less than
the Km for ARG in our study, it is likely that arginine transport is not saturated and that a relative deficiency in tissue
ARG levels could result. Our measurement of vascular tissue
ARG levels supports this hypothesis. The decrease in tissue
ARG level also is not likely to produce a generalized decrease
in amino acids as a consequence of streptozotocin-induced
diabetes, because we have noted either no change or increases in other amino acids in vascular tissue (unpublished
observations). This observation is similar to that obtained for
other amino acids in the brain tissue of streptozotocin-induced diabetic rats (Mans et al., 1987).
Furthermore, the decrease in vascular tissue ARG is not
likely to be caused by intrinsic defects in the cationic amino
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Fig. 5. cGMP in control and diabetic aortic rings (with or without
endothelium) stimulated with 10 mM ACH. Bars represent: (a) control
rings pretreated with L-NA (n 5 4); (b) control rings without endothelium
(n 5 4); (c) L-ARG-treated control rings without endothelium (n 5 4); (d)
control rings with endothelium (n 5 12); (e) L-ARG-treated control rings
with endothelium (n 5 11); (f) diabetic rings pretreated with L-NA (n 5
4); (g) diabetic rings without endothelium (n 5 4); (h) L-ARG-treated
diabetic rings without endothelium (n 5 4); (i) diabetic rings with endothelium (n 5 6); (j) L-ARG-treated diabetic rings with endothelium (n 5
6) *P , .05 vs. corresponding ring with L-NA and (*)P , .05 vs. untreated
control rings.
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689
1996). This suggests that the beneficial effects of arginine
supplementation are independent of the model of diabetes
chosen for evaluation. In contrast, topical application of LARG in situ failed to enhance relaxation to ACH and bradykinin in diabetic rat basilar arteries (Mayhan et al., 1996).
This divergent observation could be explained by the possibility that multiple factors contribute to endothelial dysfunction in diabetes, and the contribution of individual factors
might vary from one vessel type to another. In this regard,
because the specific contribution to defective NO-mediated
relaxation in control and diabetic basilar artery is unknown,
defects in NO-independent factors may contribute to defective relaxation of diabetic rat basilar artery and may not be
amenable to restoration by ARG supplementation. Indeed,
Kamata and Kondoh (1996) have shown that indomethacin
normalizes relaxation to ACH in diabetic rat basilar arteries.
In contrast, we have observed that ACH-mediated relaxation
in both control and diabetic aortic rings of two independent
rat strains is completely abrogated by incubation with NO
synthase inhibitors (Pieper and Peltier, 1995; Pieper et al.,
1995), which indicates that relaxation is specific for NO. This
makes this preparation ideal for studying diabetes-induced
defects in relaxation which are specific for NO.
An alternative and equally plausible explanation for these
differences is that various factors may contribute to dysfunction at different stages of the disease. The fact that topical
application of L-ARG failed to augment relaxation in the
study with the basilar artery of animals with diabetes of 3
months duration is consistent with our results showing that
L-ARG restores relaxation to ACH in the aortas of rats with
diabetes 2 months duration but not in the aortas of rats with
diabetes of 3 months duration (Pieper et al., 1995). This
underscores the importance in appreciating the time-dependent progression in the etiology of diabetes-induced endothelial dysfunction and that factors that contribute to dysfunction at one stage may no longer be important or
surmountable at later stages of the disease.
We also showed that the improved relaxation in response
to addition of L-ARG is specific for diseased blood vessels in
that responses to ACH were not improved in control rings.
This salient action is stereospecific in that we previously
demonstrated that D-ARG failed to augment relaxation to
ACH in diabetic rings (Pieper and Peltier, 1995; Pieper et al.,
1995). Furthermore, we also showed that L-ARG effects are
specific for endothelium-dependent processes because responses to the endothelium-independent vasodilator NTG
was unaltered by L-ARG treatment of diabetic rings (Pieper
and Peltier, 1995; Pieper et al., 1995). The mechanism by
which L-ARG provides this effect was not provided in our
previous studies. In the present study, experiments conducted in the presence of indomethacin or TEA suggest that
changes in prostanoid release or K1 activation cannot account for the improved relaxation elicited by L-ARG in diabetic rings.
Consistent with the notion that tissue availability of ARG
for synthesis of NO by diabetic aortic endothelium may be
compromised is our analysis of ACH-stimulated cGMP production which was observed to be decreased in untreated
diabetic aortas compared with untreated control aortas.
These findings are consistent with previous measurements of
decreased ACH-stimulated cGMP production in streptozotocin-induced diabetic rat aortas (Kamata et al., 1989; Miller et
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acid transporter for ARG per se because in vitro studies have
shown that uptake is unaltered in isolated coronary endothelial cells from the diabetic BB rat (Wu and Meininger, 1995).
Although these same authors reported an actual increase in
ARG content of diabetic endothelial cells despite impaired
NO synthesis, it is uncertain what occurs under ambient
conditions because these results were obtained in passaged
cells with medium containing 0.4 mM ARG. In fact, a recent
study has even shown increased ARG transport in gastric
glands of the diabetic rabbit at 2 to 3 days after alloxan
administration (Contreras et al., 1997). These discrepancies
may be accounted for by variances in models of diabetes,
duration of disease, species and tissue. Thus, the relevancy of
these other studies to our model is not known with certainty.
The question is raised whether this reduction of tissue
ARG levels is of sufficient magnitude to be of significant
consequence for NO production by endothelial cell NO synthase. It is not known what the exact molar concentration of
tissue ARG was in our preparation, because our tissue values
are not reported in these units; however, diabetes decreased
ARG content by .40%. The intracellular ARG concentration
in normal bovine aortic endothelial cells has been estimated
to range from 122 mM (Mitchell et al., 1990) to 250 mM
(Preik-Steinhoff et al., 1995).
The Km for ARG for purified NO synthase has been reported to be 6 mM and the activity appears to be maximized
at ARG concentrations between 30 and 100 mM (Mayer et al.,
1989). The appropriate intracellular Km for ARG for NO
synthase in intact endothelial cells is unclear, because all
previous estimates have been performed in cell-free systems
under ideal conditions. Thus, it is likely that the intracellular
Km for ARG is likely to be higher than that reported for
purified enzymes.
Other factors or conditions present under intact cell conditions could also modulate the Km for ARG. This includes
such factors as the concentration of cofactor, tetrahydrobiopterin, which in suboptimal concentrations increases the Km
for ARG (Klatt et al., 1994a, b). In this regard, the concentration of tetrahydrobiopterin is known to be reduced in the
brain of diabetic rats (Hamon et al., 1989), and we have found
that acute incubation with a derivative of tetrahydrobiopterin restores relaxation to ACH in diabetic blood vessels
(Pieper, 1997). Thus, it is theoretically possible that supplementing cofactor may improve relaxation by increasing NO
despite lower ARG content in diabetic tissue even through a
process of decreasing the Km for ARG.
We assume that the concentration of tissue ARG measured
in our study is uniformly distributed between all cell types
and within the endothelial cell itself, but this is not known
with any certainty. Nevertheless, we cannot exclude the possibility because of the decreased overall ARG content, a localized ARG deficiency in endothelial cells could place the
concentration at or near the optimal intracellular Km for
ARG for the purified constitutive NO synthase.
The fact that L-ARG improved relaxation to ACH in diabetic aortic rings is consistent with previous studies in our
laboratory using two different strains of rat (Pieper and
Peltier, 1995; Pieper et al., 1995) and in our genetic model of
the spontaneously diabetic BB rat (Pieper et al., 1997). This
agrees also with others showing improved relaxation after
intravenous ARG infusion in canine coronary arteries in
short-term alloxan-induced diabetes (Matsunaga et al.,
Arginine Augments NO in Diabetes
690
Pieper and Dondlinger
Acknowledgments
The shared research facilities of the Cancer Center of the Medical
College of Wisconsin were used for the amino acid analysis.
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al., 1994) and in alloxan-diabetic rabbit aortas (Abiru et al.,
1990).
Our cGMP studies extend these initial observations in
several important ways. First, we demonstrated that the
ACH-stimulated cGMP production is specific for NO production in both control and diabetic rat aortas because they are
blocked by the NO synthase inhibitor, L-NA. Second, our data
showing that L-ARG augmented ACH-stimulated cGMP production in diabetic (but not control aortas) is also consistent
with a deficiency in ARG supply in diabetic vascular endothelium and cannot be accounted by supply of ARG for any
NO synthase in nonendothelial tissue. Third, the additional
studies in which D-ARG failed to alter cGMP production in
diabetic aortas indicate that this is stereospecific. Fourth, the
lack of effect of L-ARG on cGMP generated by NTG suggests
that this cannot be explained by an L-ARG-induced change in
guanylate cyclase reactivity. To our knowledge the present
study is the first report to show improved cGMP production
in diabetic blood vessels by any type of pharmacological intervention and the first to show that the action of L-ARG to
improve endothelium-dependent relaxation in any diseased
model is specific for enhanced cGMP generation.
Finally, it has been hypothesized that abnormal endothelial function in patients with insulin-dependent diabetic mellitus may be caused by a defect in ARG-derived NO synthesis
(Calver et al., 1993). Accordingly, L-ARG given to diabetic
patients might increase NO production. Indeed, there is an
isolated report in which L-ARG augmented plasma concentrations of nitrite in patients with non-insulin-dependent
diabetes (Das et al., 1993). Unfortunately, it is unknown
whether healthy individuals respond similarly to L-ARG.
Vol. 283
1997
691
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Send reprint requests to: Dr. Galen M. Pieper, Dept. of Transplant Surgery,
Medical College of Wisconsin, Froedtert Memorial Hospital, 9200 West Wisconsin Avenue, Milwaukee WI 53226.
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Arginine Augments NO in Diabetes