© 1995 American Heart Association, Inc.
Articles |
Megakaryocytes From Patients With Coronary Atherosclerosis Express the Inducible Nitric Oxide Synthase
From the Department of Cardiology (A. de B., A.B.), King's College Hospital, and the Department of Medicine (A. de B., A.B., J.M.), King's College School of Medicine and Dentistry, London; and the Wellcome Research Labs (M.R., V.H., S.M., J.M.), Beckenham, Kent, UK.
Correspondence to Professor J.F. Martin, MD, FRCP, Department of Medicine, King's College School of Medicine and Dentistry, Bessemer Rd, London SE5 9PJ, UK.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Endothelial and platelet generation of nitric oxide (NO) plays an important role in the regulation of hemostasis. Alterations in NO biosynthesis are described in atherosclerosis. We have investigated the NO pathway in megakaryocytes and platelets from patients with atherosclerosis and age-matched control subjects. Megakaryocytes and platelets were isolated from patients with severe coronary atherosclerosis (n=19) and normal coronary arteries (n=9) as demonstrated by selective angiography. Constitutive (Ca2+-dependent) and inducible (Ca2+-independent) NO synthase (cNOS and iNOS, respectively) activities were measured by using the citrulline assay and by immunostaining techniques using an anti-peptide antibody to iNOS. Megakaryocytes from patients with atherosclerosis expressed significantly greater amounts of iNOS (1.28±0.46 pmol citrulline · mg-1 · min-1) than cNOS (0.29±0.40 pmol · mg-1 · min-1). In contrast, megakaryocytes from patients with normal coronary arteries expressed significantly more cNOS (1.48±0.23 pmol · mg-1 · min-1) than iNOS (0.49±0.40 pmol · mg-1 · min-1). Platelets isolated from both groups showed no significant difference in cNOS expression, and no iNOS was seen in either group. Immunostaining confirmed the presence of the iNOS in megakaryocytes. These results suggest there is a link between the expression of iNOS in the megakaryocyte and atherosclerosis.
Key Words: atherosclerosis • nitric oxide • megakaryocytes • platelets
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Nitric oxide (NO) is an important physiological and, under certain conditions, pathological mediator in human biology. The continuous generation of NO by the vascular endothelium is crucial to the maintenance of blood pressure and flow.1 This endothelial NO generation also inhibits platelet aggregation2 and adhesion.3 In addition, platelets themselves are able to generate NO via a constitutive NO synthase (cNOS) that acts as a negative feedback system to regulate the extent of platelet aggregation.4 The presence of this platelet cNOS has been confirmed by bioassay,5 6 7 8 spin trapping/electron paramagnetic resonance studies,9 and by specific NO electrode measurement.8 10
NO can also be generated by an immunologically expressed isoform of NO synthase (iNOS). The expression of this isoform requires induction by appropriate cytokines, and iNOS releases large amounts of NO over long periods of time. There is no known posttranslational regulatory control system of this enzyme once expressed. However, its induction can be prevented by inhibitors of protein synthesis, such as cycloheximide and glucocorticoids.1
iNOS has been demonstrated in a number of animal tissues and cells11 including macrophages,12 13 vascular endothelial and smooth muscle cells,14 15 and myocardium.16 17 The expression of iNOS is believed to form a part of the nonspecific host defense mechanism and, when exaggerated, may account for the clinical manifestations of acute and chronic inflammatory conditions.11
Since NO has a significant role in the regulation of platelet
hemostasis,4 it is probable that altered synthesis and
release of this molecule may be associated with thrombotic and
atherosclerotic disorders. As platelets are anucleate and contain no
DNA and only residual mRNA, they derive their proteins largely by
transfer from the megakaryocyte.18 We have found that the
human megakaryoblastic cell line (Meg-0119 ) contains cNOS,
and when stimulated with interleukin-1ß (IL-1ß) and tumor necrosis
factor–
(TNF-), expresses iNOS.20 The vascular
lesion of atherosclerosis is a chronic systemic inflammatory process,
and these cytokines have been clearly implicated in its
pathogenesis.21 Thus, we investigated the hypothesis that
in patients with severe coronary atherosclerosis there is expression of
iNOS in the human megakaryocyte that is passed onto the platelet.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This investigation was approved by King's College Hospital Research Ethics Committee. Informed consent was obtained from all patients.
Patients
Sternal bone marrow was aspirated under sterile conditions from
anesthetized age-matched patients just prior to either cardiopulmonary
bypass for coronary artery (n=19) or valvular heart surgery (n=9). The
patient characteristics of these two groups are shown in the
Table
. All patients had undergone selective
high-resolution coronary angiography; bypass patients were receiving at
least three bypass conduits, whereas the valvular heart surgery group
had normal coronary arteries. Although the appearance of normal
coronary epicardial vessels on angiography does not guarantee normal
physiological and pharmacological behavior, the disparity in coronary
anatomy between the two groups provided the most appropriate
comparative data for this investigation.
|
Bone Marrow Aspiration
Bone marrow was aspirated into modified phosphate-buffered
saline (PBS), pH 6.5, at 4°C containing 7.5 mmol/L glucose, 3.15%
(wt/vol) trisodium citrate, 2 mmol/L potassium chloride, 0.5 mmol/L
sodium sulfate, 0.1% (wt/vol) bovine serum albumin, and 1 nmol/L
prostaglandin E1. All procedures were performed in plastic
tubes on ice. The sample was monodispersed by gentle pipetting and
filtered through a 100-µm nylon gauze.
Megakaryocyte Isolation
Megakaryocyte isolation was performed by the method of Gladwin
et al.18 Briefly, the sample was incubated for 30 minutes
at 4°C with 5 µL Plt-1, a mouse monoclonal IgM that specifically
recognizes a human platelet/megakaryocyte differentiation antigen
(Coulter Clone). The samples were then incubated with 10 µL diluted
(1:10) magnetizable particles (goat anti-mouse IgG) (Dynal UK Ltd) for
a further 30 minutes at 4°C. The cells labeled with magnetizable
particles were harvested by placing the plastic tube in a magnetic
field (Dynal UK Ltd) for 2 minutes. The supernatant was removed gently
to avoid disturbing the cells in contact with the magnet. The magnet
was removed, and the cells were resuspended in modified PBS and washed
twice by concentrating with the magnet. Finally the cells were
resuspended in 1 mL modified PBS. The cells were confirmed as
megakaryocytes by phase-contrast microscopy (x40 objective) after
dilution with Trypan blue stain (GIBCO) and counted by using a Neubauer
counting chamber. The sample was then centrifuged at 2000g
for 2 minutes, the supernatant was removed, and the pellet was stored
at -70°C until the assay and immunostaining procedures were
performed. In four samples isolation of megakaryocytes was performed in
duplicate, one as described above and the other in the presence of
dexamethasone (1 µmol/L).
Platelet Preparation
Blood was obtained from eight of the patients with severe
atherosclerosis and five of the patients undergoing valve surgery just
prior to obtaining the bone marrow aspirate. Platelets were isolated by
the prostacyclin method22 and were stored at -70°C
until assayed.
NOS Assay
The megakaryocyte and platelet pellets were homogenized in
buffer (HEPES 10 mmol/L, sucrose 0.32 mol/L, dithiothreitol 1 mmol/L,
soybean trypsin inhibitor 10 µg/mL, leupeptin 10 µg/mL, and
aprotinin 2 µg/mL) by freezing in liquid nitrogen and thawing on ice
(two cycles). The sample was centrifuged at 11 000g for 20
minutes at 4°C. NOS activity was measured by the conversion of
L-[14C]arginine to
L-[14C]citrulline and expressed as picomoles
per microgram per protein per minute.23 Briefly, Eppendorf
tubes were prewarmed to 37°C with 100 µL buffer consisting of 50
mmol/L valine, 100 µmol/L L-citrulline, 20 µmol/L
arginine, 100 µmol/L NADPH, 10 µmol/L tetrahydrobiopterin (Dr
Schircks laboratories), 1.2 mmol/L MgCl2, 0.24
mmol/L CaCl2, and
L-[14C]arginine (150 000 dpm; Amersham). The
specificity of the conversion of
L-[14C]arginine to
L-[14C]citrulline has been confirmed by
high-performance liquid chromatography (HPLC)25 and
independent spectrophotometric analysis.26 Duplicate
incubations for 20 minutes at 37°C were performed for each cell
sample in the presence or absence of either EGTA (1 mmol/L) or EGTA
plus an NOS inhibitor,
NG-monomethyl-L-arginine (1 mmol/L
each), to determine the level of the Ca2+-dependent
and Ca2+-independent formation of citrulline. This
specific NOS inhibitor allows differentiation between NOS-mediated and
possible NOS-unrelated formation of citrulline. In megakaryocytic cells
the Ca2+ requirement differentiates between cNOS
(Ca2+-dependent) and iNOS
(Ca2+-independent) activities.20 The
reaction was terminated by removal of substrate and dilution by
addition of 1 mL H2O/Dowex AF 50W-X8 cationic resin (1:1,
vol/vol; BioRad), pH 7.2; 0.5 mL H2O was then added to the
incubation mix and centrifuged for 3 minutes at 10 000g,
and 0.4 mL supernatant was removed and examined for the presence of
[14C]citrulline by liquid scintillation counting.
Protein Assay
The protein content of the homogenized cell preparations was
analyzed by using the differential color change of Coomassie
brilliant blue C-250 in response to various concentrations of
protein.26
All reagents were from Sigma unless stated otherwise.
Immunocytochemistry of iNOS
To confirm that the Ca2+-independent
citrulline formation by megakaryocytes was an expression of iNOS,
immunocytochemistry was performed by using a specific iNOS antipeptide
antibody.27
iNOS Antipeptide Antibodies
The antibodies used were obtained from the murine macrophage
iNOS amino acid sequence 47 through 71 (49M), with the addition of a
C-terminal cysteine residue to facilitate an ordered chemical binding
to a carrier protein. The peptide was purified by reverse-phase HPLC
and coupled to limpet hemocyanin (Calbiochem). Rabbits were immunized,
and antibodies were harvested.27 This antibody was chosen
as its specificity to human tissue has been
characterized.28
Immunostaining
Megakaryocytes from four patients with severe coronary
atherosclerosis and three patients with normal coronary arteries
underwent the immunostaining procedures, which were performed by an
investigator blinded to the biochemical results and clinical details.
The cells were resuspended in 70% ethanol, pelleted, and stored at
4°C until used. They were placed on gel-alum–coated slides and left
to dry for 3 hours. After washing in PBS, the cells were permeabilized
by using 0.01% (vol/vol) Triton X-100 for 5 minutes. After further
washing in PBS, 50 µL of 1% (wt/vol) blocking agent (BCl) was added
to each slide and incubated at 37°C for 30 minutes. The cells were
then incubated with 30 µL of the primary antibody (rabbit anti-iNOS,
dilution 1:100 in 1% BCl/PBS) at 37°C for 1 hour. After two washes
the cells were incubated with a secondary antibody (goat anti-rabbit
fluorescein isothiocyanate diluted 1:100 as for the primary
antibody) and incubated for 40 minutesat 37°C. After a final
wash in PBS, the slides were mounted in glycerol, and the coverslips
were sealed with cowgum. Megakaryocytes harvested by a
megakaryocyte-specific monoclonal antibody were identified by their
large size and the presence of a single multilobar nucleus on confocal
electron microscopy.
As positive controls for expression of iNOS, cultured murine macrophage J774 cells were induced with 75 U/mL recombinant mouse interferon gamma (Genzyme) and 10 µg/mL Escherichia coli 026:B6 lipopolysaccharide (DIFCO). The cells were stored at -70°C until use.
Statistics
The results are presented as mean±SEM of n determinations.
Significance was calculated by using ANOVA, and P<.05 was
considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Patients
There were no significant differences in age, hematologic profile, including platelet count, or lipid profile between the two patient groups. All patients had normal renal and liver function. Four of the patients undergoing bypass surgery were taking lipid-lowering agents. The drugs the patients were taking are shown in the Table
.
Megakaryocyte Isolation
The megakaryocyte isolation technique harvested a mean of
1.78±0.52x105 cells for each patient. There was no
significant difference in purity between the two groups. This isolation
compares with the original description by Gladwin et al.18
In suspension, morphologically recognizable megakaryocytes at different
maturation stages comprised approximately 98% of all nucleated cells.
The few cells that did not appear characteristic of megakaryocytes did
label with magnetizable particles and probably represented
immature megakaryocytes. Again, there was no significant difference in
the number of cells that were not characteristic of mature
megakaryocytes between the two groups.
NOS Assay
Megakaryocytes from patients with severe coronary atherosclerosis
showed significantly higher iNOS (1.28±0.46 pmol
citrulline · mg-1 · min-1) than cNOS
(0.29±0.40 pmol · mg-1 · min-1)
activity (n=19) (Fig 1A). In contrast, megakaryocytes
from patients with valvular heart disease and normal coronary arteries
showed significantly more cNOS (1.48±0.23
pmol · mg-1 · min-1) than iNOS
(0.49±0.40 pmol · mg-1 · min-1)
activity (n=9) (Fig 1B). In individual cases, if the activity of one
enzyme was increased, it was accompanied by a decreased activity of the
other (Fig 1C and 1D). Incubation with dexamethasone made no difference
to NOS activity (results not shown). In six patients (four from the
atherosclerosis group and two with normal coronary arteries), there was
no detectable citrulline formation (<0.1 pmol · mg
protein-1 · min-1).
|
Platelets isolated from patients with severe coronary atherosclerosis and normal coronary arteries expressed cNOS only (0.54±0.17 pmol · mg-1 · min-1 [n=8] and 0.38±0.22 pmol · mg-1 · min-1 [n=5], respectively). There was no detectable platelet iNOS activity seen in either group.
Immunostaining
The J774 macrophage cell line showed no fluorescence when
unstimulated (Fig 2A). After incubation with interferon
gamma and lipopolysaccharide, J774 macrophages showed intense
fluorescence using the iNOS anti-peptide antibody 49M (Fig 2B).
Megakaryocytes that had been isolated from atherosclerotic patients and
that showed biochemical evidence for
Ca2+-independent citrulline formation showed
positive fluorescence, indicating the presence of the first antibody in
the cytosol of these cells (n=4) (Fig 2C and 2D). However, there was no
fluorescence of these cells when the first antibody was excluded from
the labeling protocol. Megakaryocytes isolated from three patients with
normal coronary arteries showed no Ca2+-independent
citrulline formation and did not show fluorescence when incubated with
the iNOS antipeptide antibody (Fig 2E and 2F).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The understanding of platelet behavior in health and disease has been aided by the relative ease by which these cells can be obtained and investigated. Yet little is known about the biology of its progenitor cell, the megakaryocyte, which makes up approximately 0.01% of human bone marrow. This is because isolating sufficient numbers of purified cells for investigation has been extremely difficult.29 The use of an immunomagnetic separation technique18 to obtain pure populations of megakaryocytes has allowed us to investigate further the biochemical properties of this cell.
We have shown, by biochemical assay and immunostaining, that human megakaryocytes have the capacity to express both cNOS and iNOS. In patients with severe coronary atherosclerosis there was increased iNOS activity, whereas in those undergoing valvular heart surgery but with normal coronary arteries the cNOS activity was higher. Since platelet cytoplasmic proteins are transcribed in the megakaryocyte, the presence of cNOS in megakaryocytes is to be expected in view of the presence of cNOS in platelets.4 However, the presence of iNOS in megakaryocytes of patients with severe atherosclerosis is intriguing. Dexamethasone had no effect on the expression of iNOS activity, making it unlikely that the isolation technique induced the iNOS. The Ca2+-independent NOS activity could be derived from active immunocompetent cells, but this possibility is unlikely both because of the purity of the megakaryocyte preparation (>98%), with a similar degree of nonmature megakaryocytes in the cell populations of both groups, and because of the presence of iNOS in the immunostained megakaryocyte. Thus, our results suggest that there is an association between the expression of iNOS in the megakaryocyte and atherosclerosis.
The total citrulline formation by megakaryocytes was the same in the two groups of patients, the only difference being the dependency on Ca2+. The expression of iNOS by cells of megakaryocytic lineage is associated with a downregulation of cNOS activity.20 There is growing evidence that these reciprocal interactions between iNOS and cNOS occur in other cells such as bovine aortic endothelial cells30 and human umbilical vein cells,31 and this phenomenon may be due to the downregulation of cNOS mRNA by increasing the rate of its degradation.31 Thus, it is not surprising that the total NOS activity may not change upon induction of iNOS.
It is well recognized that induction of iNOS in human cell culture has proved difficult; iNOS appears less sensitive to cytokine stimulation than, for example, rodent cell culture. However, the expression of this enzyme is readily measured in human pathological situations, eg, in human tissue from patients with inflammatory heart,17 bowel,32 and lung28 disease, suggesting that the immunological conditions already exist for induction of iNOS in vivo.
A possible explanation for our findings probably lies in the
pathogenesis of the atherosclerotic lesion. Atherosclerosis is a
chronic inflammatory process, and the presence of several immune
cell-derived cytokines within these lesions21 has been
clearly demonstrated. Indeed, there is little doubt that these products
of immune activation influence the pathogenetic process of
atherosclerosis.33 Interestingly, two such cytokines
(TNF- and IL-1ß either singly or together) stimulated expression
of this enzyme in Meg-01 cells.20 Thus, it is conceivable
that the presence of iNOS in the human megakaryocyte is directly linked
to the atherosclerotic process, as has been shown in the lungs of
rabbits with experimental atherosclerosis.34 If the
function of megakaryocyte generation of iNOS is to modify the platelet
response to the inflamed atheromatous plaque, then iNOS would have to
be identified in platelets. Our investigations failed to confirm this.
The inability to demonstrate iNOS in platelets may be due to a number
of reasons: (1) the presence of iNOS is below the sensitivity of the
assay, (2) the platelets have the capacity to express the enzyme but
require the appropriate conditions to do so, or (3) iNOS is not passed
onto the platelet, and its function in megakaryocytes is limited to
megakaryocytopoiesis. Indeed, a role for NO in megakaryocyte maturation
is possible since NO has been shown to influence erythropoiesis in
hypoxic mice.35
Interestingly, patients with renal failure and liver cirrhosis, both conditions associated with increased cytokine production, have platelets with impaired aggregatory and adhesive properties, which may be due to increased NO generation within platelets by the iNOS.36 37 38
In Meg-01 cells, we have found that cytokine induction of iNOS resulted in a time-dependent decrease in cNOS and that inhibition of iNOS activity was accompanied by the upregulation of cNOS.20 This is supported by our present findings, which showed that in our patient groups there was a clear predominance of either iNOS or cNOS. These phenomena might be due to the regulation of enzyme activities by cytokines39 or even cytokine-mediated gene regulation of expression of both enzymes.
In conclusion, we have demonstrated the presence of cNOS and iNOS in the human megakaryocyte. Future work will determine whether the balance of these enzymes influences hemostatic potential in the circulating platelet in atherosclerosis.
|
Acknowledgments |
|---|
Dr de Belder is an Intermediate and Dr Brown is a Junior Research Fellow funded by the British Heart Foundation. Professor Martin is a British Heart Foundation Professor of Cardiovascular Science.
Received June 28, 1994; accepted February 1, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev. 1991;43:109-142. [Medline] [Order article via Infotrieve]
2. Radomski MW, Palmer RMJ, Moncada S. Comparative pharmacology of endothelial-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol. 1987;92:181-187. [Medline] [Order article via Infotrieve]
3. Radomski MW, Palmer RMJ, Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun. 1987;148:1482-1489. [Medline] [Order article via Infotrieve]
4.
Radomski MW, Palmer RMJ, Moncada S. An L-arginine:nitric
oxide pathway present in human platelets regulates aggregation.
Proc Natl Acad Sci U S A. 1990;87:5193-5197.
5.
Yao SK, Ober JC, Krishnaswami A, Ferguson JJ, Anderson HV,
Golino P, Buja LM, Willerson JT. Endogenous nitric oxide protects
against platelet aggregation and cyclic flow variations in stenosed and
endothelium-injured arteries.
Circulation. 1992;86:1302-1309.
6. Schini VB, Durante W, Elizondo E, Scott-Burden T, Juncquero DC, Schafer AI, Vanhoutte PM. The induction of nitric oxide synthase activity is inhibited by TGF-beta1, PDGF-AB and PDGF-BB in vascular smooth muscle cells. Eur J Pharmacol. 1992;216:379-383. [Medline] [Order article via Infotrieve]
7. Cadgwan TM, Benjamin N. Evidence for altered platelet nitric oxide synthesis in essential hypertension. J Hypertens. 1993;11:417-420. [Medline] [Order article via Infotrieve]
8. Berkels R, Klaus W, Rosen R. Nifedipine induced inhibition of platelet aggregation: a nitric oxide mediated process. Endothelium. 1993;1:S69. Abstract.
9. Pronai L, Ichimori K, Nozaki H, Nakazawa H, Okino H, Carmichael AJ, Arroyo CM. Investigation of the existence and biological role of L-arginine/nitric oxide pathway in human platelets by spin trapping/EPR studies. Eur J Biochem. 1991;202:923. [Medline] [Order article via Infotrieve]
10. Malinski T, Radomski MW, Taha Z, Moncada S. Direct electrochemical measurement of nitric oxide released from human platelets. Biochem Biophys Res Commun. 1993;194:960-965.[Medline] [Order article via Infotrieve]
11. Moncada S. The 1991 Ulf von Euler Lecture: the L-arginine:nitric oxide pathway. Acta Physiol Scand. 1992;145:201-227. [Medline] [Order article via Infotrieve]
12. Hibbs JB Jr, Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun. 1988;157:87-94. [Medline] [Order article via Infotrieve]
13.
Stuehr DJ, Nathan CF. Nitric oxide: a macrophage product
responsible for cytostasis and respiratory inhibition in tumor target
cells. J Exp Med. 1989;169:1543-1555.
14.
Radomski MW, Palmer RMJ, Moncada S. Glucocorticoids inhibit
the expression of an inducible but not the constitutive, nitric oxide
synthase in vascular endothelial cells. Proc Natl Acad Sci
U S A. 1990;87:10043-10047.
15. Rees DD, Cellek S, Palmer RMJ, Moncada S. Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock. Biochem Biophys Res Commun. 1990;173:541-547. [Medline] [Order article via Infotrieve]
16. Schulz R, Nava E, Moncada S. Induction and potential biological relevance of a Ca2+-independent nitric oxide synthase in the myocardium. Br J Pharmacol. 1992;105:575-580. [Medline] [Order article via Infotrieve]
17. de Belder AJ, Radomski MW, Why HJF, Richardson PJ, Bucknall CA, Salas E, Martin JF, Moncada S. Nitric oxide synthase activities in human myocardium. Lancet. 1993;341:84-85. [Medline] [Order article via Infotrieve]
18. Gladwin AM, Carrier MJ, Beesley JE, Lelchuk R, Hancock V, Martin JF. Identification of mRNA for PDGF B-chain in human megakaryocytes isolated using a novel immunomagnetic separation method. Br J Haematol. 1990;76:333-339. [Medline] [Order article via Infotrieve]
19.
Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi W, Nagura H,
Saito HS. Establishment of a novel human megakaryocyte leukemia cell
line, Meg-01, with a positive philadelphia chromosome.
Blood. 1985;66:1384-1392.
20.
Lelchuk R, Radomski MW, Martin JF, Moncada S. Constitutive and
inducible nitric oxide synthases in human megakaryoblastic cells.
J Pharmacol Exp Ther. 1992;262:1220-1224.
21. Arbustini EA, Grasso M, Diegoli M, Pucci A, Bramerio M, Ardissino D, Angoli L, de Servi S, Bramucci E, Mussini A, Minzioni G, Vigano M, Specchia G. Coronary atherosclerotic plaques with and without thrombus in ischemic heart syndromes: a morphologic, immunohistochemical and biochemical study. Am J Cardiol. 1991;68:36B-50B. [Medline] [Order article via Infotrieve]
22. Radomski MW, Moncada S. An improved method for washing of human platelets with prostacyclin. Thromb Res. 1983;30:383-389. [Medline] [Order article via Infotrieve]
23.
Radomski MW, Vallance P, Whitley G, Foxwell N, Moncada S.
Platelet adhesion to human vascular endothelium is modulated by
constitutive and cytokine-induced nitric oxide. Cardiovasc
Res. 1993;27:1380-1383.
24. Salter M, Knowles R, Moncada S. Widespread species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett. 1991;291:145-149. [Medline] [Order article via Infotrieve]
25. Knowles R, Merrett M, Salter M, Moncada S. Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat. Biochem J. 1990;270:833-836. [Medline] [Order article via Infotrieve]
26. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
27. Riveros-Moreno V, Beddell C, Moncada S. Nitric oxide synthase: structural studies using anti-peptide antibodies. Eur J Biochem. 1993;215:801-808. [Medline] [Order article via Infotrieve]
28. Hamid Q, Springall DR, Riveros-Moreno V, Chanez P, Howarth P, Redington A, Bousquet J, Godard J, Holgate S, Polak JM. Induction of nitric oxide synthase in asthma. Lancet. 1993;342:1510-1513. [Medline] [Order article via Infotrieve]
29. Shoff PK, Levine RF. Elutriation for isolation of megakaryocytes. Blood Cells. 1989;15:285-305. [Medline] [Order article via Infotrieve]
30.
Lamas S, Michel T, Brenner BM, Marsden PA. Nitric oxide
synthesis in endothelial cells: evidence for a pathway inducible by
TNF-alpha. Am J Physiol. 1991;261:C634-C641.
31. Yoshizumi M, Perrella MA, Burnett JC Jr, Lee M-E. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res. 1993;73:205-209. [Abstract]
32. Boughton-Smith NK, Evans SM, Hawkey CJ, Cole AT, Balsitis M, Whittle BJR, Moncada S. Nitric oxide synthase activity in ulcerative colitis. Lancet. 1993;342:338-340. [Medline] [Order article via Infotrieve]
33.
Nilsson J. Cytokines and smooth muscle cells in
atherosclerosis. Cardiovasc Res. 1993;27:1184-1190.
34. Lang D, Smith JA, Lewis MJ. Induction of a calcium-independent NO synthase by hypercholesterolaemia in the rabbit. Br J Pharmacol. 1993;108:290-292. [Medline] [Order article via Infotrieve]
35. Ohigashi T, Brookins J, Fisher JW. Interaction of nitric oxide and cyclic guanosine 3',5'-monophosphate in erythropoetin production. J Clin Invest. 1993;92:1587-1591.
36. Laffi G, Marra F, Failli P, Ruggiero M, Cecchi E, Carloni V, Giotti A, Gentilini P. Defective signal transduction in platelets from cirrhotics is associated with increased cyclic nucleotides. Gastroenterology. 1993;105:148-156. [Medline] [Order article via Infotrieve]
37. Remuzzi G, Perico N, Zoja C, Coma D, Macconi D, Vigano G. Role of endothelium-derived nitric oxide in the bleeding tendency of uremia. J Clin Invest. 1990;86:1768-1771.
38. Noris M, Benigni A, Boccardo P, Aiello S, Gaspain F, Todeschini M, Figliuzzi M, Remuzzi G. Enhanced nitric oxide synthesis in uremia: implications for platelet dysfunction and dialysis hypotension. Kidney Int. 1993;44:445-450. [Medline] [Order article via Infotrieve]
39.
Forstermann U, Kuk JE, Nakane M, Pollock JS. The expression of
endothelial nitric oxide synthase is downregulated by tumor necrosis
factor (TNF-). Naunyn Schmiedebergs Arch Pharmacol.
1993;347(suppl):R61. Abstract.
This article has been cited by other articles:
 |
|
 |
|
  S. Ravalli, A. Albala, M. Ming, M. Szabolcs, A. Barbone, R. E. Michler, and P. J. Cannon Inducible Nitric Oxide Synthase Expression in Smooth Muscle Cells and Macrophages of Human Transplant Coronary Artery Disease Circulation, June 16, 1998; 97(23): 2338 - 2345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Greenberg, X. Zhao, J.-F. Wang, L. Hua, and J. Ouyang cAMP and purinergic P2y receptors upregulate and enhance inducible NO synthase mRNA and protein in vivo Am J Physiol Lung Cell Mol Physiol, November 1, 1997; 273(5): L967 - L979. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||