Division of Nephrology and Hypertension, Department of Medicine, University of California, Irvine, California 92697
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ABSTRACT |
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Diabetes is
associated with endothelial dysfunction and increased risk of
hypertension, cardiovascular disease, and renal complications. Earlier
studies have revealed that hyperglycemia impairs nitric oxide (NO)
production and diabetes causes endothelial dysfunction in humans and
experimental animals. This study was designed to test the effects of
altered concentrations of glucose, insulin, and glucagon, the principal
variables in types I and II diabetes, on NO production and endothelial
NO synthase (eNOS) expression in cultured human coronary endothelial
cells. Cultured endothelial cells were incubated in the presence of
glucose at either normal (5.6 mM) or high (25 mM) concentrations for 7 days. The rates of basal and bradykinin-stimulated NO production
(nitrate + nitrite) and eNOS protein expression (Western blot)
were then determined at the basal condition and in the presence of
insulin (108 and 10
7 M), glucagon
(10
8 and 10
7 M), or both. Incubation with a
high-glucose concentration for 7 days significantly downregulated,
whereas insulin significantly upregulated, basal and
bradykinin-stimulated NO production and eNOS expression in cultured
endothelial cells. The stimulatory action of insulin was mitigated by
high-glucose concentration and abolished by cotreatment of cells with
glucagon. Thus hyperglycemia, insulinopenia, and hyperglucagonemia,
which frequently coexist in diabetes, can work in concert to suppress
NO production by human coronary artery endothelial cells.
endothelial cells; diabetes; coronary artery; hypertension; arteriosclerosis; endothelium-derived relaxation
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INTRODUCTION |
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NITRIC OXIDE (NO) is an endogenous modulator with diverse biological functions. It is the most potent endogenous vasodilator and, as such, plays an important role in the regulation of renal and systemic vascular resistance (20, 37, 48). In addition, by inhibiting tubular Na+ reabsorption, NO promotes natriuresis (7, 16, 28, 35, 40). Thus constitutively produced NO plays an important role in blood pressure homeostasis. The physiological importance of NO in the regulation of blood pressure is evidenced by the fact that pharmacological inhibition of NO synthases leads to severe hypertension, vascular injury, and glomerulosclerosis in experimental animals (50). Moreover, endothelial NO synthase (eNOS) knockout mice exhibit hypertension (32), thus providing further support for the importance of NO in the regulation of blood pressure. In addition to being a potent vasodilator and a natriuretic agent, NO inhibits platelet and leukocyte adhesion, cell migration and proliferation, and matrix accumulation (1, 30, 36). Because these events are intimately involved in the pathogenesis of arteriosclerosis, atherosclerosis, and glomerulosclerosis, their inhibition by NO protects cardiovascular and renal function. Occurrence of these lesions with NOS inhibitors in experimental animals strongly supports this contention.
Diabetes is one of the major risk factors for ischemic cardiovascular complications and the leading cause of end-stage renal disease (19, 45). Advanced diabetes is frequently complicated by hypertension, premature arteriosclerosis, and endothelial dysfunction, suggesting depressed NO availability (8, 22, 39, 55). The effect of diabetes on NO metabolism is controversial. Several studies have demonstrated impaired endothelium-dependent vasorelaxation in diabetic humans (18, 33, 41, 54, 58) and experimental animals (4, 15, 43, 49). In contrast, a number of other studies have found no discernible impairment of endothelium-dependent vasodilation (6, 9, 31, 38, 53). In fact, several recent studies have revealed increased NO production in diabetes (11-14, 24-27, 42, 62). However, avid inactivation of NO by reactive oxygen species has been shown to reduce its bioavailability in diabetes (10-13, 23, 56, 57). Support for the latter contention comes from several studies demonstrating improvement of endothelial dysfunction with antioxidant therapy in diabetes (4, 13, 15, 43, 58). Accordingly, endothelial dysfunction can occur as a result of increased NO inactivation by reactive oxygen species despite enhanced NO production. It should be noted that most of the available studies reporting endothelial dysfunction have evaluated humans and animals with long-term diabetes as opposed to exploring the direct effects of altered glucose or glucose regulatory hormones.
The untreated type I diabetes is characterized by hyperglycemia and
insulinopenia (51). Treatment of type I diabetes with intermittent insulin administration leads to variable glycemia control.
In addition, subcutaneously administered insulin exposes the systemic
vasculature to higher levels of insulin than those occurring normally
when insulin is released by the pancreas into the hepatic portal venous
circulation (47). Type II diabetes is marked by insulin
resistance and, as such, hyperglycemia is coupled with elevated insulin
levels in these subjects (44). Administration of oral
hypoglycemic agents or exogenous insulin further raises insulin levels
in patients with type II diabetes. In advanced stages of type II
diabetes, exhaustion of insulin-producing -cells leads to
insulinopenia and exogenous insulin dependence. In addition to insulin
deficiency or resistance, diabetes is frequently associated with
increased production of glucagon, which is a potent insulin antagonist
(21, 60).
The present study was designed to explore the effects of the principal variables in diabetes, namely, glucose concentration, insulin level, and the insulin antagonist glucagon on endothelial NOS protein expression and NO production in cultured human coronary endothelial cells.
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METHODS |
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Cell culture. Human coronary artery endothelial cells (Biowhittaker, San Diego, CA) were cultured in a manner that was precisely the same as that described in our previous studies (61). Cells obtained on the third and fourth passages were used.
Study protocol.
Cultured endothelial cells were incubated in the medium containing
glucose at either a high (25 mM) or normal (5.6 mM) concentration for 7 days. Subsets of the cells were then treated with insulin at either
108 M or 10
7 M alone, with glucagon at
either 10
7 M or 10
8 M alone, or with
insulin plus glucagon for 24 h. Cells treated with inactive
vehicle served as controls. Both insulin and glucagon were purchased
from Sigma Chemical (St. Louis, MO). At the conclusion of the 24-h
treatment period, cells were harvested and processed for measurement of
eNOS protein abundance by Western blot analysis, and the medium was
used for determination of total nitrate plus nitrite (NOx). On each
occasion, cell viability was determined by Trypan blue exclusion test
and optical inspection and was found to be >95%.
Measurement of NO production. NO production was determined from the NOx recovered in the culture medium. NOx was quantified by use of the purge system of the Sievers NO Analyzer (model 270 B NOA, Sievers Instruments, Boulder, CO). The amount of NOx produced was normalized against total cellular protein. The procedures involved in this assay have been described in detail in our earlier studies (17).
Measurement of eNOS protein. Endothelial cells were processed for determination of eNOS protein abundance by Western analysis, as described in our earlier studies (17). Briefly, cells were washed with PBS and extracted directly into the sample buffer (2% SDS, 10% glycerol, 0.0025% bromphenol, and 63 mmol/l Tris · HCl, pH 6.8), and total protein was determined by using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Fifty micrograms of cell lysate protein were size-fractionated on 4-12% Tris-glycine gel at 130 V for 3 h. In preliminary experiments, the given protein concentrations were found to fall within the linear range of detection for our Western blot technique. After electrophoresis, proteins were transferred onto Hybond enhanced chemiluminescence (ECL) membrane at 400 mA for 120 min by use of the Novex transfer system (Novex, San Diego, CA). The membrane was prehybridized in 10 ml of buffer A (10 mM Tris · Hcl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 10% nonfat milk powder) for 1 h and then hybridized for an additional 1 h in the same buffer containing 10 µl of the anti-eNOS monoclonal antibody (Transduction Laboratories, Lexington, KY) at 1:1,000 dilution. Thereafter, the membrane was washed for 30 min in a shaking bath, with the wash buffer (buffer A without nonfat milk) changed every 5 min before a 1-h incubation in buffer A plus goat anti-mouse IgG-horseradish peroxidase at the final titer of 1:1,000. Experiments were carried out at room temperature. The washes were repeated before the membrane was developed with a light-emitting nonradioactive method using ECL reagent (Amersham Life Science, Arlington Heights, IL). The membrane was then subjected to autoluminography for 1-5 min. The autoluminographs were scanned with a laser densitometer (model PD1211, Molecular Dynamics, Sunnyvale, CA) to determine the relative optical densities of the bands. In all instances, the membranes were stained with Ponceau stain before prehybridization to verify the uniformity of protein load and transfer efficiency across the test samples.
Data analysis.
Analysis of variance (ANOVA), regression analysis, and Student's
t-test were used in evaluation of the data, which are
presented as means ± SE. P values 0.05 were
considered significant.
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RESULTS |
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Effect of glucose concentration.
Exposure to high-glucose concentration for 7 days resulted in a
significant downregulation of eNOS expression in cultured human
coronary artery endothelial cells. This was accompanied by a marked
reduction in basal NO production as discerned from NOx recovered in the
extracellular medium (Fig. 1).
Stimulation with 106 M bradykinin for 1 h resulted
in an expected rise in NOx generation in cells exposed to normal
glucose concentration (0.047 ± 0.01 vs. 0.072 ± 0.01 nmol/µg protein, P < 0.02, vehicle vs.
bradykinin-treated cells). Similarly, bradykinin augmented NOx
production in cells exposed to simulated hyperglycemia (0.031 ± 0.003 vs. 0.054 ± 0.007 nmol/µg protein, P < 0.001). However, both basal and bradykinin-stimulated NOx productions
were significantly lower in cells exposed to simulated hyperglycemia
than their counterparts kept in the medium containing normal glucose
concentration (P < 0.03 for both).
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Effect of insulin.
Addition of insulin at 108 and 10
7 M to
endothelial cells, maintained in a medium with normal-glucose level,
led to a concentration-dependent rise in eNOS protein expression and NO
production. Similarly, insulin dose dependently raised eNOS expression
and NO production in endothelial cells subjected to simulated
hyperglycemia. However, the values obtained for each insulin
concentration were lower in cells exposed to high-glucose than in those
exposed to normal-glucose levels. Thus high-glucose concentration
depressed the stimulatory action of insulin on the NO system in
coronary endothelial cells (Fig. 2).
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Effect of glucagon.
Addition of glucagon at either 107 or
10
8 M concentration failed to alter eNOS protein
expression in endothelial cells maintained in either normal- or
high-glucose media. Similarly, glucagon had no effect on NO production
by endothelial cells under either condition (Fig.
3).
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Effect of glucagon on insulin action.
Although glucagon alone had no discernible effect on the endothelial NO
system, it virtually abrogated the upregulatory action of insulin on
both eNOS expression and NO production by endothelial cells (Fig.
4). This phenomenon was independent of
extracellular glucose concentration and was seen with both normal- and
high-glucose concentrations.
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Correlation.
A significant direct correlation was found between eNOS protein
abundance and NO production in all experiments (r = 0.803, P < 0.01). However, glucose concentration
showed an inverse correlation with eNOS expression (r = 0.816, P < 0.01) and NO production (r =
0.787, P < 0.01).
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DISCUSSION |
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Several earlier studies have revealed that both short- and
long-term exposure to hyperglycemia impairs endothelial function in
humans and experimental animals (13, 34).
This can be due to impaired production of endothelium-derived NO,
enhanced inactivation of NO, or increased release of
endothelium-derived vasoconstrictive factors. Earlier studies have
demonstrated a marked increase in production of the superoxide radical
(O2), which is an avid scavenger of NO, in cultured
aorta endothelial cells exposed to simulated hyperglycemia for 5 days
(13). These observations support the contribution of
enhanced NO inactivation in the pathogenesis of endothelial dysfunction
associated with chronic hyperglycemia. In addition to inactivating NO,
oxygen free radicals can promote generation of vasocontractile
prostanoids (12, 42, 56,
57). Thus elevated glucose concentration can contribute to
endothelial dysfunction through an enhanced oxygen free
radical-mediated NO inactivation and increased generation of
vasoconstrictive prostanoids. The present study was carried out to
explore the direct effects of simulated hyperglycemia, insulin, and
glucagon on the NO system in cultured endothelial cells.
Exposure to simulated hyperglycemia for 7 days resulted in a significant downregulation of eNOS expression as well as basal and bradykinin-stimulated NO production in cultured human coronary endothelial cells. The downregulatory effect of high glucose concentration was seen at different levels of insulin in the culture media. On the basis of these observations, it appears that hyperglycemia can contribute to coronary artery endothelial cell dysfunction at a wide range of insulin levels. We caution against extension of these findings in human coronary artery endothelial cells to endothelial cells of other origins that may behave differently. In fact, studies conducted by Cosentino et al. (13) with cultured aorta endothelial cells revealed increased NO production and eNOS expression in cells exposed for 5 days to a high-glucose concentration. The reason for the observed difference in the effect of simulated hyperglycemia on the NO system in the two studies is not entirely clear. It should be noted that Cosentino et al. used aorta endothelial cells, whereas the present study employed coronary artery endothelial cells. In addition, glucose concentration used to simulate hyperglycemia (22.2 mmol/l) and duration exposure (5 days) in the former study (13) were less than those employed here (25 mmol/l and 7 days, respectively). Moreover, cells up to passage 6 were used in the former study, whereas only cells from the third and fourth passages were used here. It should be noted that increased NO production in aorta endothelial cells reported by Cosentino et al. was coupled with an even greater increase in generation of superoxide, which is known to inactivate NO. The imbalance between NO production and superoxide generation shown in the latter study can clearly contribute to endothelial dysfunction associated with high glucose levels (15).
Incubation with insulin at 108 and 10
7 M
for 24 h resulted in a dose-dependent upregulation of eNOS
expression and NO production by cultured coronary endothelial cells. It
should be noted that, on each occasion, the stimulatory action of
insulin on the NO system was dampened by high-glucose concentration.
Thus it appears that insulinopenia, and perhaps insulin resistance, may
contribute to coronary artery endothelial dysfunction, compounding the
adverse effect of hyperglycemia. In a recent study, Kawaguchi et al.
(34) demonstrated that administration of insulin for 4 wk
to diabetic obese Zucker rats ameliorated hypertension and raised
plasma concentration and urinary excretion of NO metabolites,
suggesting enhanced NO production. Similarly, Zeng and Quon
(63) have shown increased NO production with insulin. The
results of the present in vitro studies are consistent with the
findings of in vivo studies reported by Kawaguchi et al. and Zeng and
Quon. A number of previous studies have demonstrated a reduction in
blood pressure with insulin therapy in diabetic humans (2,
3, 5). Enhanced NO production and decreased
NO inactivation by superoxide with insulin replacement and amelioration
of hyperglycemia may play a part in this process. Further studies are
needed to explore this possibility.
Glucagon is a peptide hormone produced by the -cells of the
pancreas. By promoting glycogenolysis and gluconeogenesis from amino
acids, glycerol, and pyruvate, glucagon raises glucose concentration. Thus glucagon serves as a natural insulin antagonist. Glucagon levels
have been shown to be elevated in both type I and type II diabetes
mellitus (21, 60). Therefore, we sought to
explore the possible effect of glucagon on the NO system in coronary
endothelial cells. This study revealed that glucagon, per se, has no
discernible effect on either eNOS expression or NO production in
isolated human coronary endothelial cells. However, glucagon virtually abolished the stimulatory effect of insulin on eNOS protein expression and NO production in these cells. These observations revealed another
anti-insulin action of glucagon beyond its known effects on glucose
metabolism. Moreover, the results suggest that the associated
hyperglucagonemia may potentially contribute to coronary artery
endothelial dysfunction in diabetes. The inhibitory action of glucagon
on insulin-mediated upregulation of eNOS expression and NO production
in endothelial cells shown here parallels the results of earlier
studies showing that glucagon mitigated the cytokine-mediated induction
of inducible NOS in cultured hepatocytes (29,
52). Therefore, the downregulatory action of glucagon on
the NO system is not limited to eNOS. Interestingly, glucagon has been
shown to cause a fast-acting renal vasodilation in rat in vivo and
vasorelaxation in dog arterial and venous preparations in vitro
(46, 59). This effect was cAMP mediated and
was present in endothelium-denuded as well as endothelium-bearing
preparations. It should be noted that the fast-acting direct
vasodilatory action of glucagon shown in the latter studies is distinct
from its late effect on eNOS expression via inhibition of insulin's
action shown here.
In conclusion, simulated hyperglycemia resulted in a significant downregulation of eNOS expression and NO production by cultured human coronary endothelial cells. In contrast, insulin caused a dose-dependent upregulation of eNOS expression and NO production in this system. The stimulatory action of insulin was mitigated by simulated hyperglycemia and was abrogated by glucagon administration. Thus hyperglycemia, insulinopenia, and hyperglucagonemia, which frequently coexist in diabetes, can suppress the NO system in coronary endothelial cells in vitro. Caution should be exercised in extending the present in vitro findings to the highly complex clinical setting in vivo.
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ACKNOWLEDGEMENTS |
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This work was supported by an award from the American Heart Association, Florida/Puerto Rico Affiliate (MN845B).
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. D. Vaziri, Division of Nephrology and Hypertension, UCI Medical Center, 101 The City Drive, Orange, CA 92868.
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. §1734 solely to indicate this fact.
Received 6 October 1999; accepted in final form 18 January 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, MR,
Jessup W,
Hailstones D,
and
Celermajer DS.
L-Arginine reduces human monocyte adhesion to vascular endothelium and endothelial expression of cell adhesion molecules.
Circulation
95:
662-668,
1997
2.
Anderson, EA,
Hoffman RP,
Balon TW,
Sinkey CA,
and
Mark AL.
Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans.
J Clin Invest
87:
2246-2252,
1991[ISI][Medline].
3.
Anderson, EA,
and
Mark AL.
The vasodilator action of insulin. Implications for the insulin hypothesis of hypertension.
Hypertension
21:
136-141,
1993[ISI][Medline].
4.
Angulo, J,
Sanchez-Ferrer CF,
Peiro C,
Marin J,
and
Rodriguez-Manas L.
Impairment of endothelium-dependent relaxation by increasing percentages of glycosylated human hemoglobin. Possible mechanisms involved.
Hypertension
28:
583-592,
1996
5.
Baron, AD.
Hemodynamic actions of insulin.
Am J Physiol Endocrinol Metab
267:
E187-E202,
1994
6.
Bohlen, HG,
and
Lash JM.
Endothelial-dependent vasodilation is preserved in non-insulin-dependent Zucker fatty diabetic rats.
Am J Physiol Heart Circ Physiol
268:
H2366-H2374,
1995
7.
Broere, A,
Van Den Meiracker AH,
Boomsma F,
Derkx FH,
Veld AJ,
and
Schalekamp MA.
Human renal and systemic hemodynamic, natriuretic, and neurohumoral responses to different doses of L-NAME.
Am J Physiol Renal Physiol
275:
F870-F877,
1998
8.
Busse, R,
and
Fleming I.
Regulation and functional consequences of endothelial nitric oxide formation.
Ann Med
27:
331-340,
1995[ISI][Medline].
9.
Calver, A,
Collier J,
and
Vallance P.
Inhibition and stimulation of nitric oxide synthesis in the human forearm arterial bed of patients with insulin-dependent diabetes.
J Clin Invest
90:
2548-2554,
1992[ISI][Medline].
10.
Ceriello, A,
Quatraro A,
and
Giugliano D.
Diabetes mellitus and hypertension: the possible role of hyperglycaemia through oxidative stress.
Diabetologia
36:
265-266,
1993[ISI][Medline].
11.
Cohen, RA.
Dysfunction of vascular endothelium.
Circulation
87, Suppl V:
V67-V76,
1993.
12.
Cohen, RA.
The potential clinical impact of 20 years of nitric oxide research.
Am J Physiol Heart Circ Physiol
276:
H1404-H1407,
1999
13.
Cosentino, F,
Hishikawa K,
Katusic ZS,
and
Luscher TF.
High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells.
Circulation
96:
25-28,
1997
14.
Craven, PA,
DeRubertis FR,
and
Melhem M.
Nitric oxide in diabetic nephropathy.
Kidney Int Suppl
60:
S46-S53,
1997[Medline].
15.
Dai, FX,
Diederich A,
Skopec J,
and
Diederich D.
Diabetes-induced endothelial dysfunction in streptozotocin-treated rats: role of prostaglandin endoperoxides and free radicals.
J Am Soc Nephrol
4:
1327-1336,
1993[Abstract].
16.
Dijkhorst-Oei, LT,
and
Koomans HA.
Effects of a nitric oxide synthesis inhibitor on renal sodium handling and diluting capacity in humans.
Nephrol Dial Transplant
13:
587-593,
1998[Abstract].
17.
Ding, Y,
and
Vaziri ND.
Calcium channel blockade enhances nitric oxide synthase expression by cultured endothelial cells.
Hypertension
32:
718-723,
1998
18.
Elliott, TG,
Cockcroft JR,
Groop PH,
Viberti GC,
and
Ritter JM.
Inhibition of nitric oxide synthesis in forearm vasculature of insulin-dependent diabetic patients: blunted vasoconstriction in patients with microalbuminuria.
Clin Sci (Colch)
85:
687-693,
1993[ISI][Medline].
19.
Excerpts from United States Renal Data System 1991 Annual Data Report.
Am J Kidney Dis
18:
1-127,
1991.
20.
Gabbai, FB,
and
Blantz RC.
Role of nitric oxide in renal hemodynamics.
Semin Nephrol
19:
242-250,
1999[ISI][Medline].
21.
Gerich, JE.
Physiology of glucagon.
Int Rev Physiol
24:
243-275,
1981[Medline].
22.
Gimbrone, MA, Jr.
Vascular endothelium: an integrator of pathophysiologic stimuli in atherosclerosis.
Am J Cardiol
75:
67B-70B,
1995[Medline].
23.
Giugliano, D,
Ceriello A,
and
Paolisso G.
Diabetes mellitus, hypertension, and cardiovascular disease: which role for oxidative stress?
Metabolism
44:
363-368,
1995[ISI][Medline].
24.
Graier, WF,
Posch K,
Wascher TC,
and
Kostner GM.
Role of superoxide anions in changes of endothelial vasoactive response during acute hyperglycemia.
Horm Metab Res
29:
622-626,
1997[ISI][Medline].
25.
Graier, WF,
Simecek S,
Hoebel BG,
Wascher TC,
Dittrich P,
and
Kostner GM.
Antioxidants prevent high-D-glucose-enhanced endothelial Ca2+/cGMP response by scavenging superoxide anions.
Eur J Pharmacol
322:
113-122,
1997[ISI][Medline].
26.
Graier, WF,
Simecek S,
Kukovetz WR,
and
Kostner GM.
High D-glucose-induced changes in endothelial Ca2+/EDRF signaling are due to generation of superoxide anions.
Diabetes
45:
1386-1395,
1996[Abstract].
27.
Graier, WF,
Wascher TC,
Lackner L,
Toplak H,
Krejs GJ,
and
Kukovetz WR.
Exposure to elevated D-glucose concentrations modulates vascular endothelial cell vasodilatory response.
Diabetes
42:
1497-1505,
1993[Abstract].
28.
Granger, JP,
Kassab S,
Novak J,
Reckelhoff JF,
Tucker B,
and
Miller MT.
Role of nitric oxide in modulating renal function and arterial pressure during chronic aldosterone excess.
Am J Physiol Regulatory Integrative Comp Physiol
276:
R197-R202,
1999
29.
Harbrecht, BG,
Wirant EM,
Kim YM,
and
Billiar TR.
Glucagon inhibits hepatocyte nitric oxide synthesis.
Arch Surg
131:
1266-1272,
1996[Abstract].
30.
Honing, ML,
Morrison PJ,
Banga JD,
Stroes ES,
and
Rabelink TJ.
Nitric oxide availability in diabetes mellitus.
Diabetes Metab Rev
14:
241-249,
1998[ISI][Medline].
31.
Houben, AJHM,
Schaper NC,
de Haan CHA,
Huvers FC,
Slaaf DW,
de Leeuw PW,
and
Nieuwenhuijzen Kruseman AC.
Local 24-h hyperglycemia does not affect endothelium-dependent or -independent vasoreactivity in humans.
Am J Physiol Heart Circ Physiol
270:
H2014-H2020,
1996
32.
Huang, PL,
Huang Z,
Mashimo H,
Bloch KD,
Moskowitz MA,
Bevan JA,
and
Fishman MC.
Hypertension in mice lacking the gene for endothelial nitric oxide synthase.
Nature
377:
239-242,
1995[ISI][Medline].
33.
Johnstone, MT,
Creager SJ,
Scales KM,
Cusco JA,
Lee BK,
and
Creager MA.
Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus.
Circulation
88:
2510-2516,
1993[Abstract].
34.
Kawaguchi, M,
Koshimura K,
Murakami Y,
Tsumori M,
Gonda T,
and
Kato Y.
Antihypertensive effect of insulin via nitric oxide production in the Zucker diabetic fatty rat, an animal model for non-insulin-dependent diabetes mellitus.
Eur J Endocrinol
140:
341-349,
1999[ISI][Medline].
35.
Komers, R,
and
Cooper ME.
Renal sodium handling in experimental diabetes: role of NO.
Nephrol Dial Transplant
11:
2170-2177,
1996[Abstract].
36.
Kubes, P,
Suzuki M,
and
Granger DN.
Nitric oxide: an endogenous modulator of leukocyte adhesion.
Proc Natl Acad Sci USA
88:
4651-4655,
1991[Abstract].
37.
Lahera, V,
Navarro-Cid J,
Cachofeiro V,
Garcia-Estan J,
and
Ruilope LM.
Nitric oxide, the kidney, and hypertension.
Am J Hypertens
10:
129-140,
1997[ISI][Medline].
38.
Lambert, J,
Aarsen M,
Donker AJ,
and
Stehouwer CD.
Endothelium-dependent and -independent vasodilation of large arteries in normoalbuminuric insulin-dependent diabetes mellitus.
Arterioscler Thromb Vasc Biol
16:
705-711,
1996
39.
Loscalzo, J.
Nitric oxide and vascular disease.
N Engl J Med
333:
251-253,
1995
40.
Majid, DS,
Omoro SA,
Chin SY,
and
Navar LG.
Intrarenal nitric oxide activity and pressure natriuresis in anesthetized dogs.
Hypertension
32:
266-272,
1998
41.
Mäkimattila, S,
Virkamaki A,
Groop PH,
Cockcroft J,
Utriainen T,
Fagerudd J,
and
Yki-Järvinen H.
Chronic hyperglycemia impairs endothelial function and insulin sensitivity via different mechanisms in insulin-dependent diabetes mellitus.
Circulation
94:
1276-1282,
1996
42.
Mattar, AL,
Fujihara CK,
Ribeiro MO,
de Nucci G,
and
Zatz R.
Renal effects of acute and chronic nitric oxide inhibition in experimental diabetes.
Nephron
74:
136-143,
1996[ISI][Medline].
43.
Mayhan, WG,
and
Patel KP.
Treatment with dimethylthiourea prevents impaired dilatation of the basilar artery during diabetes mellitus.
Am J Physiol Heart Circ Physiol
274:
H1895-H1901,
1998
44.
Moller, DE,
and
Flier JS.
Insulin resistancemechanisms, syndromes, and implications.
N Engl J Med
325:
938-948,
1991[ISI][Medline].
45.
Nathan, DM.
Long-term complications of diabetes mellitus.
N Engl J Med
328:
1676-1685,
1993
46.
Okamura, T,
Miyazaki M,
and
Toda N.
Responses of isolated dog blood vessels to glucagon.
Eur J Pharmacol
125:
395-401,
1986[ISI][Medline].
47.
Orci, L.
Macro- and micro-domains in the endocrine pancreas.
Diabetes
31:
538-565,
1982[ISI][Medline].
48.
Panza, JA,
Casino PR,
Badar DM,
and
Quyyumi AA.
Effect of increased availability of endothelium-derived nitric oxide precursor on endothelium-dependent vascular relaxation in normal subjects and in patients with essential hypertension.
Circulation
87:
1475-1481,
1993[Abstract].
49.
Pflueger, AC,
Osswald H,
and
Knox FG.
Adenosine-induced renal vasoconstriction in diabetes mellitus rats: role of nitric oxide.
Am J Physiol Renal Physiol
276:
F340-F346,
1999
50.
Qiu, C,
Muchant D,
Beierwaltes WH,
Racusen L,
and
Baylis C.
Evolution of chronic nitric oxide inhibition hypertension: relationship to renal function.
Hypertension
31:
21-26,
1998
51.
Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus.
Diabetes Care
20:
1183-1197,
1997[ISI][Medline].
52.
Smith, FS,
Ceppi ED,
and
Titheradge MA.
Inhibition of cytokine-induced inducible nitric oxide synthase expression by glucagon and cAMP in cultured hepatocytes.
Biochem J
326:
187-192,
1997[ISI][Medline].
53.
Smits, P,
Kapma JA,
Jacobs MC,
Lutterman J,
and
Thien T.
Endothelium-dependent vascular relaxation in patients with type I diabetes.
Diabetes
42:
148-153,
1993[Abstract].
54.
Steinberg, HO,
Chaker H,
Leaming R,
Johnson A,
Brechtel G,
and
Baron AD.
Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance.
J Clin Invest
97:
2601-2610,
1996
55.
Stevens, RB,
Sutherland DE,
Ansite JD,
Saxena M,
Rossini TJ,
Levay-Young BK,
Hering BJ,
and
Mills CD.
Insulin down-regulates the inducible nitric oxide synthase pathway: nitric oxide as cause and effect of diabetes?
J Immunol
159:
5329-5335,
1997[Abstract].
56.
Tesfamariam, B.
Free radicals in diabetic endothelial cell dysfunction.
Free Radic Biol Med
16:
383-391,
1994[ISI][Medline].
57.
Tesfamariam, B,
and
Cohen RA.
Free radicals mediate endothelial cell dysfunction caused by elevated glucose.
Am J Physiol Heart Circ Physiol
263:
H321-H326,
1992
58.
Ting, HH,
Timimi FK,
Boles KS,
Creager SJ,
Ganz P,
and
Creager MA.
Vitamin C improves endothelium-dependent vasodilation in patients with non-insulin-dependent diabetes mellitus.
J Clin Invest
97:
22-28,
1996
59.
Tolins, JP.
Mechanisms of glucagon-induced renal vasodilation: role of prostaglandins and endothelium-derived relaxing factor.
J Lab Clin Med
120:
941-948,
1992[ISI][Medline].
60.
Unger, RH,
and
Orci L.
Role of glucagon in diabetes.
Arch Intern Med
137:
482-491,
1977[Abstract].
61.
Wang, XQ,
and
Vaziri ND.
Erythropoietin depresses nitric oxide synthase expression by human endothelial cells.
Hypertension
33:
894-899,
1999
62.
Wascher, TC,
Toplak H,
Krejs GJ,
Simecek S,
Kukovetz WR,
and
Graier WF.
Intracellular mechanisms involved in D-glucose-mediated amplification of agonist-induced Ca2+ response and EDRF formation in vascular endothelial cells.
Diabetes
43:
984-991,
1994[Abstract].
63.
Zeng, G,
and
Quon MJ.
Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells.
J Clin Invest
98:
894-898,
1996