Post on 16-May-2023
Journal of the American Society of Nephrology 2067
Expression and Preferential Inhibition of Inducible NitricOxide Synthase in Aortas of Endotoxemic Rats1Andr#{233}1. Weigert, Elisa M.S. Higa, Michel Niederberger, Ivan F. McMurtry, Mary Raynolds, and
Robert W. Schrier�
AL. Weigert. EMS. Higa. M. Niederberger. R.W.Schrier. Division of Renal Diseases and Hypertension.Department of Medicine. University of Colorado. Den-
ver, CO
IF. McMurtry, Cardiovascular-Pulmonary Laboratory,Department of Medicine. University of Colorado. Den-ver, CO.
M. Raynolds, Division of Cardiology. Department ofMedicine. University of Colorado. Denver, CO.
(J. Am. Soc. Nephrol. 1995; 5:2067-2072)
ABSTRACTSeptic shock is associated with high mortality. There isin vitro evidence that the induction of nitric oxide
synthase (iNOS) in vascular smooth muscle cells maybe an important mediator of the systemic vasodila-tion and hypotension associated with sepsis. In this
study, an in vivo murine model of sepsis was used tofurther examine this important question. Lipopolysac-charide (LPS), the major wall component of gram-negative bacteria, was administered to rats. By theuse of a selective cDNA probe for iNOS, mRNA foriNOS was demonstrated In the aortas of these rats. Thefunctional significance of this iNOS was then exam-med with aminoguanidine, a preferential inhibitor ofINOS. Aminoguanidine reversed the blunted phe-nylephrine-evoked contraction of endothelium-de-nuded aortlc rings from LPS-treated rats or rings ex-posed to LPS in vitro. Ammnoguanidmne did not Impairthe relaxation of aortic rings with endothelium to�cetylcholine, a known stimulator of endothelial NOS.The reversal of LPS-induced vascular hyporesponsive-ness by aminoguanidine therefore strongly supportsthe functional importance of INOS mRNA expressionin the aorta of endotoxemic rats. Future clinical trials
in treating septic shock should therefore consider thepreferential inhibition of INOS while maintaining theintegrily of endothelial NOS.
Key Words: Endothelium, llpopolysaccharide. nitric oxide syn-
Those, vascular smooth muscle
C Received September 12, 1994. Accepted March 21 , 1995.
2 Dr. R.W. Schrler, Department of Medicine, University of colorado Health Sd-
ences center, 4200 East Ninth Ave., Denver, co 80262.
1046.6673/0512-2067$03.00/0Journal of the American Society of Nephrologycopv�ght C 1995 by the American Society of Nephrology
S eptic shock is associated with a high mortality
that correlates with the degree of systemic vaso-
dilation and hypotension. There is In vitro evidence
that endotoxin or lipopolysaccharide (LPS), the major
component of gram-negative bacteria. induces nitric
oxide synthase (1NOS) in endothelium and vascular
smooth muscle and therefore may be of primary im-
portance in septic shock ( 1-13). However, although
mRNA has been detected in cytokine-stimulated en-
dothelial ( 1 4) and vascular smooth muscle cells (15-
20; [VSMCI) in culture, there are no in viva studies
examining this question. Several recent advances
make possible the further study of this important
question. First, a specific cDNA probe for VSMC iNOS
has been developed ( 1 9). Second, in contrast to the
widely used L-aginine analogues, aminoguanidine
(AG) has been shown to inhibit iNOS preferentially
while maintaining the integrity of constitutive endo-
thelial NOS (eNOS) (8,2 1 ,22). Thus, to further advance
the understanding of this Important Issue, aortas
from LPS-treated rats, an in vivo model ofsepsis, were
examined for the presence ofiNOS mRNA by the use of
the selective probe from an iNOS cDNA cloned from
VSMC. The functional significance of the expressed
iNOS mRNA was then investigated by studying the
effect of AG on the phenylephrine-evoked contractionin aortic rings from LPS-treated rats. The preferential
effect of AG In iNOS was confirmed with aortic rings
with endothelium tested with acetylcholine, a known
stimulator of endothelial NOS.
MATERIALS AND METHODS
Molecular Biology
Male Sprague-Dawley rats (250 to 300 g; Sasco, Omaha,NE) were injected with LPS (from Escherichla coil 0 1 27:B8),20 mg/kg, or with only 1 mL of saline (vehicle) ip as prey-ously described (9). Four hours after the animals were anes-
thetlzed with pentobarbital (50 mg/kg ip), their descendingthoracic aortas were isolated, placed in cold physiologicsaline, cleaned ofclots, and frozen in liquid nitrogen. In someaortas, the endothelium was mechanically removed and aring of these aortas was cut to confirm the absence of
functional endothelium with acetylcholine ( 1 �.tM) after con-
traction with phenylephrine ( 1 .tM; as described below). Afterpulverization under liquid nitrogen, total RNA was extractedby the use of a simplified guanidinium thiocyanate protocol(23: RNAzol-B method (Teltest, Inc., Friendswood, lxi). Aor-tas of two anImals were pooled for each extraction; threepairs of aortas from each of the experimental groups wereused. Extracted RNA was quantified by absorbance at 260nm and frozen at -80#{176}Cuntil northern analysis. Twentymicrograms of total RNA was size fractionated by formalde-hyde-i .2% agarose gel electrophoresis. electrically trans-ferred to Hybond-N membrane (Amersham. Arlington
Aortic NOS Expression
2068 Volume 5 . Number 12 ‘ 1995
Heights, IL), and cross-linked by UV radiation. Pictures of the
ethidium bromide-stained rRNA were obtained before trans-fer to confirm equal RNA loading in all lanes. The membraneswere prehybridIzed at 42#{176}Cfor 2 to 3 h in a buffer of 50%formamide, 5X sodium saline-citrate buffer (SSC), 5x Den-
hardt’s solution, 0.2% sodium dodecyl sulfate (SDS), 50 mMsodium phosphate (pH 7.0), and 200 �g/mL heat-denaturedherring sperm DNA and then hybridized for 18 h at 42#{176}Cinthe same solution with the radiolabeled cDNA probe. An
(a32PIdCTP-labeled cDNA probe (specific activity of0.5 x i09to i X iO� cpm/�g DNA) was prepared by random priming(Megaprime; Amersham) from a cDNA fragment correspond-ing to the 5-end (nucleotides 1 to 830) ofthe 3441-base-pair
VSMC NOS ( 1 9). This cDNA was kindly provided by Dr.Nunokawa (19). The membranes were washed four times atroom temperature in 2X SSC. 0. 1% SDS solution and twice
at 55#{176}Cin 0. 1 x SSC. 0. 1 % SDS solution. Then, X-OMAT ifim(Kodak. Rochester, NY) was exposed to the blots at -80#{176}Cwith an intensifying screen for 1 2 to 48 h.
Organ Chamber Studies
Three groups of male Sprague-Dawley rats (250 to 300 g)were used: rats treated with ip LPS (20 mg/kg; 4 h); ratstreated with its vehicle (ip saline); and untreated rats. Theywere anesthesized with pentobarbital (50 mg/kg ip). In eachgroup, the thoracic aortas were isolated and placed In a coldphysiologic salt solution with the following milhimolar com-position: NaC1, 1 16.3; KC1, 5.4; MgSO4, 0.83; NaH2PO4, 1.04;
CaCl (H20)2, 1.8; NaHCO3, 19.0; glucose, 5.5; phenol redsodium was present at 0.01 1 g/L as a pH indicator (controlsolution). The aortas were cleaned of surrounding tissuesand cut Into four to six rIngs (2 to 3 mm in length). Theendothelium was removed by gentle mechanical abrasion.The rings were suspended between two stainless-steel stir-nips in 10 mL ofcontrol solution (37#{176}C)and gassed with 74%N2, 21% 02, and 5% CO2 (pH 7.4). One of the stirrups wasanchored, and the other was connected to a strain gauge(Grass FT03; Grass Instruments, Qulncy. MA) to record
changes in Isometric force. After equilibration and stepwiseincreases in resting tension to 2 g in 30 mm, two sequentialcontractions were evoked with phenylephrine hydrochloride( 1 tiM) and the tissues were tested for the presence of theendothelium with acetylcholine hydrochloride ( 1 �.tM). All
rings were washed with control solution and treated withindomethacin (I 10 �iMI prepared with equimolar sodiumcarbonate and sonicated until dissolved) to inhibit the pro-duction of endogenous prostanoids. The rings from the threegroups ofrats were incubated for 30 min with the preferentialinhibitor ofiNOS, AG hemisulfate (300 �M (8)), with equimo-lar sodium sulfate (150 �tM) or with vehicle (distilled water,50 �L (control ringsl). Then, concentration-response curvesto phenylephrine ( 1 nM to 10 �M) were performed. Additionalrings from control rats were Incubated for 6 h in controlsolution in the presence or absence of LPS ( 1 �g/mL) beforecontraction with phenylephrine. Some of the latter rings.exposed and unexposed to LPS, were incubated with AG (300�M) during the last 30 mm. The rings were then removedfrom the organ chambers, air dried for 48 h, and weighed.The following parameters (expressed as means ± SE) werecompared: the maximal contraction to phenylephrine (Em�;
in grams of force per milligram of dried tissue) and thelogarithm of the effective molar concentration of phenyleph-rine causing 50% of maximal contraction (log EC5O). A two-tailed unpaired t test was used to compare the above param-
eters between control rings of LPS- and saline-treated rats:
the effect of AG between rings of the same rat, either LPS orsaline treated, was compared by a two-tailed paired t test.The effect of control solution, LPS, and/or AG in ringsincubated for 6 h was evaluated by analysis of variance(Scheff#{233}’s test). Differences between treatments were consid-
ered significant when P < 0.05.
In other experiments, aortic rings with a functional endo-thelium obtained from control rats and not subjected todenudation were incubated with and without AG (300 j.�M; 30min) and contracted with 1 �M phenylephrine. Once a pla-teau was reached, a concentration-response curve to acetyl-choline (1 nM to 10 �M) was performed to test if thisconcentration of AG affected endothelium-dependent relax-ation. Results are expressed as percentage of phenylephrine-evoked contraction ± SE. Maximal relaxation to acetylcho-
line and the logarithm of the effective molar concentration ofacetylcholine causing 50% of maximal relaxation (log EC5O)were compared by a paired t test.
All experiments were performed in parallel with a singleconcentration-response curve per ring. and N is the numberof rings from different rats. All reagents and drugs used inmolecular biology and physiologic studies were purchasedfrom Sigma (St. Louis, MO) unless otherwise stated.
RESULTS
Molecular Evidence for INOS Expression inAortas From LPS-Treated Rats
Northern analysis of total RNA extracted from aortas
with and without functional endothelium obtained
from rats treated with LPS (20 mg/kg ip; 4 h) indicated
that iNOS mRNA was expressed in these blood yes-
sels; however, total RNA obtained from aortas with or
without endothelium from control rats did not express
iNOS mRNA (Figure 1 ). By comparison with 285 rRNA
and an RNA ladder, the calculated size of the ex-
pressed iNOS is approximately 4.4 kilobase pairs.
Functional Evidence for iNOS Activity in AortasFrom LPS-Treated Rats
Endothelium-denuded aortic rings from LPS-
treated rats (20 mg/kg Ip; 4 h) showed a marked
reduction in reactivity and contractility to phenyleph-
rine (Table 1 and Figure 2b) when compared with rings
obtained from saline-treated rats (Table 1 and Figure
2a). The incubation ofrings from LPS-treated rats with
AG (300 �M; 30 mm) significantly enhanced both
reactivity and contractility to phenylephrine (Table 1
and Figure 2b). However, AG did not affect either the
reactivity or the contractility to phenylephrine in rings
obtained from saline-treated rats (Table 1 and Figure
2a). Sodium sulfate ( 150 jIM) had no effect on the
phenylephrine-evoked contractions in rings from LPS-
or saline-treated rats (N = 6; data not shown).
The in vitro incubation of aortic rings without endo-
thelium from untreated rats with LPS caused a shift to
the right In the concentration-response curve to phe-
nylephrlne relative to rings incubated in the control
solution (log EC50 control versus LPS: -7.5 ± 0.1
versus -7. 1 ± 0. 1 ; P < 0.05; N 7) and a nonsignif-
CTLP CTLP A
-8 .7 .6 -5 -4
Phenylephrine log (M)
B
DOEDO
0,
L.C
0,
DOEDO
0,Cs1�C
Cs
DOEDO
Cs
C
-9 -8 .7 -6
Phenylephrine log (M)
C
.5 .4
3
2
a p < 0.0001 when compared with control from saline-treated rats.b p < 0.001 when compared with control from LPS-treated rats.
1
0-10 -9 -8 -7 -6 -5 -4
Weigert et al
Journal of the American Society of Nephrology 2069
- INOS � � 28s
� - 1
CT LP
, -28s
�i-18s
without withEndothelium Endothelium
Figure 1. Northern blots of total RNA (20 � extracted fromaortas of IPS (U’; 20 mg/kg ip; 4 h)- or normal saline-treatedrats (CT). Left pair, aortas without endothelium; right pair,aortas with endothelium. The RNA was electrically transferredto a Hybond-N membrane and hybridized with an 830-base-pair a32P-Iabeled probe obtained from the 5-end of theVSMC iNOS. The positions of 18S and 28S rRNA are depictedon the right side of the blot. A picture of the 18S and 28S RNAbands on the formaldehyde/agarose gel is depicted todemonstrate equal loading of RNA In both lanes.
TABLE 1 . Phenylephrine-evoked contraction of aorticrings without endothelium from saline- andLPS-treated (Rx) rats in the presence and absence ofAG (300 �M; 30 mm)
Group log EC� Emax N
Saline-Rx Control -7.9 ± 0.1 3.5 ± 0.2 12
Saline-Rx AG -7.8 ± 0.1 3.2 ± 0.2 12LPS-Rx Control -6.7 ± 0.2#{176} 1 .0 ± 0.2#{176} 8LPS-Rx AG -7.3 ± 0.2k’ 2.8 ± 0.2k’ 8
I’henylephrinc lug (M)
Icant decrease in maximal contraction (Em� control
versus LPS: 3.0 ± 0.2 versus 2.4 ± 0.2 g/mg). In other
endothelium-denuded aortic rings incubated with
LPS, the addition ofAG caused a significant enhance-
ment in vascular reactivity (log EC50 LPS + AG versus
LPS: -7.7 ± 0.1; P < 0.01; N - 7) and contractility
(Em� LPS + AG: 3.6 ± 0.2 g/mg; P < 0.0 1 ; [Figure
2c]). AG did not significantly affect the vascular reac-
tivity or contractility of aortic rings without endothe-
hum incubated for 6 h in control solution (N = 7; data
not shown).
Figure 2. Phenylephrine-evoked contraction of rat aorticrings without endothelium from: (a) saline-treated rats (1 mLip; 4 h) In the presence (squares) or absence (circles) of AG(300 jIM; 30 mm; N = 12); (b) LPS-treated rats (20 mg/kg Ip; 4h) in the presence (squares) or absence (circles) of AG (300�M; 30 mm; N = 8); (c) aortic rings without endothelium fromcontrol rats treated in vitro with LPS (1 �g/mL; for 6 h) in thepresence (squares) or absence (circles) of AG (300 �M; 30mm; N = 7). Data are shown as means ± SE.
120
100
80
60
40
20
C0
U
C0U
V
0
CC
0
C
a,a,
Aortic NOS Expression
2070 Volume 5 . Number 12 ‘ 1995
Absence of Effect of AG Either onPhenylephrine-Evoked Contraction or onAcetylcholine-Evoked Relaxation in Aortas WithEndothelium From Control Rats
In aortic rings with endothelium from saline-treated
rats, AG (300 �M) did not affect the contraction evoked
by 1 pM phenylephrine (control versus AG: 1 .3 ± 0.2
versus 1 .5 ± 0.2 g/mg). The endothelium-dependent
relaxation to acetylcholine ( 1 nM to 10 jtM) was not
affected by AG either (Figure 3). The log EC50 values
for control versus AG were -7.0 ± 0. 1 versus -7. 1 ±
0. 1 ; the maximal relaxations were 86 ± 5 versus 86 ±
4% ( 1 4 ± 5 versus 1 4 ± 4% of phenylephrine-evoked
contraction [N = 51).
DISCUSSION
The increased production of nitric oxide has been
suggested to be important in septic shock. Biochemi-
cal and pharmacologic data suggest that LPS and
cytokines (e.g., interleukin- 1f3 and tumor necrosis
factor-a) increase nitric oxide production In isolated
blood vessels (1,2,4-6,8-10,12,13,22) and in both
endothelial ( 1 ,24) and VSMC in culture (7, 1 1,25).
Julou-Schaeffer et at. (9) demonstrated that the hypo-
responsiveness of aortas from endotoxemic rats was
reversed by L-aginlne analogues, which nonselec-
tively inhibit NOS activity. Using a similar murine
model of sepsis, we have extended these phamaco-
logic observations by demonstrating with northern
analysis the expression of iNOS mRNA in aortas of
endotoxemic rats. The in vitro expression of iNOS
mRNA has been observed previously in RNA extracted
from LPS- or interleukin 1 j3-stimulated endothelial
cells and VSMC in culture ( 1 4-20). The observation
-10 -9 -8 -7 -6 -5 -4
Aeelylt’holine log I %1)
Figure 3. Absence of effect of AG (300 �M; 30 mm) on theacetylchollne-evoked relaxation In aortlc rings with endo-thellum contracted with 1 �M phenylephrine. Circles repre-sent control rings, and Squares represent rings Incubatedwith AG. Data are shown as means ± SE and are expressedas the percentage ofthe phenylephrine-evoked contractionof force per milligram of tissue (N = 5).
presented here that INOS mRNA expression occurs invivo extends the findings in stimulated cultured cells,
thus supporting the potential pathophysiologic corre-
late of this in vitro observation. The size ofiNOS mRNA
extracted from blood vessels of endotoxemic rats is
similar to that observed in studies using extracts from
cultured endothelial cells and VSMC stimulated with
cytokines (approximately 4.4 kilobases; Figure 1).
The VSMC iNOS has been cloned and shows 93%
homology with the isoform found in macrophages but
only 50% homology with eNOS (19). The 830-base-
pair cDNA fragment corresponding to the 5’-end of the
VSMC 1NOS used to generate our probe therefore
shares minimal sequence similarity with eNOS. If this
probe was not specific for iNOS, we should have found
hybridization with eNOS in the RNA extracted from
aortas with endothellum of control animals; however,
the iNOS message was only detected in aortic total
RNA extracted from rats exposed to LPS. Interleukin
1 f3-activated rat aortic endothellal cells can also ex-
press iNOS ( 1 4) in addition to constitutive eNOS. In
this study, the persistence of iNOS expression in
aortas without functional endothelium suggests that
VSMC can express iNOS in viva; the results, however,
do not exclude that resident or invading macrophages
or other cells may express 1NOS in the blood vessels ofLPS-treated rats.
There are studies that suggest that LPS may stim-
ulate iNOS in organs other than blood vessels. For
example, rats treated with Propionibacterium acnes
and LPS expressed mRNA for the hepatic (Ca2�-de-
pendent) iNOS isoform and Ca2�-dependent NOS ac-
tivity in the liver, lung, spleen, and colon; blood
vessels were not evaluated (26). Moreover, the NOS
activity induced by LPS in blood vessels is Ca2�
independent (4). An immunohistochemical study us-
Ing a polyclonal antibody for hepatic iNOS isoform
reported positive staining of the aortic endothelial
cells from LPS-stimulated rats (27), but detection wasequivocal in VSMC (27). LPS treatment In viva resulted
in the expression of INOS mRNA in lung, liver, spleen,
skeletal muscle, and kidney, but again, blood vessels
were not reported (28). These results are therefore thefirst to demonstrate the expression of iNOS mRNA in
the vascular tissues of endotoxemic animals.
Next, in this study, AG, a preferential inhibitor of
iNOS (8,2 1 ,22), was used to test the functional signif-
icance of iNOS mRNA expression in the rat aorta. AG
enhanced the phenylephrine-evoked response in en-
dothelium-denuded aortic rings of LPS-treated rats.
This pharmacologic fmding with AG extends previous
experiments using nonselective L-aginine analogues
(2,4,6,7,9,10,12,13,25), which inhibit both INOS and
eNOS. These results of the reversal by AG of the
hyporesponsiveness observed in aortic rings of un-
treated rats incubated in vitro with LPS are consistentwith recently published studies that compared AG
with L-arginine analogues (22). Those authors found
AG equipotent to the L-aginine analogues in endothe-
hum-denuded rings exposed to LPS but detected no
Weigert et al
Journal of the American Society of Nephrology 2071
action on control rings with endothelium. Equimolar
L-arglnine prevented the action of AG. In this study,
AG also did not affect the responsiveness of control
rings without endothelium from saline-treated rats.
Previous observations have also shown that the hypo-
responsiveness of the pulmonary arteries of endotox-
emic rats is reversed by AG (8). Additionally, in this
study, AG did not affect endothelium-dependent re-
laxation to acetylcholine in aortic rings. A similar
finding was observed in the pulmonary arteries of
control rats (8) and in the rat aorta with a lower
concentration of AG (22). Other observations have
shown the preferential inhibition of INOS relative to
the constitutive neuronal and endothelial NOS by AG
using purified enzyme preparations (2 1 ). These re-
sults from septic rats demonstrating the preferential
functional inhibition of iNOS has potential clinical
implications. Specifically, AG’s preferential inhibition
of iNOS is appealing as compared with the nonselec-
tive effects of L-aginine analogues because the regu-
lated release of NO by endothelial cells is maintained
while the production of nitric oxide by iNOS is
blocked. Despite some reports suggesting that LPS or
cytokines may impair endothelium-dependent relax-
ation ( 13,29,30) or cause increased degradation of
eNOS mRNA ( 1 7,3 1 ), the microvasculature of endo-
toxemic rats shows normal acetylcholine relaxation
(5). This suggests that the ability of local blood flow
regulation by the endothelium is preserved in these
animals. AG’s preferential action on iNOS, while
maintaining eNOS integrity, may therefore be advan-
tageous in the therapy of sepsis and other states
associated with the overproduction of Inflammatory
cytokines. For example, in spite of the data supporting
the marked Increase of nitric oxide production in
animal models of sepsis and in septic patients, both
the biologic significance of this hyperproduction andthe desirability in intervening in that pathway with
nonselective inhibitors, i.e. , L-aginine analogues, are
controversial. The unexpected increased mortality ob-
served in septic dogs treated with L-arginine ama-
logues (32), after encouraging initial reports (3), is
particularly disturbing. The nonselective effects of
L-aginine analogues to impair eNOS integrity could
counterbalance and obscure a beneficial effect of
blocking iNOS. Therefore, survival studies and sys-
temic and regional hemodynamic evaluations are nec-
essary to further evaluate the usefulness of AG in
these states. This more selective approach would
block iNOS while preserving the endothelial regulation
of the microvasculature by the constitutive eNOS.
In summary, we have demonstrated in aortas ofLPS-treated animals both the In viva expression of
iNOS mRNA and the reversal of LPS-induced hypore-
sponsiveness to phenylephrine by AG, a preferential
inhibitor of 1NOS. These data extend previous blo-
chemical and pharmacologic evidence of the expres-
sian of iNOS activity by LPS. In addition, this model
may be an excellent positive control to evaluate the
expression of iNOS in other diseases thought to be
associated with induction of iNOS in viva. e.g., hepatic
cirrhosis. Finally, the results confirm the usefulness
of AG in the study of diseases where iNOS is ex-
pressed. Further studies will be necessary to examine
the mechanisms involved in the LPS-medlated iNOS
expression in viva and whether INOS mRNA abun-
dance increases by transcriptional activation or by
reduced mRNA degradation.
ACKNOWLEDGMENTSWe thank Benjamin Perryman. PhD, from the Division of Cardiology.Department of Medicine. UCHSC for invaluable advice; CarolynBurke for graphical assistance; and Dr. Nunokawa. Suntory Institute
for Biomedical Research. Osaka. Japan. for providing the cDNA used
in the northern analysis.
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