Nitrotyrosine formation with endotoxin-induced kidney injury
detected by immunohistochemistry
Ka
Bian1,
Karen
Davis1,
Jeff
Kuret2,
Lester
Binder2, and
Ferid
Murad1
1 Department of Integrative
Biology, Physiology, and Pharmacology, University of Texas-Houston
Medical School, Houston, Texas 77030; and
2 Department of Cell Biology,
Northwestern University Medical School, Chicago, Illinois 60611
 |
ABSTRACT |
The presence of
nitrotyrosine in the kidney has been associated with several
pathological conditions. In the present study, we investigated
nitrotyrosine formation in rat kidney after animals received endotoxin
for 24 h. With lipopolysaccharide (LPS) treatment, immunohistochemical
data demonstrated intense nitrotyrosine staining throughout the kidney.
In spite of marked nitrotyrosine formation, the architectural
appearance of tubules, glomeruli, and capillaries remained intact when
examined by reticulin staining. Our data suggested that the marked
staining of nitrotyrosine in proximal tubular epithelial cells was in
the subapical compartment where the endocytic lysosomal apparatus is
located. Thus a large portion of nitrotyrosine may come from the
hydrolysis of nitrated proteins that are reabsorbed by the proximal
tubule during the LPS treatment. We also found the colocalization of
nitric oxide synthase (NOS-1) and nitrotyrosine within the macula densa
of LPS-treated rats by using a double fluorescence staining method. In
renal arterial vessels, vascular endothelial cells were more strongly
stained for nitrotyrosine than vascular smooth muscle cells. Control
animals without LPS treatment showed much less renal staining for
nitrotyrosine. The general distribution of nitrotyrosine staining in
control rat renal cortex is in the proximal and convoluted tubules,
whereas the endothelial cells of vasa recta are major areas of
nitrotyrosine staining in inner medulla. The renal distribution of
nitrotyrosine in control and LPS-treated animals suggests that protein
nitration may participate in renal regulation and injury in ways that
are yet to be defined.
nitric oxide; peroxynitrite; renal protein nitration; lipopolysaccharide
 |
INTRODUCTION |
NITRIC OXIDE (NO ·), a free radical gas, can
regulate an ever-growing list of biological processes [reviewed
by Murad (25, 26)]. In the normal kidney, NO · is
actively involved in the regulation of glomerular hemodynamics and
filtration and the control of tubular function and sodium excretion
(30, 36). However, excessive NO · production may contribute
to several renal diseases, including immune-mediated
glomerulonephritis, postischemic renal failure, radiocontrast
nephropathy, obstructive nephropathy, and renal allograft rejection
[reviewed by Kone (17)].
The paramagnetic NO · rapidly reacts with superoxide
(O
2) to form peroxynitrite anion
(ONOO
), a potent
nitrating and oxidizing agent, resulting in oxidative damage to
proteins, lipids, carbohydrates, DNA, and sulfhydryl groups.
ONOO
can give several
reactive products, including hydroxyl radical and nitronium ion. It can
nitrate aromatic amino acids, and nitration on the three position of
tyrosine can form free or protein-associated nitrotyrosine (13, 32,
34). Nitrotyrosine has been used as an index of
ONOO
formation. The
presence of nitrotyrosine in the kidney detected with
immunohistochemistry has been associated with several pathological conditions, such as human renal allograft rejection (19), experimental glomerulonephritis (10), and the Goldblatt animal model for hypertension (2).
Acute renal failure due to endotoxemia is a common problem in clinical
medicine. Endotoxins are released from the outer membrane of
gram-negative bacteria and are composed of lipopolysaccharide (LPS).
Endotoxins can activate macrophages and other cells that can release
cytokines and inflammatory mediators, which increase the formation of
the inducible isoform of nitric oxide synthase (NOS-2). The increased
production of NO · and subsequent formation of
ONOO
and nitrotyrosine are
thought to contribute to the inflammatory response (22, 29, 38). In the
present study, we have examined the renal distribution of nitrotyrosine
with immunohistochemical methods after rats have received LPS. With
Western immunoblots of kidney extracts, we have also found several
proteins (~39 and 70-75 kDa) that probably contributed to the
immunohistochemical staining observed.
 |
MATERIALS AND METHODS |
Animal and tissue preparation. Male
Sprague-Dawley rats (275-300 g) were obtained from Charles River.
The experiments were conducted in accordance with the National
Institutes of Health Guide for the Care and Use of
Laboratory Animals and were approved by the University
of Texas Medical School Animal Care and Use Committee. The rats were
divided into control and experimental groups. LPS (serotype 0111:B4;
Sigma) dissolved in sterile saline was administered intraperitoneally
at a dose of 20 mg/kg body wt, and rats were killed under ether
anesthesia at 6, 12, or 24 h after LPS injection. For the control
group, rats were injected with the same volume of physiological saline.
After death, the kidneys were collected and immediately frozen in
liquid nitrogen. The tissues were stored at
135°C until
further processing.
Frozen kidneys were pulverized with a pestle and mortar that contained
liquid nitrogen and then were homogenized at 4°C in 20 mM
Tris · HCl buffer (pH 7.4) containing protease
inhibitors (final concentration: 10 µg/ml soybean trypsin inhibitor,
10 µg/ml benzamidine, 5 millitissue inhibitor units/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 5 µg/ml antipain, 0.2 mM
phenylmethane sulfonate fluoride, and 0.1 mM EDTA). Each sample was
sonicated on ice with 10 pulses at 40% duty cycle and output 3 and
then was centrifuged at 1,000 g for 15 min at 4°C. The supernatant fractions were used for SDS-PAGE and
Western immunoblotting.
Immunohistochemistry. The animals were
anesthetized with ether, and kidneys were perfusion fixed via the left
ventricle with 4% paraformaldehyde. Kidneys were removed and incubated
at 4°C overnight in 30% sucrose in Tris buffer (100 mM Tris base,
pH 8.0). Tissues were embedded in optimum cutting temperature compound (Miles, Elkhart, IN) and were frozen in ethanol cooled on dry ice. The
3- to 8-µm frozen sections were prepared with a Minotome (Damon, MA)
and were thaw mounted on Superfrost Plus slides (Fisher Scientific,
Pittsburgh, PA). Tissue sections were treated with 0.1% Triton X-100
for 5 min to permeabilize cell membranes. After being rinsed with 100 mM Tris buffer (pH 8.0), slides were incubated with donkey serum (at
1:20 dilution) for 1 h at room temperature. This was followed by
overnight incubation with an anti-nitrotyrosine antibody (at 1:100 to
1:500 dilution). The anti-nitrotyrosine mouse monoclonal antibody was
prepared against a random peptide of L-nitrotyrosine,
L-alanine, and L-glycine coupled to keyhole limpet hemocyanin. Antibody purified from cell culture medium of a
selected mouse clone was utilized. To assess nonspecific staining,
control experiments were performed by incubating the slides without
primary antibody or with primary antibody preincubated with nitrated
BSA for 1 h at room temperature. Nitrotyrosine-containing BSA was
prepared by treating BSA with peroxynitrite, tetranitromethane, or
3-morpholinosydnonimine as recently described (16).
Primary antibody preincubated under the same condition with either free
L-3-nitrotyrosine (20 mM) or BSA that was not nitrated was
also tested in our experiments. Biotin-conjugated donkey anti-mouse antibody (1:2,000) was used as secondary antibody for avidin-biotin amplified 3,3'-diaminobenzidine (DAB) staining and was applied to
sections for 1 h at room temperature. Sections were also stained with
hematoxylin. Silver impregnation (Gomori's method) was used for
reticulin staining. In the immunofluorescence staining, a rabbit
polyclonal antibody against nitric oxide synthase-1 (NOS-1; bNOS
251-270; Sigma) was used as a second primary antibody. Lissamine Rhodamine (LRSC)-conjugated affinity purified donkey anti-rabbit IgG
(1:200; Jackson Immuno-chemicals, West Grove, PA) was used for
detecting NOS-1. In the same section, Fluorescein
(FITC)-conjugated affinity purified donkey anti-mouse IgM (1:200;
Jackson Immuno-chemicals) was used for staining of nitrotyrosine. After
incubation with both NOS-1 and nitrotyrosine primary antibodies
overnight, the slides were incubated with the LRSC-conjugated secondary
antibody for 1 h at room temperature, rinsed with 100 mM Tris buffer
(pH 8.0), and further incubated with the FITC-conjugated secondary antibody under the same conditions. Sections were examined with a Zeiss
Axiophot (D-7082) microscope. The fluorescence photomicrographs were
taken using Kodachrome slide film, and Kodak LoyalGolden film was used
for color pictures.
Immunoblotting. The kidney lysates
were subjected to standard SDS-PAGE and Western blotting techniques.
The samples were electrophoresed using a 5% polyacrylamide stacking
gel and a 7.5 or 10% polyacrylamide separating gel (gel size, 12 × 15 × 0.15 cm). Equal amounts of proteins (400 µg/well)
were loaded onto the gel for each experimental sample. Separated
proteins were transferred to nitrocellulose membranes, and the
membranes were treated with 5% nonfat dry milk in Tris-buffered saline
(20 mM Tris · HCl and 130 mM NaCl, pH 7.6) plus 0.1%
Tween 20 (TBS-T) and then were incubated at 4°C overnight with the anti-nitrotyrosine antibody. The membranes were
washed with TBS-T and incubated with peroxidase-conjugated goat
anti-mouse antibody. Chemiluminesence was used to identify nitrotyrosine-containing proteins according to the enhanced
chemiluminescence Western blotting detection system (Amersham Life Science).
 |
RESULTS |
Immunoblotting. Western blot analysis
indicated that there are two major groups of nitrotyrosine-containing
proteins recognized by the anti-nitrotyrosine antibody in normal
kidney. As shown in Fig. 1, the nitrated
proteins of ~70-75 kDa were markedly increased in a
time-dependent manner after LPS treatment. Similarly, another group of
proteins of ~39 kDa were found in normal kidney extracts, and these
proteins and/or their nitrotyrosine content also increased with LPS
treatment.

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Fig. 1.
Representative Western blot analysis of nitrotyrosine-containing
proteins in kidney extracts from a control rat (0 h) and rats after 6, 12, and 24 h of lipopolysaccharide (LPS, 20 mg/kg ip) treatment
(n = 3 rats).
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|
Immunohistochemistry. LPS treatment
resulted in intense renal cortical staining of nitrotyrosine (Fig.
2, B and
D). Preabsorption of the
anti-nitrotyrosine antibody with nitrated BSA prevented renal staining
(Fig. 2C). Similar inhibition of staining was observed when the antibody was pretreated with free nitrotyrosine (data not
shown). However, preabsorption of the antibody with BSA did not alter
the histochemical staining (Fig.
2D). In the LPS-treated renal
cortex, intense nitrotyrosine staining was observed in the region of
proximal and distal convoluted tubules, initial collecting tubules,
interlobular vascular endothelium, and glomeruli. Control animals
without LPS treatment showed much less renal staining for
nitrotyrosine, and the general distribution of nitrotyrosine staining
was in the area of proximal and convoluted tubules, but not in
glomeruli.

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Fig. 2.
Images of rat kidney cortex (magnification: ×200).
A: section from a control animal
that shows much less nitrotyrosine staining (dark
brown). B-D: sections from 24 h LPS
(20 mg/kg)-treated rat kidney. Nitrotyrosine staining is broadly
distributed throughout the renal cortex
(B). Preabsorption of the
anti-nitrotyrosine antibody with nitrated BSA prevented renal staining
(C). However, preabsorption of the
antibody with BSA did not alter nitrotyrosine staining
(D). Preabsorption of
anti-nitrotyrosine antibody with free nitrotyrosine also prevented
staining (data not shown). Similar findings were observed in 3 rats.
|
|
Figure 3,
A-C,
shows a general distribution of nitrotyrosine staining in LPS-treated
rat kidney outer medulla (A) and
inner medulla (B and
C). Strong nitrotyrosine staining
was found in the inner medulla, which contains the thin limb of the
loop of Henle, collecting tubules and ducts, and vasa recta. It is
notable that nitrotyrosine staining of the endothelial cells was
observed not only in the renal of LPS-treated animal but also in the
vasa recta from control rats (Fig.
3D).



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Fig. 3.
Images of kidney medulla. A-C: images
of the same section from LPS-treated rat kidney.
A: outer medulla (OM; magnification:
×100). C, cortex; B: inner
medulla (IM; magnification: ×100);
C: higher magnification (×1,000)
image of the inner medulla. D: inner
medulla (magnification: ×1,000) of the kidney without LPS
treatment. Note an intense staining of nitrotyrosine (arrows) not only
in the vasa recta of LPS-treated rats but also in the vasa recta of
untreated control animals.
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|
Despite the intense nitrotyrosine formation induced by 24 h of LPS
treatment, the reticulin framework outlining renal glomeruli and
tubules was not altered (Fig. 4) compared with normal
kidney sections (data not show). The architectural appearance of
tubules, glomeruli, and/or capillaries remained intact as demonstrated by silver staining of type III collagen, the basis of the reticulin stain (Fig. 4).

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Fig. 4.
Reticulin staining (magnification: ×400) of kidney from
LPS-treated rats. Note that despite the intense nitrotyrosine formation
induced by 24 h of LPS treatment, the reticulin framework outlining
renal glomeruli and tubules remained intact.
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As demonstrated in Fig. 2, proximal convoluted tubules represent the
major parenchymal component. A high-magnification micrograph of the
pars convoluta of LPS-treated proximal tubule is shown in Fig.
5. The initial convoluted portion of the tubule is a
direct continuation of parietal epithelium of Bowman's capsule and has a characteristic tall brush border and a well-developed endocytic lysosomal apparatus system that is located at the apical cytoplasm beneath the luminal brush border. Histologically, numerous mitochondria are distributed in the basal part of the tubular cells. The
nitrotyrosine staining was localized in the brush border and was
greatest underneath the luminal brush border where the endocytic
lysosomal apparatus is distributed. Although the staining seems much
less than that of the tubular cells, the area where glomerular basement
membrane and mesangial cells are located was also stained by
anti-nitrotyrosine antibody (Fig. 5).

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Fig. 5.
High-magnification (×1,000) image of the pars convoluta of
LPS-treated proximal tubule. Note that the nitrotyrosine staining (open
arrows) was greatest in a subapical compartment where the endocytic
lysosomal apparatus is distributed. Although the staining seems much
less than that of the tubular cells, the area where glomerular basement
membrane and mesangial cells are located was also stained by
anti-nitrotyrosine antibody (filled arrows).
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In the region of the cortical thick ascending limb adjacent to the
hilus of the glomeruli of LPS-treated rats, a group of low columnar
cells with the morphological appearance of macula densa cells (Fig.
6) demonstrated intense basolateral staining of
nitrotyrosine. We confirmed the presence of nitrotyrosine-containing proteins in macula densa cells by co-staining the same section with
anti-NOS-1 antibody (LRSC fluorescence, Fig
7A) and
anti-nitrotyrosine antibody (FITC fluorescence, Fig.
7B). Figure
7C indicated the colocalization of
NOS-1 and nitrotyrosine within the macula densa. In control animals,
there was no detectable nitrotyrosine formation in the macula densa.

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Fig. 6.
High-magnification (×1,000) image of the region of the cortical
thick ascending limb adjacent to the hilus of the glomeruli. Note a
group of low columnar cells with the morphological appearance of macula
densa cells that demonstrate intense staining of nitrotyrosine
(arrows). The section was from a rat treated with LPS and is
representative of 3 rats.
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Fig. 7.
A-C: images of the same macula densa
(magnification: ×1,000) from LPS-treated rat kidney of Fig. 6.
Co-staining macula densa with anti-nitric oxide synthase (NOS)-B1
antibody (lissamine rhodamine fluorescence,
A) and anti-nitrotyrosine antibody
(FITC fluorescence, B).
C: colocalization of NOS-B1 and
nitrotyrosine within the macula densa.
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|
The renal vasculature is a major component of the kidney and can be
divided into three major groups: arterial vessels, glomeruli, and vasa
recta (including capillaries). As described previously, the glomeruli
and vasa recta demonstrated different intensity of nitrotyrosine
staining. In renal arterial vessels, vascular endothelial cells were
more strongly stained for nitrotyrosine than vascular smooth muscle
cells (Fig.
8A).
Figure 8B depicts the transverse
course of an afferent (hilar) arteriole that connects a glomerulus and
an interlobular artery and shows an intense endothelium staining of
nitrated protein. It is notable that nitrotyrosine staining of
endothelial cells was also clearly observed in renal vasa recta from
control rats without LPS treatment (Fig.
3D).


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Fig. 8.
Nitrotyrosine staining in endothelium of arterial vessels of
LPS-treated rat kidney. A: intense
nitrotyrosine staining (arrows) in vascular endothelial cells of an
arcuate artery and an interlobular artery (magnification: ×200).
Note that vascular smooth muscle cells have much less staining.
B: transverse course of afferent
(hilar) arteriole that connects a glomerulus (G) and interlobular
artery (A); intense endothelium staining of nitrated protein can also
be seen (arrow; magnification: ×400).
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|
 |
DISCUSSION |
NOS-2 expression and activity by LPS in various organs, including
kidney, has been well documented (4, 8, 9, 11, 23, 28, 33, 37, 39, 41).
This results in the increased formation of NO · and its
oxidized products (7, 15, 44). Several studies have shown that
enzymatically generated NO · is the sole source of nitrogen
oxides in vivo and nitration of tyrosine residues is almost certainly
derived from NO · [reviewed by Ischiropoulos (12)]. In fact, inhibition of NO · synthesis
successfully prevented the increases in nitrotyrosine content of
various tissues (21, 31, 42). In the current study, the administration
of LPS has markedly increased several groups of nitrated proteins of
kidney lysates in a time-dependent manner (Fig. 1), which further
confirmed the existence of endotoxin-triggered nitrotyrosine formation
in rat kidney.
In LPS-treated rat kidney cortex, we have demonstrated that the most
intense staining of nitrotyrosine was observed in the region of
cortical labyrinth that consists of proximal and distal convoluted
tubules, initial collecting tubules, and glomeruli. In the region of
cortex, proximal tubules account for ~70% of the relative tissue
volume (14). The proximal tubule begins abruptly at the urinary pole of
the glomerulus (Fig. 5) and consists of an initial convoluted portion
(the pars convoluta) and a straight portion (the pars recta). The
luminal brush border of the proximal tubule is composed of numerous
microvilli that were stained by the anti-nitrotyrosine antibody. The
cytoplasm immediately beneath the brush border contains many
well-developed endocytic lysosomal apparatuses that demonstrated very
strong nitrotyrosine staining. However, less staining was found at the
basal part of these cells where mitochondria are located (5). Further
studies will be required, however, to define the specific intracellular
sites of nitrotyrosine expression. Our finding suggests that the
nitrotyrosine-containing proteins observed in the proximal tubule may
have come from two sources. First, nitrotyrosine is formed in the
proximal tubular cells as a consequence of LPS-induced NO · production, since the tubule epithelium is the location for both
endothelial nitric oxide synthase (NOS-3; see Ref. 43) and NOS-2 (23).
The second source may be the reabsorption of nitrated proteins by the
endocytic-lysosomal apparatus of the proximal tubule, which is
responsible for the reabsorption of ~60% of the glomerular
ultrafiltrate, including glucose, amino acids, and minerals. It is
known that ONOO
reacts
selectively and rapidly with the iron-sulfur centers of several
mitochondrial respiratory proteins (35). However, in the present study,
the strongest staining of nitrotyrosine in proximal tubular epithelial
cells is not in the basal mitochondria region. Thus the cellular
location of nitrotyrosine staining suggests that a large portion of
nitrotyrosine may come from the hydrolysis of nitrated proteins during
the 24 h of LPS treatment. The pathological implication of reabsorbed
nitrotyrosine-containing proteins is unknown. The fate of the nitrated
proteins is also unclear. They may be further metabolized in the
kidney, redistributed to the blood and body, and/or be excreted.
The current study clearly colocalized NOS-1 and nitrotyrosine to the
LPS-treated macular densa segment (MDS; Fig. 6 and 7). The concept that
MDS is the principal site of NOS-1 gene expression in the rat kidney is
generally accepted (40, 45), although NOS-1 immunoreactivity was also
observed in the structures such as endothelium of efferent arterioles,
single cells of the glomerular visceral epithelium, and perivascular
nerves [reviewed by Kone and Baylis (18)]. Although
Bachmann et al. (1) described that the NOS-1 labeling frequently
extended beyond the typical location of the MDS in rat kidney,
"perimacular" NOS-positive staining was sometimes located on the
opposite side of the MDS or at a considerable distance upstream or
downstream to the MDS when portions of the thick ascending limb were
longitudinally sectioned (1). In our cross-sectional profiles of MDS,
NOS-1 staining was largely restricted to the morphologically defined site of the MDS plaque adjacent to the extraglomerular mesangium. Several in vitro studies have indicated that phosphorylation of NOS-1
by different kinases may affect the catalytic activity of NOS-1 (3, 6,
27); however, an effect of LPS on regulation of NOS-1 expression has
not been described. Although our immunolocalization experiments should
not be viewed as quantitative, NOS-1 immunoreactivity in the MDS
appeared to be comparable between normal and LPS-treated rats. Perhaps
LPS directly or indirectly increases protein kinases activity and
stimulates the expression and/or activity of NOS-1 in MDS that results
in increased nitrotyrosine formation. NO ·, O
2, or
ONOO
might also diffuse
into the MDS from adjacent cells where abundant NO · was
produced after LPS stimulation. Although it has been demonstrated that
NOS activity in MDS is directly linked with glomerular capillary
pressure (45), the role of nitrotyrosine formation in MDS remains to be established.
Biochemical studies have indicated that the renal medulla has a greater
capacity to generate NO · than the renal cortex. The renal
medulla has a high basal NOS activity, approximately threefold greater
than that of the renal cortex (20). In the present study, our finding
demonstrated an intense staining of nitrotyrosine not only in the vasa
recta stimulated by LPS (Fig. 3C)
but also in the vasa recta of untreated control animals (Fig.
3D). Both NOS-2 and NOS-3
expressions have been detected in the vasa recta (1, 24). Although
little is known about the regulation of NOS-3 expression in kidney,
endothelial cell culture studies suggest that both shear stress and
oxygen tension influence the level of NOS-3 expression and activity
(18). Perhaps changes of shear stress and oxygen tension in vasa recta
can regulate NO · production, which enhances nitrotyrosine formation.
The kidney is an intricate structure with diverse roles of excreting
waste products, regulating body fluid and solute balance, regulating
blood pressure, and secreting hormones. The three major NOS isoforms
are broadly distributed in renal tissues, and NO plays an important
role in many renal processes. Extensive protein nitration is evident in
the kidneys of LPS-treated rats (present study), indicating that
NO ·, O
2, and
ONOO
are produced in these
kidney compartments. In addition to illustrating extensive protein
nitration in LPS-stimulated rats, this study also demonstrates tyrosine
nitration in control animals. These observations suggest both
pathophysiological and normal biological roles for nitrotyrosine in
renal regulation and cellular signal transduction. The functions and
effects of nitrotyrosine have yet to be defined, and many additional
studies are suggested from those results.
 |
FOOTNOTES |
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.
Address for reprint requests and other correspondence: F. Murad,
Dept. of Integrative Biology, Physiology, and Pharmacology, The Univ.
of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030 (E-mail: fmurad{at}girch1.med.uth.tmc.edu).
Received 9 November 1998; accepted in final form 1 April 1999.
 |
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